Use of cholesin in modulating cholesterol homeostasis

Abstract

The present disclosure provides use of cholesin in modulating cholesterol homeostasis and a composition comprising cholesin, a nucleic acid encoding cholesin, an expression vector comprising the nucleic acid, or an agent for enhancing the expression or activity of endogenous cholesin protein in a subject. Specifically, the present disclosure relates to a method for treating or preventing a disease associated with an elevated plasma cholesterol level in a subject. The method comprises: administering to the subject an effective amount of cholesin, a nucleic acid encoding cholesin, or an expression vector comprising the nucleic acid, or enhancing the expression or activity of endogenous cholesin protein in the subject.

Description

Use of cholesin in regulating cholesterol homeostasis

[0001] This international patent application claims priority to Chinese patent application No. 202311385254.5 filed on October 24, 2023, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0002] The present invention belongs to the field of biomedicine, and particularly relates to the use of the intestinal secretory hormone Cholesin in regulating cholesterol homeostasis, and its use in regulating cholesterol homeostasis after being used in combination with other cholesterol-lowering drugs. Background Art

[0003] Cholesterol homeostasis is coordinated by various tissues to maintain a balance between dietary cholesterol absorption, de novo synthesis, and bile clearance and excretion. Elevated blood cholesterol levels, particularly low-density lipoprotein cholesterol (LDL-C), contribute to the development of atherosclerosis and are considered a significant risk factor for cardiovascular disease (CVD). CVD is a pressing public health concern, accounting for over 30% of deaths worldwide. Both dietary cholesterol and bile cholesterol are taken up by the Niemann-Pick C1 like 1 (NPC1L1) protein, located on the apical surface of enterocytes in the intestine. Following absorption, dietary cholesterol is released as chylomicrons and then taken up by the liver, the primary site of cholesterol synthesis. Cholesterol molecules from de novo synthesis and intestinal absorption are transported through the circulation and used by cells. To maintain cholesterol homeostasis, excess cholesterol is excreted in the feces through bile secretion.

[0004] De novo cholesterol synthesis is an energy-consuming process, generated from acetyl-CoA under the control of a series of enzymes. HMGCR (3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase) is the rate-limiting enzyme, whose activity is controlled at both transcriptional and post-transcriptional levels. SREBP2 (sterol regulatory element binding protein 2) acts as a master transcription factor, controlling the expression of HMGCR and other genes involved in cholesterol biosynthesis and uptake. Cholesterol biosynthesis and uptake are tightly regulated through a negative feedback mechanism that senses cellular cholesterol levels. When cells are cholesterol-deficient, SREBP2, along with its escort protein SCAP (SREBP cleavage-activating protein), is transported from the endoplasmic reticulum (ER) to the Golgi apparatus in COPII vesicles. Within the Golgi, SREBP2 is sequentially cleaved by site 1 and site 2 proteases. The N-terminal structure of SREBP2, released by this cleavage, travels to the nucleus, where it acts as a transcription factor, enhancing the expression of genes involved in cholesterol biosynthesis and uptake. Conversely, when cellular cholesterol levels rise, cholesterol molecules bind to SCAP, triggering its interaction with INSIG (insulin-induced gene). This interaction retains SREBP in the ER and prevents subsequent activation of SREBP and expression of genes involved in cholesterol metabolism. This mechanism helps maintain cholesterol homeostasis. Both endogenously synthesized and exogenously acquired cholesterol are secreted into the blood in the form of very low-density lipoproteins (VLDL). After processing in the blood, VLDL gives rise to circulating LDL, which can be taken up by cells via LDL receptor (LDLR)-mediated endocytosis. Although the regulation of cholesterol homeostasis in response to cholesterol levels is well understood, our understanding of how signals other than cholesterol influence cholesterol synthesis remains limited.

[0005] The interplay between cholesterol synthesis and absorption is known to regulate the response of circulating cholesterol to dietary or cholesterol-lowering interventions. However, the specific factors responsible for orchestrating the balance between cholesterol absorption and synthesis, and how precisely these factors regulate this balance, remain poorly understood.

[0006] Currently, three major classes of cholesterol-lowering drugs are commonly used to prevent and treat hypercholesterolemia. The first class, ezetimibe, inhibits the endocytosis of NPC1L1, thereby blocking cholesterol uptake. The second class, statins, reduces cholesterol synthesis by inhibiting HMGCR and increases LDL uptake by upregulating LDLR. The third class, PCSK9 inhibitors, stabilize the LDLR and promote hepatic LDL absorption. Although ezetimibe reduces intestinal cholesterol absorption, its compensatory effect of enhancing cholesterol synthesis limits its effectiveness in lowering plasma cholesterol levels. The cholesterol-lowering effects of statins and PCSK9 inhibitors are both LDLR-dependent, posing a challenge for patients with LDLR deficiency or insufficiency. Furthermore, compensatory increases in HMGCR levels worsen outcomes in some patients who discontinue statins after long-term treatment. Furthermore, statins can significantly increase the expression of HMGCR and other cholesterol-promoting genes, raising questions about their efficacy.

[0007] Therefore, finding new cholesterol-lowering drugs has important biological significance for the treatment of diseases associated with elevated plasma cholesterol levels, including hypercholesterolemia and atherosclerosis.

[0008] Summary of the Invention

[0009] The present invention aims to provide methods for treating diseases associated with elevated plasma cholesterol levels, including hypercholesterolemia and atherosclerosis. The inventors unexpectedly discovered that cholesin, a previously unreported hormone secreted by the intestine in response to cholesterol absorption, can inhibit cholesterol synthesis and VLDL secretion in the liver, thereby lowering plasma cholesterol levels and preventing diseases associated with elevated plasma cholesterol levels, such as hypercholesterolemia and atherosclerosis. Mechanistic studies have demonstrated that cholesin inhibits PKA signaling and SREBP2-controlled cholesterol synthesis by binding to its receptor GPR146 ( FIG30 ). Therefore, the cholesin-GPR146 axis is the molecular link between intestinal cholesterol absorption and the inhibition of cholesterol synthesis in the liver. Therefore, exogenous cholesin or methods or agents that promote the expression or activity of endogenous cholesin protein in a subject can lower plasma cholesterol and / or triglyceride levels, thereby preventing or treating diseases associated with elevated plasma cholesterol levels, such as hypercholesterolemia and atherosclerosis. Compared to the three major classes of existing cholesterol-lowering drugs, Cholesin produces an LDLR-independent downregulation of the expression of genes involved in cholesterol synthesis and reduces plasma cholesterol levels, suggesting that Cholesin treatment is an effective strategy for combating conditions associated with elevated plasma cholesterol levels, such as hypercholesterolemia and atherosclerosis. Furthermore, because Cholesin can reduce weight gain and triglyceride levels, combining Cholesin with existing cholesterol-lowering drugs, such as statins, is also a promising strategy for treating conditions associated with elevated plasma cholesterol levels, including hyperlipidemia and atherosclerosis.

[0010] Accordingly, in one aspect, the present invention provides a method for lowering plasma cholesterol levels and / or triglyceride levels in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

[0011] In some embodiments, the plasma cholesterol is selected from plasma total cholesterol and plasma LDL-cholesterol.

[0012] In another aspect, the present invention provides a method for inhibiting hepatic cholesterol synthesis in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

[0013] In some embodiments, the method inhibits the expression of a gene associated with cholesterol synthesis, and the gene is selected from Hmgcr, Hmgcs1, Mvd, Mvk, Pmvk, and Sqle. In some embodiments, the method inhibits the expression of a gene associated with cholesterol uptake, and the gene is Ldlr.

[0014] In some embodiments, the method inhibits expression of a transcription factor that promotes expression of genes involved in cholesterol synthesis or uptake, and the transcription factor is SREBP2.

[0015] In another aspect, the present invention provides a method for inhibiting hepatic VLDL-cholesterol secretion in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

[0016] In another aspect, the present invention provides a method for treating or preventing a disease associated with elevated plasma cholesterol levels in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

[0017] In an embodiment of the method of the present invention, the Cholesin protein is a human Cholesin protein.

[0018] In some embodiments, the nucleic acid is DNA or RNA.

[0019] In some embodiments, the expression vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

[0020] In some embodiments, the disease associated with elevated plasma cholesterol levels is selected from hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, nephrotic syndrome, and gallstones.

[0021] In some embodiments, the disease associated with elevated plasma cholesterol levels is hypercholesterolemia.

[0022] In some embodiments, the disease associated with elevated plasma cholesterol levels is atherosclerosis.

[0023] In some embodiments, the method further comprises the step of administering a second therapeutic agent, preferably, the second therapeutic agent is selected from the group consisting of a protein or peptide, a nucleic acid, and a small molecule drug.

[0024] In some embodiments, the second therapeutic agent is a cholesterol-lowering drug selected from the group consisting of an HMGCR inhibitor, a PCSK9 inhibitor, and a cholesterol absorption inhibitor.

[0025] In some embodiments, the HMGCR inhibitor is a statin.

[0026] In some embodiments, the statin is selected from rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin, preferably rosuvastatin.

[0027] In some embodiments, the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran, and Tafolecimab.

[0028] In some embodiments, the cholesterol absorption inhibitor is an NPC1L1 inhibitor, preferably ezetimibe or hebomib.

[0029] In some embodiments, the second therapeutic agent is administered before, after, or simultaneously with the Cholesin protein, the nucleic acid, or the expression vector.

[0030] In some embodiments, the subject is a human.

[0031] In some embodiments, the subject is unresponsive or intolerant to one or more of a cholesterol absorption inhibitor, an HMGCR inhibitor, and a PCSK9 inhibitor, eg, the subject has an LDL receptor (LDLR) deficiency.

[0032] In another aspect, the present invention provides a composition comprising a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of human endogenous Cholesin protein, and a pharmaceutically acceptable carrier or excipient.

[0033] In some embodiments, the composition is used for one or more of the following:

[0034] 1) reducing plasma cholesterol levels in a subject;

[0035] 2) reducing plasma total cholesterol or plasma LDL-cholesterol levels in a subject;

[0036] 3) reducing plasma triglyceride levels in a subject;

[0037] 4) inhibiting hepatic cholesterol synthesis in a subject;

[0038] 5) inhibiting hepatic VLDL-cholesterol secretion in a subject; and

[0039] 6) Treating or preventing a disease associated with elevated plasma cholesterol levels in a subject.

[0040] In some embodiments, the Cholesin protein is a human Cholesin protein.

[0041] In some embodiments, the composition further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from proteins or peptides, nucleic acids and small molecule drugs.

[0042] In some embodiments, the second therapeutic agent is selected from cholesterol-lowering drugs.

[0043] In some embodiments, the cholesterol-lowering drug is selected from the group consisting of an HMGCR inhibitor, a PCSK9 inhibitor, and a cholesterol absorption inhibitor.

[0044] In some embodiments, the HMGCR inhibitor is a statin.

[0045] In some embodiments, the statin is selected from rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin, preferably rosuvastatin.

[0046] In some embodiments, the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran, and Tafolecimab.

[0047] In some embodiments, the cholesterol absorption inhibitor is an NPC1L1 inhibitor, preferably ezetimibe or hebomibe.

[0048] In some embodiments, the composition is formulated as an injectable preparation or an oral preparation, such as a tablet, capsule, granule, or suspension. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1: Silver staining and immunoblotting results of serum proteins from mice fed a regular diet (RD) and a Western diet (WD).

[0050] Figure 2A: Localization of human Cholesin (hCholesin) and mouse Cholesin (mCholesin) in cells detected by immunofluorescence assay.

[0051] Figure 2B: Secretion of human and mouse Cholesin in the culture medium detected by immunoblotting.

[0052] FIG3A : Plasma Cholesin levels of fasting, RD, and WD mice measured by ELISA.

[0053] Figure 3B: Changes in plasma cholesin levels in human clinical samples after fasting and 1 h feeding.

[0054] Figure 4: Cholesin protein levels in different mouse tissues. Cholesin knockout (KO) mice served as controls.

[0055] Figure 5A: Plasma cholesin levels and small intestinal total cholesterol levels in fasted and WD-fed WT and IKO mice.

[0056] FIG5B : Plasma total cholesterol levels and plasma triglyceride levels in fasted and WD-fed WT and IKO mice.

[0057] Figure 5C: Plasma cholesin levels and small intestinal total cholesterol levels in WT mice and IKO mice gavaged with corn oil (chol-) and cholesterol (chol+).

[0058] FIG5D : Plasma total cholesterol levels and plasma triglyceride levels in WT mice and IKO mice gavaged with corn oil (chol-) and cholesterol (chol+).

[0059] Figure 6: Cellular localization of Cholesin observed by immunofluorescence experiments.

[0060] Figure 7: Cholesin secreted in the culture medium and total cholesterol levels in HCT116 cells overexpressing Cholesin after cholesterol stimulation.

[0061] FIG8A : Cholesin secretion from Cholesin-overexpressing HCT116 cells with NPC1L1 knockdown (NPC1L1 KD) detected by immunoblotting. Cholesin-overexpressing HCT116 cells without NPC1L1 knockdown (KD) served as a control.

[0062] Figure 8B shows the secreted Cholesin level in the culture medium and the total cholesterol level in the cells after co-culture with cholesterol in NPC1L1 knockdown (NPC1L1 KD) Cholesin-overexpressing HCT116 cells. NPC1L1 non-knockdown (NT) Cholesin-overexpressing HCT116 cells served as a control.

[0063] Figure 9A: Plasma cholesin levels and total cholesterol levels in the small intestine of mice treated with ezetimibe (Ezetimlbe+) after oral administration of cholesterol (chol+). Mice not treated with ezetimibe (Ezetimlbe-) served as controls.

[0064] Figure 9B: Total cholesterol levels in plasma of mice treated with ezetimibe after oral administration of cholesterol. Mice not treated with ezetimibe served as controls.

[0065] FIG9C : Npc1l1 gene knockout detected by immunoblotting (Npc1l1 - / - ) mice secrete Cholesin. + / + ) mice served as controls.

[0066] Figure 9D: Npc1l1 gene knockout (Npc1l1 - / - ) mice after oral administration of cholesterol. + / + ) mice served as controls.

[0067] Figure 9E: Npc1l1 gene knockout (Npc1l1 - / - ) mice after oral administration of cholesterol. + / + ) mice served as controls.

[0068] FIG10A : Correlation between plasma Cholesin levels and plasma total cholesterol (TC) levels in humans.

[0069] FIG10B : Correlation between plasma Cholesin level and plasma low-density lipoprotein cholesterol (LDL-C) level.

[0070] Figure 11A: Plasma total cholesterol and triglyceride levels in wild-type (WT) and intestinal cholesin-specific knockout (IKO) mice after feeding RD and WD.

[0071] Figure 11B: Levels of total cholesterol and triglycerides in the liver of wild-type (WT) and intestinal cholesin-specific knockout (IKO) mice fed RD and WD.

[0072] Figure 11C: Plasma total cholesterol levels and levels of HDL, IDL / LDL, and VLDL in wild-type (WT) and intestinal cholesin-specific knockout (IKO) mice after feeding RD and WD, as measured by fast protein liquid chromatography.

[0073] FIG. 12A : Body weight of wild-type (WT) and intestinal cholesin-specific knockout (IKO) mice after feeding RD and WD.

[0074] FIG12B : Body fat percentage of wild-type (WT) and intestinal cholesin-specific knockout (IKO) mice after feeding RD and WD.

[0075] FIG. 12C : Food intake of wild-type (WT) and intestinal cholesin-specific knockout (IKO) mice fed RD and WD.

[0076] FIG. 12D : Water intake of wild-type (WT) and intestinal cholesin-specific knockout (IKO) mice fed RD and WD.

[0077] Figure 12E: Small intestinal total cholesterol and triglyceride levels in wild-type (WT) and intestinal cholesin-specific knockout (IKO) mice after feeding RD and WD.

[0078] FIG12F : Chylomicron breakdown rates in the small intestine of wild-type (WT) and intestinal cholesin-specific knockout (IKO) mice fed RD and WD, as assessed by lipid tolerance assay.

[0079] FIG13A : Gallbladder volume, bile total cholesterol concentration, and bile acid concentration in wild-type (WT) and intestinal cholesin-specific knockout (IKO) mice fed RD and WD.

[0080] FIG. 13B : Fecal total cholesterol and triglyceride levels in wild-type (WT) and intestinal cholesin-specific knockout (IKO) mice after feeding RD and WD.

[0081] FIG. 14A : mRNA levels of cholesterol synthesis-related genes in the livers of WT and IKO mice fed RD and WD, determined by qPCR.

[0082] FIG14B : Protein expression levels of SREBP2, HMGCR, HMGCS1 and LDLR in the livers of WT and IKO mice fed RD and WD as determined by immunoblotting.

[0083] FIG15 : Poloxamer 407-injected plasma VLDL secretion rates in WT and IKO mice fed RD and WD.

[0084] Figure 16A: Cholesin and wild type (Gpr146 + / + ) and Gpr146 knockout (Gpr146 - / - ) Combined immunofluorescence staining of various mouse tissues.

[0085] Figure 16B: Wild type (Gpr146 + / + ) and Gpr146 knockout (Gpr146 - / - ) Immunofluorescence staining of mouse primary hepatocytes binding to Cholesin.

[0086] FIG16C : Wild type (Gpr146 + / + ) and Gpr146 knockout (Gpr146 - / - ) Binding saturation curve of Cholesin between primary mouse hepatocytes.

[0087] FIG17 : Binding affinity curves of Cholesin to wild-type (WT) and mutant (Mut) GPR146.

[0088] FIG18 : Interaction of GPR146 with GNAI1 detected by co-immunoprecipitation in the presence and absence of Cholesin.

[0089] Figure 19: Wild type (Gpr146) treated with Cholesin + / + ) and Gpr146 knockout (Gpr146 - / - ) cAMP levels in mouse primary hepatocytes.

[0090] FIG20 : Hmgcr mRNA levels in mouse liver after intraperitoneal injection of Cholesin.

[0091] FIG21A : Experimental scheme for intraperitoneal injection of Cholesin into mice.

[0092] FIG. 21B : Hmgcr mRNA levels in the livers of mice injected intraperitoneally with Cholesin.

[0093] FIG. 21C : Total cholesterol levels in plasma of mice injected intraperitoneally with Cholesin.

[0094] FIG. 21D : Total cholesterol levels in the livers of mice injected intraperitoneally with Cholesin.

[0095] FIG22A : Experimental scheme for injection of adenovirus and Cholesin into mice.

[0096] FIG22B : Wild type (Gpr146 fl / fl mRNA levels of cholesterol synthesis-related genes Hmgcs1, Hmgcr, Mvd, Mvk, and Pmvk in the liver of ) mice and Gpr146 knockout (Gpr146 LKO) mice injected with adenovirus control (Vec), adenovirus expressing wild-type GPR146 (WT), or adenovirus expressing mutant GPR146 (Mut).

[0097] FIG22C : Wild type (Gpr146 fl / fl Figure 3. Protein levels of SREBP2, HMGCR, HMGCS1, LDLR, and pKA Sub in the livers of 146 (100, 157) and Gpr146 knockout (Gpr146 LKO) mice injected with adenovirus control (Vec), adenovirus expressing wild-type GPR146 (WT), or adenovirus expressing mutant GPR146 (Mut). HSP90 expression was used as a loading control.

[0098] FIG22D : Wild type (Gpr146 fl / fl Total cholesterol levels in the liver of ) mice and Gpr146 knockout (Gpr146 LKO) mice after injection of adenovirus control (Vec), adenovirus expressing wild-type GPR146 (WT), or adenovirus expressing mutant GPR146 (Mut).

[0099] FIG22E : Wild type (Gpr146 fl / fl Total cholesterol levels in plasma of ) mice and Gpr146 knockout (Gpr146 LKO) mice after injection of adenovirus control (Vec), adenovirus expressing wild-type GPR146 (WT), or adenovirus expressing mutant GPR146 (Mut).

[0100] Figure 23: Ldlr gene knockout (Ldlr - / - ) Schematic diagram of the dosing regimen for mice.

[0101] Figure 24A: Ldlr under different dosing regimens - / - Changes in total cholesterol levels in mouse plasma and its area under the curve (AUC) statistics.

[0102] Figure 24B: Ldlr under different dosing regimens - / -Total cholesterol levels in the liver of mice after 8 weeks of drug administration.

[0103] Figure 24C: Ldlr under different dosing regimens - / - Changes in plasma triglyceride levels in mice and statistics of its area under the curve (AUC).

[0104] Figure 24D: Ldlr under different dosing regimens - / - Liver triglyceride levels in mice after 8 weeks of drug administration.

[0105] Figure 25A: Ldlr under different dosing regimens - / - mRNA levels of cholesterol synthesis-related genes in the liver of mice after 8 weeks of drug administration.

[0106] Figure 25B: Ldlr under different dosing regimens - / - The expression levels of liver cholesterol synthesis-related genes Hmgcs1, Hmgcr and transcription factor SREBP2 in mice after 8 weeks of drug administration.

[0107] Figure 26: Ldlr under different dosing regimens - / - Oil red staining of the whole aorta of mice after 8 weeks of drug administration and its statistical results.

[0108] Figure 27A: Ldlr under different dosing regimens - / - Body weight changes of mice.

[0109] Figure 27B: Ldlr under different dosing regimens - / - Changes in food intake in mice.

[0110] Figure 28: Ldlr under different dosing regimens - / - HE staining of liver tissue of mice after 8 weeks of drug administration.

[0111] Figure 29A: Ldlr under different dosing regimens - / - Plasma alanine aminotransferase (ALT) levels in mice after 8 weeks of administration.

[0112] Figure 29B: Ldlr under different dosing regimens - / - Plasma aspartate aminotransferase (AST) levels in mice after 8 weeks of drug administration.

[0113] Figure 30: Schematic diagram of the mechanism by which Cholesin regulates cholesterol metabolism through its receptor GPR146. DETAILED DESCRIPTION

[0114] The above features and advantages of the present invention and their additional features and advantages will be more clearly understood below through the detailed description of the following embodiments in conjunction with the accompanying drawings. The embodiments described herein with reference to the accompanying drawings are illustrative and illustrative and are used for a general understanding of the present invention. The embodiments should not be construed as limiting the scope of the present invention.

[0115] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For example, terms used herein such as "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Definitions as described in H., ed. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0116] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, the terms "a," "an," "one or more," and "at least one" can be used interchangeably. Similarly, the terms "comprising," "including," and "having" can be used interchangeably.

[0117] When the term "comprising" is used herein and in the appended claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising or containing at least a certain number of embodiments, it should also be understood to disclose a group that preferably consists only of these embodiments.

[0118] As used herein, the term "and / or" is considered a specific disclosure of each of the two specified features or components with or without the other. Thus, as used in phrases such as "A and / or B," the term "and / or" is intended to include A and B; A or B; A (alone); and B (alone). Likewise, as used in phrases such as "A, B, and / or C," the term "and / or" is intended to encompass each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0119] The ranges recited herein should be understood to include all values ​​within the range (including the recited endpoints). For example, a range of 1 to 100 should be understood to include any value, combination of values, or sub-range therebetween.

[0120] As used herein, the term "Cholesin" or "Cholesin protein" refers to a homolog of the unknown human protein C7orf50. The human Cholesin gene is located in the p22 region of human chromosome 7. The gene encoding human Cholesin has the NCBI gene ID 84310, but its specific function has not yet been reported. The inventors characterized this protein and discovered its inhibitory effect on cholesterol synthesis, hence the name "Cholesin." The amino acid sequence of the Cholesin protein is highly conserved across species. The amino acid sequences of Cholesin proteins from different species are shown below:

[0121] Homo sapiens (Gene ID 84310)

[0122] Mouse (Mus musculus) (Gene ID 73212)

[0123] Rhesus monkey (Macaca mulatta) (Gene ID 696867)

[0124] Domestic cattle (Bos taurus) (Gene ID 522840)

[0125] Brown rat (Rattus norvegicus) (Gene ID 498154)

[0126] Jungle fowl (Gallus gallus) (Gene ID 416455)

[0127] In this article, Cholesin protein should be understood in its broadest sense, including wild-type, isolated or purified, or recombinant Cholesin protein, its variants or derivatives, as long as the desired biological activity (e.g., the activity of lowering plasma cholesterol level and / or triglyceride level in a subject) is retained. Variants or derivatives of Cholesin protein encompass, for example, variants or derivatives of the Cholesin protein described herein that retain the ability to lower plasma cholesterol level and / or triglyceride level in a subject to a similar degree as wild-type Cholesin protein, to the same degree as wild-type Cholesin protein, or to a greater degree than wild-type Cholesin protein. Variants or derivatives of the Cholesin protein retain at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% of the biological activity of the wild-type Cholesin protein, or have greater biological activity than the wild-type Cholesin protein, particularly the activity of lowering plasma cholesterol levels and / or triglyceride levels in a subject.

[0128] Variants of the Cholesin protein include polypeptides that have significant sequence identity to the wild-type Cholesin protein and retain the biological activity of the Cholesin protein (e.g., activity in reducing plasma cholesterol levels and / or triglyceride levels in a subject). Variants can have one or more amino acid mutations compared to the wild-type Cholesin protein. Mutations can include, for example, amino acid insertions, deletions, or substitutions. The amino acid sequence of the variant can, for example, be at least about 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 98.2%, 98.4%, 98.6%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to the amino acid sequence of the wild-type Cholesin protein.

[0129] A derivative of a cholesin protein generally refers to a compound that can be prepared from a wild-type cholesin protein or a variant thereof by chemical modification, particularly by covalent attachment of one or more substituents. Therefore, a derivative of a cholesin protein refers to a chemically modified cholesin protein or a variant thereof. Typical derivatives include, for example, amidated, glycosylated, alkylated, acylated, esterified, or pegylated cholesin proteins or variants thereof.

[0130] As used herein, the terms "step of enhancing the expression or activity of an endogenous Cholesin protein in a subject," "method of enhancing the expression or activity of an endogenous Cholesin protein in a subject," and "agent for enhancing the expression or activity of an endogenous Cholesin protein in a subject" refer to any method or substance that can enhance the stability or expression of a nucleic acid encoding a Cholesin protein, enhance the transcription and translation of Cholesin, increase Cholesin protein levels, enhance the activity of a Cholesin protein, enhance the stability of a Cholesin protein, or increase the effective duration of action of a Cholesin protein. For example, an agent for enhancing the expression or activity of an endogenous Cholesin protein in a subject can be a small molecule, protein, polypeptide, nucleic acid, or the like.

[0131] The term "GPR146" as used herein is a G protein-coupled receptor (GPCR) that is involved in the regulation of cholesterol metabolism and inhibits cholesterol synthesis controlled by PKA signaling and SREBP2. Previous studies have found that GPR146 promotes the activity of hepatic sterol regulatory element binding protein 2 (SREBP2) by activating the extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating the secretion of very low-density lipoprotein (VLDL) in the liver and then regulating the levels of low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) in the circulation. The GPR146 / ERK axis has a regulatory role in systemic cholesterol metabolism, and inhibiting GPR146 may be an effective strategy to reduce plasma cholesterol levels and atherosclerosis.

[0132] The human GPR146 gene ID is 115330, and the amino acid sequence is: MWSCSWFNGTGLVEELPACQDLQLGLSLLSLLGLVVGVPVGLCYNALLVLANLHSKASMTMPDVYFVNMAVAGLVLSALAPVHLLGPPSSRWALWSVGGEVHVALQIPFNVSSLVAMYSTALLSLDHYIERALPRTYMASVYNTRHVCGFVWGGA LLTSFSSLLFYICSHVSTRALECAKMQNAEAADATLVFIGYVVPALATLYALVLLSRVRREDTPLDRDTGRLEPSAHRLLVATVCTQFGLWTPHYLILLGHTVIISRGKPVDAHYLGLLHFVKDFSKLLAFSSSFVTPLLYRYMNQSFPSKLQRLMKKLPCGDRHCSPDHMGVQQVLA(SEQ ID NO:7)

[0133] The mouse GPR146 gene ID is 80290, and the amino acid sequence is: MWSCGPLNSTAWAEEPLCRNLRLGLWVLSLLYLGAGVPVSLGYNALLVLANLASKNTMTMPDVYFVNMAVAGLVLTALAPAYLLGPAHSRWALWSLSSEAHVTLLILFNVASLVTMYSTALLSLDYYIERALPRTYMASVYNTRHVCGFVWGGAV LTSFSSLLFYICSHVSSRIAECARMQNTEAADAILVLIGYVVPGLAVLYALALISRIGKEDTPLDQDTSRLDPSVHRLLVATVCTQFGLWTPYYLSLGHTVLTSRGRTVEGHYLGILQVAKDLAKFLAFSSSSVTPLLYRYINKAFPGKLRRLMKKMHCGRRHCSPDPSGIQQVMAQA(SEQ ID NO:8).

[0134] Cholesterol is a major component of cell membranes and an important raw material for the synthesis of physiologically active substances such as adrenocortical hormones, sex hormones, bile acids, and vitamin D. The concentration of cholesterol (including total cholesterol and / or low-density lipoprotein cholesterol) in human blood can be used as an indicator of lipid metabolism to assess the risk of cardiovascular and cerebrovascular diseases, hyperlipidemia, atherosclerosis, and other diseases.

[0135] Cholesterol is commonly found in the blood in the form of lipoproteins. Lipoproteins are classified into five types based on particle size and density: chylomicrons, very-low-density lipoproteins, intermediate-density lipoproteins, low-density lipoproteins, and high-density lipoproteins. Very-low-density lipoproteins synthesized in the liver transport triglycerides into the bloodstream, where they are broken down by lipase, producing very-low-density lipoprotein remnants, also known as intermediate-density lipoproteins. Some intermediate-density lipoproteins are metabolized by the liver; others are further converted to low-density lipoproteins in the blood by lipase and hepatic lipase. Low-density lipoproteins act throughout the body, binding to low-density lipoprotein receptors on cells and tissues for utilization.

[0136] Low-density lipoprotein (LDL) in plasma is the primary carrier for the transport of endogenous cholesterol, which is degraded and converted by binding to the low-density lipoprotein receptor (LDLR) on its cell membrane. LDLR dysfunction reduces the ability to clear plasma LDL, ultimately leading to the formation of atherosclerotic plaques in the arterial intima. Low-density lipoprotein cholesterol (LDL-C) is the major lipoprotein in fasting plasma, accounting for approximately two-thirds of plasma lipoproteins. It is the primary vehicle for transporting cholesterol to extrahepatic tissues. Therefore, LDL-C levels are correlated with the incidence and severity of cardiovascular disease, hyperlipidemia, and atherosclerosis. It is considered the primary pathogenic factor for atherosclerosis. Its concentration has a significant positive correlation with the incidence of coronary heart disease and is an important indicator for assessing individual risk factors for coronary heart disease. In this article, "LDL" and "LDL-C" are used interchangeably.

[0137] Very low-density lipoprotein (VLDL) primarily consists of triglycerides, cholesterol, and lipoproteins. VLDL is synthesized in the liver and transports triglycerides and cholesterol to tissues via the bloodstream. Because this type of lipoprotein carries a relatively small amount of cholesterol and its particles are relatively large, they are less likely to penetrate the vascular endothelium. Therefore, normal VLDL generally does not have an atherogenic effect. However, because triglycerides account for 50%-70% of VLDL and cholesterol accounts for 8%-12%, once VLDL levels increase significantly, in addition to elevated triglycerides in the plasma, cholesterol levels also increase, which has an atherogenic effect. In this article, "VLDL" and "VLDL-C" are used interchangeably.

[0138] Intermediate-density lipoprotein (IDL) is primarily an intermediate metabolite of very-low-density lipoprotein (VLDL), and is therefore also called residual VLDL. IDL can also be directly secreted by the liver, but in minute quantities. Among apolipoprotein B-containing lipoproteins, it is the last lipoprotein to be broken down by lipoprotein lipase (LPL).

[0139] High-density lipoproteins (HDL) are the smallest and densest group of plasma lipoproteins. In normal plasma, most HDL particles are spherical, composed of a hydrophobic core surrounded by phospholipids, unacylated cholesterol, and apolipoproteins. High-density lipoprotein cholesterol (HDL-C) transports cholesterol from extrahepatic tissues to the liver for metabolism and excretion in the bile. Throughout this article, "HDL" and "HDL-C" are used interchangeably.

[0140] Chylomicrons (CMs) are the largest lipoprotein particles in plasma and the primary lipoprotein transporter of exogenous triglycerides. Cholesterol and triglycerides contained in food are absorbed in the small intestine, through the lymphatic vessels, and then through chylomicrons. Once in the bloodstream, chylomicrons mature into mature chylomicrons. They are then converted into free fatty acids by the enzyme lipase. These free fatty acids are converted into energy by the body and further synthesized and stored in fat.

[0141] Cholesterol synthesis occurs primarily in the liver. Cholesterol is produced from acetyl-CoA under the control of a series of enzymes. HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) is the rate-limiting enzyme, whose activity is controlled at both transcriptional and post-transcriptional levels. SREBP2 (sterol regulatory element-binding protein 2) acts as a master transcription factor, controlling the expression of HMGCR and other genes involved in cholesterol synthesis and uptake. Cholesterol biosynthesis and uptake are tightly regulated through a negative feedback mechanism that senses cellular cholesterol levels. When cells are cholesterol-deficient, SREBP2, along with its escort protein SCAP (SREBP cleavage-activating protein), is transported from the endoplasmic reticulum (ER) to the Golgi apparatus in COPII vesicles. Within the Golgi apparatus, SREBP2 is sequentially cleaved by site 1 and site 2 proteases. The N-terminal structure of SREBP2 released by this cleavage travels to the cell nucleus, where it acts as a transcription factor, enhancing the expression of genes involved in cholesterol synthesis (such as Hmgcr, Hmgcs1, Mvd, Mvk, Pmvk, and Sqle). Conversely, when cellular cholesterol levels rise, cholesterol molecules bind to SCAP, triggering its interaction with INSIG (insulin-induced gene). This interaction retains SREBP in the ER and prevents subsequent SREBP activation and expression of genes involved in cholesterol metabolism. This mechanism helps maintain cholesterol homeostasis.

[0142] Both endogenously synthesized and exogenously acquired cholesterol are secreted into the blood as very low-density lipoproteins (VLDL). After processing in the blood, VLDL produces circulating LDL, which can be taken up by cells via LDL receptor (LDLR)-mediated endocytosis.

[0143] As used herein, the term "expression vector" is a nucleic acid molecule used as a medium for transferring (exogenous) genetic material into a host cell where it can, for example, be replicated and / or expressed as a vector. The term "expression vector" encompasses, but is not limited to, plasmids, viral vectors (including, for example, retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adenovirus-associated vectors (AAV)), phages, phagemids, cosmids, and artificial chromosomes (including, for example, BACs and YACs).

[0144] As used herein, the term "treatment" includes therapeutic or prophylactic treatment in a subject in need thereof. "Therapeutic or prophylactic treatment" includes prophylactic treatment intended to completely prevent clinical and / or pathological manifestations or therapeutic treatment intended to ameliorate or alleviate clinical and / or pathological manifestations. Thus, the term "treatment" also includes ameliorating or preventing a disease.

[0145] As used herein, the term "effective amount" means an amount of a therapeutic agent that, when administered to a subject for the treatment or prevention of a disease, is sufficient to achieve such treatment or prevention. The "effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated. A "therapeutically effective amount" refers to an effective amount for therapeutic treatment. A "prophylactically effective amount" refers to an effective amount for preventive treatment.

[0146] As used herein, the terms "subject" or "individual" or "animal" or "patient" are used interchangeably herein to refer to any subject in need of treatment, particularly a mammalian subject. Generally speaking, mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc.

[0147] As used herein, the term "diseases associated with elevated plasma cholesterol levels" refers to diseases, conditions or disorders caused by elevated plasma cholesterol levels, including but not limited to diseases caused by dyslipidemia and lipodystrophy-related diseases.

[0148] Dyslipidemia is an abnormality in lipoprotein metabolism in the human body, primarily including elevated total cholesterol, low-density lipoprotein cholesterol, and triglycerides and / or decreased high-density lipoprotein cholesterol. Dyslipidemia can lead to diseases or symptoms such as hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease, angina pectoris, malignant arrhythmias), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, liver failure, nephrotic syndrome, gallstones, and pancreatitis.

[0149] Diseases related to fat metabolism disorders are a general term for related diseases caused by fat metabolism disorders, including, for example: hyperlipidemia, hypertension, atherosclerosis, obesity, fatty liver, cirrhosis, coronary heart disease, angina pectoris, myocardial infarction, inflammatory bowel disease, indigestion and gastrointestinal ulcers.

[0150] Hyperlipidemia generally refers to elevated plasma cholesterol and / or triglyceride levels. Hyperlipidemia is a manifestation of abnormal fat metabolism and is primarily categorized into three types: hypercholesterolemia, hypertriglyceridemia, and combined hyperlipidemia. Hypercholesterolemia refers to elevated plasma total cholesterol or low-density lipoprotein cholesterol levels. Hypercholesterolemia can lead to various life-threatening complications, including cardiovascular disease and atherosclerosis.

[0151] Atherosclerosis is the most common and important type of vascular disease known as arteriosclerosis. Impaired lipid metabolism underlies the pathological process of atherosclerosis, and its characteristic manifestation is that lesions begin in the intima of the affected arteries. Generally, there is accumulation of lipids and complex carbohydrates, followed by bleeding and thrombosis, fibrosis, and calcification, followed by gradual degeneration and calcification of the arterial media. The lesions often affect elastic and large and medium-sized muscular arteries. Once the disease progresses to the point of obstructing the arterial lumen, the tissues or organs supplied by the arteries will suffer ischemia or necrosis. The lipid accumulation in the arterial intima gives it a yellow, porridge-like appearance, hence the name atherosclerosis. Atherosclerosis is often associated with conditions such as hypertension, diabetes, and obesity.

[0152] As used herein, the term "HMGCR inhibitor," also known as an HMG-CoA reductase inhibitor, refers to a compound capable of inhibiting HMG-CoA reductase. It inhibits the biosynthesis of isoprenoids by inhibiting HMG-CoA reductase, blocking protein prenylation and thereby lowering plasma cholesterol levels.

[0153] As used herein, the term "PCSK9 inhibitor" refers to a class of compounds that inhibit PCSK9 (member 9 of the proconvertase subtilisin / kexin family). PCSK9 is a liver-derived secretory protein that reduces the number of LDLRs on hepatocytes, affecting LDL internalization and preventing LDL clearance from the blood, leading to hypercholesterolemia. Therefore, inhibiting PCSK9 stabilizes the LDLR, promoting LDL absorption by the liver, and thus lowering LDL levels in the blood.

[0154] As used herein, the term "cholesterol absorption inhibitor" refers to a compound that is capable of inhibiting the absorption of cholesterol, for example, by inhibiting NPC1L1-mediated endocytosis, thereby blocking the uptake of cholesterol.

[0155] The term "composition" particularly refers to a composition suitable for administration to humans. However, the term also generally encompasses compositions suitable for administration to non-human animals. The composition and its components (i.e., the active agent and optional carrier or excipient) are preferably pharmaceutically acceptable, i.e., capable of eliciting the desired therapeutic effect in the recipient without causing any undesirable local or systemic effects.

[0156] The term "pharmaceutically acceptable" means that the carrier or excipient is compatible with the other ingredients of the composition and not substantially deleterious to the recipient thereof, and / or such carrier or excipient is approved or available for inclusion in the composition.

[0157] The inventors unexpectedly discovered that cholesin, a hormone secreted by the intestine in response to cholesterol absorption, can inhibit cholesterol synthesis and VLDL secretion in the liver, thereby lowering plasma cholesterol and / or triglyceride levels and preventing diseases associated with elevated plasma cholesterol levels, such as hypercholesterolemia and atherosclerosis. Mechanistic studies have shown that cholesin inhibits cholesterol synthesis controlled by PKA signaling and SREBP2 by binding to its receptor GPR146. Cholesin downregulates the expression of genes involved in cholesterol synthesis independently of LDLR and reduces plasma cholesterol levels, suggesting that cholesin treatment is an effective strategy for combating diseases associated with elevated plasma cholesterol levels, such as hypercholesterolemia and atherosclerosis. Therefore, exogenous cholesin or methods or agents that promote the expression or activity of endogenous cholesin protein in a subject can lower plasma cholesterol and / or triglyceride levels, thereby preventing or treating diseases associated with elevated plasma cholesterol levels, such as hypercholesterolemia and atherosclerosis. In addition, combining exogenous Cholesin or methods or agents that promote the expression or activity of endogenous Cholesin protein in a subject with existing cholesterol-lowering drugs such as statins is also a promising strategy for treating diseases associated with elevated plasma cholesterol levels, including hyperlipidemia and atherosclerosis.

[0158] Accordingly, in one aspect, the present invention provides a method for lowering plasma cholesterol levels and / or triglyceride levels in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

[0159] In some embodiments, plasma cholesterol is selected from plasma total cholesterol and plasma LDL-cholesterol. In some embodiments, plasma cholesterol is plasma total cholesterol. In some embodiments, plasma cholesterol is plasma LDL-cholesterol. In some embodiments, plasma cholesterol is plasma total cholesterol and plasma LDL-cholesterol.

[0160] In another aspect, the present invention provides a method for inhibiting hepatic cholesterol synthesis in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

[0161] In some embodiments, the method inhibits the expression of genes related to cholesterol synthesis. In some embodiments, the genes related to cholesterol synthesis are selected from one or more of Hmgcr, Hmgcs1, Mvd, Mvk, Pmvk, and Sqle. In some embodiments, the gene related to cholesterol synthesis is Hmgcr. In some embodiments, the gene related to cholesterol synthesis is Hmgcs1. In some embodiments, the gene related to cholesterol synthesis is Mvd. In some embodiments, the gene related to cholesterol synthesis is Mvk. In some embodiments, the gene related to cholesterol synthesis is Pmvk. In some embodiments, the gene related to cholesterol synthesis is Sqle. In some embodiments, the genes related to cholesterol synthesis are Hmgcr, Hmgcs1, Mvd, Mvk, Pmvk, and Sqle.

[0162] In some embodiments, the method inhibits the expression of a gene associated with cholesterol uptake. In some embodiments, the gene associated with cholesterol synthesis and uptake is Ldlr.

[0163] In some embodiments, the method inhibits the expression of genes associated with cholesterol synthesis and uptake. In some embodiments, the genes associated with cholesterol synthesis are Hmgcr, Hmgcs1, Mvd, Mvk, Pmvk, and Sqle; and the gene associated with cholesterol synthesis and uptake is Ldlr.

[0164] In some embodiments, the method inhibits the expression of a transcription factor that promotes the expression of a gene related to cholesterol synthesis. In some embodiments, the method inhibits the expression of a transcription factor that promotes the expression of a gene related to cholesterol uptake. In some embodiments, the transcription factor that promotes the expression of a gene related to cholesterol synthesis and / or uptake is SREBP2.

[0165] In another aspect, the present invention provides a method for inhibiting hepatic VLDL-cholesterol secretion in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

[0166] In another aspect, the present invention provides a method for treating or preventing a disease associated with elevated plasma cholesterol levels in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

[0167] In embodiments of the methods of the present invention, the Cholesin protein is isolated or purified. In some embodiments, the Cholesin protein is a mammalian Cholesin protein. Examples of mammalian Cholesin proteins include, but are not limited to, Cholesin proteins from humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cows. In some embodiments, the Cholesin protein is a non-human primate Cholesin protein. In some embodiments, the Cholesin protein is a human Cholesin protein. In some embodiments, the human Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:1.

[0168] In other embodiments, the Cholesin protein is a mouse Cholesin protein. In some embodiments, the mouse Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:2.

[0169] In other embodiments, the Cholesin protein is a macaque Cholesin protein. In some embodiments, the macaque Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:3.

[0170] In other embodiments, the Cholesin protein is a bovine Cholesin protein. In some embodiments, the bovine Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:4.

[0171] In other embodiments, the Cholesin protein is a Rattus norvegicus Cholesin protein. In some embodiments, the Rattus norvegicus Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:5.

[0172] In other embodiments, the Cholesin protein is a Gallus gallus Cholesin protein. In some embodiments, the Gallus gallus Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:6.

[0173] In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid encodes human Cholesin protein.

[0174] In some embodiments, the expression vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

[0175] The expression vector itself is typically a nucleotide sequence, typically a DNA sequence comprising an insert (transgene) and a larger sequence as a vector "backbone". Engineered vectors are typically included in a starting point for autonomous replication in a host cell (if stable expression of the polynucleotide is desired), a selective marker, and a restriction enzyme cleavage site (such as a multiple cloning site, MCS). The vector may additionally comprise a promoter, a genetic marker, a reporter gene, a targeting sequence, and / or a protein purification tag. As known to those skilled in the art, a large number of suitable expression vectors are known to those skilled in the art, and many are commercially available.

[0176] The Cholesin protein disclosed herein, the nucleic acid encoding the Cholesin protein, the expression vector comprising the nucleic acid, or the agent that enhances the expression or activity of the endogenous Cholesin protein in a subject can be used to treat and / or prevent diseases associated with elevated plasma cholesterol levels, i.e., diseases, conditions or disorders caused by elevated plasma cholesterol levels, for example, for treating and / or preventing:

[0177] 1. Dyslipidemia and its sequelae, such as atherosclerosis, cardiovascular and cerebrovascular diseases, especially those characterized by one or more of the following factors (but not limited to):

[0178] - high plasma total cholesterol concentration,

[0179] - High plasma triglyceride concentration, high postprandial plasma triglyceride concentration,

[0180] - low HDL cholesterol concentration,

[0181] - low ApoA lipoprotein concentration,

[0182] - high LDL cholesterol concentration,

[0183] - low-density LDL cholesterol particles,

[0184] - High ApoB lipoprotein concentration;

[0185] 2. A variety of other conditions that may be associated with metabolic syndrome, such as:

[0186] - Obesity (overweight), including central obesity,

[0187] - thrombosis, hypercoagulability and prothrombotic states (arterial and venous),

[0188] -hypertension,

[0189] - Heart failure, such as (but not limited to) heart failure following myocardial infarction, hypertensive heart disease, or cardiomyopathy;

[0190] 3. Fat metabolism disorder

[0191] - Hyperlipidemia,

[0192] - atherosclerosis,

[0193] -hypertension,

[0194] - obesity,

[0195] - Fatty liver, cirrhosis, liver failure,

[0196] 4. Diabetes, especially type 2 diabetes, including its related sequelae

[0197] Specifically, it involves:

[0198] - Diabetes and diabetic complications, such as diabetic vascular disease, such as damage to large blood vessels and microvessels leading to lesions of tissues and organs such as the heart, brain, kidneys, peripheral nerves, eyes, and feet, including diabetic eye disease, diabetic heart disease, diabetic nephropathy, diabetic neuropathy, and distal limb necrosis,

[0199] - Diseases that are highly prevalent in the presence of diabetes, occur with diabetes, or are aggravated by diabetes, such as atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, osteoporosis, etc.

[0200] - Fat metabolism disorders and related diseases that are highly prevalent in the setting of diabetes, occur with diabetes, or are aggravated by diabetes, including hyperlipidemia, hypertension, atherosclerosis, obesity, fatty liver, and cirrhosis;

[0201] 5. Diseases or conditions involving inflammatory responses:

[0202] - atherosclerosis, such as (but not limited to) coronary artery atherosclerosis, including angina pectoris or myocardial infarction, stroke,

[0203] - restenosis or reocclusion of the blood vessel,

[0204] - Chronic inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis,

[0205] -asthma,

[0206] - Other inflammatory conditions;

[0207] 6. Other obstacles:

[0208] - nephrotic syndrome,

[0209] -pancreatitis,

[0210] - Gallstones,

[0211] - vasculitis,

[0212] -Ischemia / reperfusion syndrome.

[0213] In some embodiments, the disease associated with elevated plasma cholesterol levels is selected from hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, nephrotic syndrome, and gallstones.

[0214] In a preferred embodiment, the disease associated with elevated plasma cholesterol levels is hypercholesterolemia. In other preferred embodiments, the disease associated with elevated plasma cholesterol levels is atherosclerosis.

[0215] In some embodiments, the method further comprises the step of administering a second therapeutic agent. In preferred embodiments, the second therapeutic agent is selected from the group consisting of a protein or peptide, a nucleic acid, and a small molecule drug.

[0216] In some embodiments, the second therapeutic agent is a cholesterol-lowering drug. In some embodiments, the cholesterol-lowering drug is selected from the group consisting of: an HMGCR inhibitor, a PCSK9 inhibitor, and a cholesterol absorption inhibitor.

[0217] In some embodiments, the cholesterol-lowering drug is an HMGCR inhibitor. In some embodiments, the HMGCR inhibitor is a statin. In some embodiments, the statin is selected from rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin, and pitavastatin. In a preferred embodiment, the second therapeutic agent is rosuvastatin.

[0218] In some embodiments, the cholesterol-lowering drug is a PCSK9 inhibitor. In some embodiments, the PCSK9 inhibitor is selected from evolocumab, alirocumab, inclisiran, and tafolecimab. In some embodiments, the second therapeutic agent is evolocumab. In some embodiments, the second therapeutic agent is alirocumab. In some embodiments, the second therapeutic agent is inclisiran. In some embodiments, the second therapeutic agent is tafolecimab.

[0219] Evolocumab (trade name: ) is a fully human IgG2 monoclonal antibody. Evolocumab can bind to PCSK9 and inhibit the binding of circulating PCSK9 to the low-density lipoprotein receptor (LDLR), thereby preventing PCSK9-mediated low-density lipoprotein receptor degradation. Its approved indications are hypercholesterolemia and mixed dyslipidemia.

[0220] Alirocumab (Alirocumab, trade name ) is a fully human IgG1 monoclonal antibody that binds to PCSK9 and inhibits the binding of circulating PCSK9 to the low-density lipoprotein receptor (LDLR), thereby preventing PCSK9-mediated LDL receptor degradation. It is used to treat adults with heterozygous familial hypercholesterolemia and clinical atherosclerotic cardiovascular disease (e.g., heart attack or stroke requiring LDL cholesterol lowering).

[0221] Inclisiran (Inclisiran, trade name ) is a small interfering nucleic acid (siRNA) drug targeting PCSK9 for lowering low-density lipoprotein cholesterol. It has been approved for the treatment of adult patients with primary hypercholesterolemia (heterozygous familial and non-familial) or mixed dyslipidemia.

[0222] Tafolecimab (trade name: ) is a fully human IgG2 monoclonal antibody that can specifically bind to the PCSK9 molecule, increase the low-density lipoprotein receptor (LDLR) level by reducing PCSK9-mediated LDLR endocytosis, and then increase the clearance of low-density lipoprotein cholesterol (LDL-C) and lower LDL-C levels. It is used to reduce LDL-C targets in adult patients with primary hypercholesterolemia (including heterozygous familial and non-familial hypercholesterolemia) and mixed dyslipidemia who are unable to achieve LDL-C targets after receiving moderate or higher doses of statins, to reduce LDL-C, total cholesterol (TC) and apolipoprotein B (ApoB) levels.

[0223] In some embodiments, the cholesterol-lowering drug is a cholesterol absorption inhibitor. In some embodiments, the cholesterol absorption inhibitor is an NPC1L1 inhibitor. NPC1L1 inhibitors target the cholesterol transporter NPC1L1. By acting on NPC1L1, a key transporter for cholesterol uptake and absorption on the brush border of the small intestinal villi, they inhibit the intracellular transport of cholesterol, reduce the absorption of cholesterol from food and bile in the small intestine, hinder the transport of cholesterol from the small intestine to the liver, reduce liver cholesterol storage, and accelerate cholesterol clearance.

[0224] In some embodiments, the NPC1L1 inhibitor is selected from Ezetimibe and Hybutimibe. In some embodiments, the second therapeutic agent is Ezetimibe. In some embodiments, the second therapeutic agent is Hybutimibe.

[0225] In some embodiments, the second therapeutic agent is administered before, after, or simultaneously with the Cholesin protein, the nucleic acid, or the expression vector, or the agent that enhances the expression or activity of the subject's endogenous Cholesin protein. In some embodiments, the second therapeutic agent is administered before the Cholesin protein, the nucleic acid, or the expression vector, or the agent that enhances the expression or activity of the subject's endogenous Cholesin protein. In some embodiments, the second therapeutic agent is administered after the Cholesin protein, the nucleic acid, or the expression vector, or the agent that enhances the expression or activity of the subject's endogenous Cholesin protein. In some embodiments, the second therapeutic agent is administered simultaneously with the Cholesin protein, the nucleic acid, or the expression vector, or the agent that enhances the expression or activity of the subject's endogenous Cholesin protein.

[0226] It will be appreciated that treatment may require a single administration of a therapeutically effective dose or multiple administrations of a therapeutically effective dose of an active agent of the invention. For example, depending on the type, half-life, and clearance rate of the active agent, the active agent may be administered one to three times a day, once every two days, once every 3 to 4 days, once a week, or once every two weeks, or once in a month.

[0227] The active agents disclosed herein (Cholesin protein, nucleic acid encoding Cholesin protein, or expression vector comprising said nucleic acid, or agent that enhances the expression or activity of endogenous Cholesin protein in a subject) can be administered in a variety of ways. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intravenous, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0228] In some embodiments, the subject is a mammal. In a preferred embodiment, the subject is a human. In some embodiments, the subject is unresponsive or intolerant to one or more of a cholesterol absorption inhibitor, an HMGCR inhibitor, and a PCSK9 inhibitor, for example, the subject has an LDL receptor (LDLR) defect.

[0229] An effective amount of a Cholesin protein disclosed herein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject can be determined by one skilled in the art using known techniques. An appropriate dosage provides a sufficient amount of the active agent of the invention and is preferably therapeutically effective, i.e., sufficient to elicit, for example, a therapeutic or prophylactic response in a subject or animal within a reasonable timeframe. For example, a dose of a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject should be sufficient to elicit a therapeutic or prophylactic response within a period of about 20 minutes, 30 minutes, 1 hour, 2 hours, or longer, such as 12 hours to 24 hours or longer (e.g., 1 month, 2 months, 3 months, 6 months, 12 months, 24 months, etc.) from the time of administration. In certain embodiments, the time period can be even longer. As is known in the art, adjustments may be necessary for therapeutic purposes, route, timing and frequency of administration, timing and frequency of administration of formulations, age, weight, general health, sex, diet, severity of the disease state, drug combinations, reaction sensitivities and tolerance / response to treatment.

[0230] Many assays for determining the dosage to be administered are known in the art. Typically, the attending physician determines the dosage of the disclosed active agent to be administered to each individual patient, taking into account a variety of factors such as age, weight, general health, diet, sex, the active agent to be administered, the route of administration, and the severity of the condition being treated.

[0231] In another aspect, the present invention provides a composition comprising a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of human endogenous Cholesin protein, and a pharmaceutically acceptable carrier or excipient.

[0232] In some embodiments, the composition is used for one or more of the following:

[0233] 1) reducing plasma cholesterol levels in a subject;

[0234] 2) reducing plasma total cholesterol or plasma LDL-cholesterol levels in a subject;

[0235] 3) reducing plasma triglyceride levels in a subject;

[0236] 4) inhibiting hepatic cholesterol synthesis in a subject;

[0237] 5) inhibiting hepatic VLDL-cholesterol secretion in a subject; and

[0238] 6) Treating or preventing a disease associated with elevated plasma cholesterol levels in a subject.

[0239] In embodiments of the compositions of the present invention, the Cholesin protein is isolated or purified. In some embodiments, the Cholesin protein is a mammalian Cholesin protein. Examples of mammalian Cholesin proteins include, but are not limited to, Cholesin proteins from humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cows. In some embodiments, the Cholesin protein is a non-human primate Cholesin protein. In some embodiments, the Cholesin protein is a human Cholesin protein. In some embodiments, the human Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:1.

[0240] In other embodiments, the Cholesin protein is a mouse Cholesin protein. In some embodiments, the mouse Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:2. In other embodiments, the Cholesin protein is a macaque Cholesin protein. In some embodiments, the macaque Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:3. In other embodiments, the Cholesin protein is a bovine Cholesin protein. In some embodiments, the bovine Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:4. In other embodiments, the Cholesin protein is a Rattus norvegicus Cholesin protein. In some embodiments, the Rattus norvegicus Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:5. In other embodiments, the Cholesin protein is a Gallus gallus Cholesin protein. In some embodiments, the Gallus gallus Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:6.

[0241] In some embodiments, the composition further comprises a second therapeutic agent. In a preferred embodiment, the second therapeutic agent is selected from a protein or peptide, a nucleic acid, and a small molecule drug.

[0242] In some embodiments, the second therapeutic agent is a cholesterol-lowering drug. In some embodiments, the cholesterol-lowering drug is selected from the group consisting of: an HMGCR inhibitor, a PCSK9 inhibitor, and a cholesterol absorption inhibitor.

[0243] In some embodiments, the HMGCR inhibitor is a statin. In some embodiments, the statin is selected from rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin. In a preferred embodiment, the second therapeutic agent is rosuvastatin.

[0244] In some embodiments, the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran, and Tafolecimab.

[0245] In some embodiments, the cholesterol absorption inhibitor is an NPC1L1 inhibitor, preferably ezetimibe or hebomibe. In a preferred embodiment, the second therapeutic agent is ezetimibe.

[0246] Depending on the active agent employed (e.g., Cholesin protein), the compositions of the present disclosure can be prepared in various forms, such as solid, liquid, gaseous, or lyophilized forms, for example, in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures, or fluid extracts, or in a form specifically adapted for the desired method of administration. Known processes for producing pharmaceuticals are shown in the 22nd edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa., 2012), and may include, for example, conventional mixing, dissolving, granulating, sugar-coating, grinding, emulsifying, encapsulating, embedding, or lyophilizing processes. Compositions comprising, for example, a Cholesin protein as described herein, an expression vector encoding the Cholesin protein, or comprising the nucleic acid, or an agent for enhancing the expression or activity of an endogenous Cholesin protein in a subject are typically provided in liquid form and preferably contain a pharmaceutically acceptable buffer. The liquid formulation may be a solution or a suspension.

[0247] In some embodiments, the compositions of the present invention are formulated as injection preparations or oral preparations. Examples of oral preparations include, but are not limited to, tablets, capsules, granules, and suspensions. In some embodiments, the compositions of the present invention can be used for multiple routes of administration, such as parenteral administration. In some embodiments, the compositions of the present invention can be administered by routes such as intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In some embodiments, the compositions of the present invention can be administered orally.

[0248] In the compositions of the present invention, the example of carrier and excipient includes but is not limited to filler, binding agent, disintegrant, coating agent, adsorbent, antiadherent, glidant, preservative, antioxidant, flavoring, coloring agent, sweetener, solvent, cosolvent, buffer, chelating agent, viscosity imparting agent, surfactant, diluent, wetting agent, diluent, preservative, emulsifying agent, stabilizer and tension regulator. It is known to those skilled in the art that suitable excipient is selected to prepare the compositions of the present invention. The exemplary carrier used for the compositions of the present invention comprises saline, buffered saline, glucose and water. Usually, the selection of suitable excipient especially depends on the desired dosage form of employed activating agent, disease to be treated and compositions.

[0249] In another aspect, the present invention provides use of a composition as described herein in the preparation of a medicament for one or more of:

[0250] 1) reducing plasma cholesterol levels in a subject;

[0251] 2) reducing plasma total cholesterol or plasma LDL-cholesterol levels in a subject;

[0252] 3) reducing plasma triglyceride levels in a subject;

[0253] 4) inhibiting hepatic cholesterol synthesis in a subject;

[0254] 5) inhibiting hepatic VLDL-cholesterol secretion in a subject; and

[0255] 6) Treating or preventing a disease associated with elevated plasma cholesterol levels in a subject.

[0256] In yet another aspect, the present invention provides a method for treating a disease associated with elevated plasma cholesterol levels in a subject, comprising the step of administering to the subject an effective amount of a composition of the present invention.

[0257] In another aspect, the present invention provides a method for diagnosing a disease associated with elevated plasma cholesterol levels in a subject, comprising the steps of:

[0258] a. obtaining a blood sample from the subject,

[0259] b. determining the level of Cholesin protein in the sample,

[0260] Wherein an increased level of Cholesin protein in the sample compared to a healthy control indicates that the subject is at risk for a disease associated with increased plasma cholesterol levels.

[0261] In an embodiment of the diagnostic method of the present invention, the disease associated with elevated plasma cholesterol levels is selected from hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, nephrotic syndrome and gallstones.

[0262] In some embodiments, the disease associated with elevated plasma cholesterol levels is hypercholesterolemia. In some embodiments, the disease associated with elevated plasma cholesterol levels is atherosclerosis.

[0263] In some embodiments, the blood sample is selected from blood, serum, and plasma. In certain embodiments, the method is performed in vitro. In some embodiments, the method further comprises the step of isolating Cholesin protein from the sample. Methods for isolating Cholesin protein are known in the art.

[0264] Methods for determining the level of Cholesin protein in a sample are known in the art. For example, in step b, the level of Cholesin protein in the sample can be determined by a method selected from the group consisting of mass spectrometry, fluorescence detection, chemiluminescence, ELISA, Western blotting, radioimmunoassay, immunohistochemical staining, multiple immunoassay, and dot blot assay.

[0265] In some embodiments, the level of Cholesin protein in the sample can be determined using an anti-Cholesin protein antibody, preferably a monoclonal antibody, but can also be a Fab, Fab', (Fab')2, Fv, scFv, bi-scFv, bispecific antibody, trispecific antibody, tetraspecific antibody, etc.

[0266] In yet another aspect, the present invention provides use of a reagent for determining the level of Cholesin protein in a blood sample from a subject in the preparation of a kit for diagnosing a disease associated with elevated plasma cholesterol levels in the subject.

[0267] In another aspect, the present invention provides a kit for diagnosing a disease associated with elevated plasma cholesterol levels in a subject, comprising reagents for determining the level of Cholesin protein in a blood sample from the subject.

[0268] In embodiments of the diagnostic use and kits of the present invention, the reagent is used to determine the level of Cholesin protein in the sample using a method selected from the group consisting of mass spectrometry, fluorescence detection, chemiluminescence, ELISA, Western blotting, radioimmunoassay, immunohistochemical staining, multiplex immunoassay, and dot blot assay. In some embodiments, the reagent is an anti-Cholesin protein antibody. The antibody is preferably a monoclonal antibody, but may also be a Fab, Fab', (Fab')2, Fv, scFv, bi-scFv, bispecific antibody, trispecific antibody, tetraspecific antibody, and the like.

[0269] In some embodiments, the disease associated with elevated plasma cholesterol levels is selected from hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, nephrotic syndrome, and gallstones. In some embodiments, the disease associated with elevated plasma cholesterol levels is hypercholesterolemia. In some embodiments, the disease associated with elevated plasma cholesterol levels is atherosclerosis.

[0270] In some embodiments, the blood sample is selected from the group consisting of blood, serum, and plasma.

[0271] In another aspect, the present disclosure provides the use of a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject in the preparation of a composition for lowering plasma cholesterol and / or triglyceride levels in a subject. In some embodiments, the plasma cholesterol is plasma total cholesterol or plasma LDL-cholesterol.

[0272] In another aspect, the present disclosure provides use of a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject in preparing a composition for inhibiting hepatic cholesterol synthesis in a subject.

[0273] In another aspect, the present disclosure provides use of a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject in the preparation of a composition for inhibiting hepatic VLDL-cholesterol secretion in a subject.

[0274] In another aspect, the present disclosure provides use of a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject in the preparation of a composition for treating or preventing a disease associated with elevated plasma cholesterol levels in a subject.

[0275] In another aspect, the present disclosure provides a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject, for use in lowering plasma cholesterol and / or triglyceride levels in a subject. In some embodiments, the plasma cholesterol is plasma total cholesterol or plasma LDL-cholesterol.

[0276] In another aspect, the present disclosure provides a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject, for use in inhibiting hepatic cholesterol synthesis in a subject.

[0277] In another aspect, the present disclosure provides a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject, for use in inhibiting hepatic VLDL-cholesterol secretion in a subject.

[0278] In another aspect, the present disclosure provides a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent for enhancing the expression or activity of an endogenous Cholesin protein in a subject, for use in treating or preventing a disease associated with elevated plasma cholesterol levels in a subject.

[0279] In embodiments of the uses of the present invention, the Cholesin protein is isolated or purified. In some embodiments, the Cholesin protein is a mammalian Cholesin protein. Examples of mammalian Cholesin proteins include, but are not limited to, Cholesin proteins from humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cows. In some embodiments, the Cholesin protein is a non-human primate Cholesin protein. In some embodiments, the Cholesin protein is a human Cholesin protein. In some embodiments, the human Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:1.

[0280] In other embodiments, the Cholesin protein is a mouse Cholesin protein. In some embodiments, the mouse Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:2. In other embodiments, the Cholesin protein is a macaque Cholesin protein. In some embodiments, the macaque Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:3. In other embodiments, the Cholesin protein is a bovine Cholesin protein. In some embodiments, the bovine Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:4. In other embodiments, the Cholesin protein is a Rattus norvegicus Cholesin protein. In some embodiments, the Rattus norvegicus Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:5. In other embodiments, the Cholesin protein is a Gallus gallus Cholesin protein. In some embodiments, the Gallus gallus Cholesin protein comprises the amino acid sequence set forth in SEQ ID NO:6.

[0281] In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the nucleic acid is DNA.

[0282] In some embodiments, the expression vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

[0283] In some embodiments, the disease associated with elevated plasma cholesterol levels is selected from hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, nephrotic syndrome, and gallstones. In some embodiments, the disease associated with elevated plasma cholesterol levels is hypercholesterolemia. In some embodiments, the disease associated with elevated plasma cholesterol levels is atherosclerosis.

[0284] In some embodiments, the Cholesin protein described herein, the nucleic acid encoding the Cholesin protein, or the expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of the subject's endogenous Cholesin protein is combined with a second therapeutic agent. In preferred embodiments, the second therapeutic agent is selected from a protein or peptide, a nucleic acid, and a small molecule drug.

[0285] In some embodiments, the second therapeutic agent is a cholesterol-lowering drug. In some embodiments, the cholesterol-lowering drug is selected from the group consisting of: an HMGCR inhibitor, a PCSK9 inhibitor, and a cholesterol absorption inhibitor.

[0286] In some embodiments, the HMGCR inhibitor is a statin. In some embodiments, the statin is selected from rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin. In a preferred embodiment, the second therapeutic agent is rosuvastatin.

[0287] In some embodiments, the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran, and Tafolecimab.

[0288] In some embodiments, the cholesterol absorption inhibitor is an NPC1L1 inhibitor, preferably ezetimibe or hebomibe. In a preferred embodiment, the second therapeutic agent is ezetimibe.

[0289] In some embodiments, the subject is a mammal. In a preferred embodiment, the subject is a human. In some embodiments, the subject is unresponsive or intolerant to one or more of a cholesterol absorption inhibitor, an HMGCR inhibitor, and a PCSK9 inhibitor, for example, the subject has an LDL receptor (LDLR) defect.

[0290] Example

[0291] The present invention is further illustrated by the following specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental methods in the following examples, where specific conditions are not specified, were performed in accordance with conventional conditions in the art or the conditions recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available.

[0292] Example 1: Cholesin is a cholesterol-induced intestinal secretory hormone

[0293] 1.1 Discovery and identification of cholesin

[0294] In this example, 10-12-week-old wild-type male mice with a C57BL / 6J genetic background were used to identify hormones that regulate cholesterol homeostasis. Serum proteins were analyzed after an overnight fast followed by a 1-hour diet (RD, containing 0.02% cholesterol) or a Western diet (WD, containing 1.25% cholesterol). The mice were randomly divided into two groups, each consisting of six mice. The first group of mice was fasted overnight and then fed a RD diet (RD group); the second group of mice was fasted overnight and then fed a Western diet (WD group). Blood was collected from both groups using lithium heparinized tubes and centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant, plasma, was transferred to a fresh centrifuge tube. The plasma was treated with a column-based albumin / immunoglobulin depletion kit (Sangon Biotech) to remove IgG and albumin. High-abundance proteins were then diluted and low-abundance proteins were concentrated using the ProteoMiner Protein Enrichment Kit (BIO-RAD). The treated plasma was mixed with 5× SDS loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-ME), boiled at 100°C for 10 minutes, and subjected to SDS-PAGE protein electrophoresis. After electrophoresis, silver staining was performed. The results showed an enhanced band at approximately 23 kDa in the Western diet group compared to the standard diet group (Figure 1).

[0295] Mass spectrometry analysis of the differential bands identified them as a protein encoded by the gene 3110082I17Rik (the mouse homolog of C7ORF50), which had not been previously reported. Based on subsequent work demonstrating its inhibitory effect on cholesterol synthesis, the inventors named it Cholesin.

[0296] 1.2 Cholesin is a secreted protein

[0297] In order to explore the localization of Cholesin in cells, immunofluorescence experiments were performed. The specific methods are as follows: 1) Place an appropriate amount of 12mm diameter cell slides in the well plate, and place HEK293T cells in the well plate at a ratio of 1:6. After the cells adhere, overexpress human Cholesin (hCholesin) with a Flag tag at the C-terminus, mouse Cholesin (mCholesin) and a control empty plasmid; 2) After 36 hours of transfection, aspirate the culture medium, wash the cells once with PBS preheated at 37°C, and then fix the cells with 4% paraformaldehyde / PBS solution equilibrated to room temperature for 20 minutes at room temperature; 3) After fixation, aspirate the 4% paraformaldehyde / PBS solution, wash once with PBS, and then rinse with 0.2% Triton Treat cells with X-100 in PBS for 5-7 minutes at room temperature to permeabilize the cell membrane; 4) Immediately aspirate and discard the permeabilization solution, wash the cells once with PBS, and block the cells with 5% BSA / TBST solution for 30 minutes at room temperature; 5) Dilute the primary antibody in 5% BSA / TBST solution and incubate with the primary antibody at room temperature for 1 hour after blocking; 6) Recover the primary antibody and wash the slides with TBST three times for 10 minutes each; 7) Dilute the fluorescent secondary antibody and DAPI (final concentration 0.5 mg / ml) in 5% BSA / TBST solution and stain at room temperature for 1 hour in the dark; 8) Discard the secondary antibody and wash the slides with TBST three times for 10 minutes each; 9) Mount the slides and image the cholesin localization using a laser confocal microscope. Immunofluorescence results show that cholesin is primarily localized in the cytoplasm (Figure 2A).

[0298] Western blotting was then used to investigate the secretion of cholesin. HEK293T cells were overexpressed with C-terminally Flag-tagged human cholesin, mouse cholesin, and a control empty plasmid. 48 hours after transfection, the cells were cultured overnight in serum-free medium, and the culture medium was harvested. Adherent cells in a 6-well plate were treated with 200 μl of RIPA lysis buffer (25 mM Tris, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 100× protease lysis buffer, pH 7.4) per well. Cells were scraped from the bottom of the dish with a cell scraper and collected into an EP tube. The cells were then sonicated on ice using a 6-second on / off cycle for 18 seconds total at 40% power. The cells were centrifuged at maximum speed for 15 minutes at 4°C. The supernatant was then added with 5× SDS loading buffer and boiled at 100°C for 10 minutes to obtain a total protein extract. Cholesin in the culture medium and lysis supernatant was detected by Western blotting. The results showed that Cholesin secretion could be detected in the culture medium of cells overexpressing human or mouse Cholesin (Figure 2B), indicating that Cholesin also has the ability to be secreted under in vitro conditions.

[0299] To characterize secreted Cholesin, a baculovirus overexpressing mouse Cholesin with a Flag tag at the C-terminus was constructed and used to infect insect Hi5 cells. The specific method is as follows: 1) Culture 1L of Hi5 cells to a density of 2-3×10 6 1) 1) 10 cells / ml, baculovirus was added at a ratio of 1:50, cultured for 48-72 hours, and the culture supernatant was collected by centrifugation and filtered through a 0.22 μm filter to remove cell debris. 2) A HisTrap excel (Cytiva) chromatography column was equilibrated with equilibration buffer (20 mM NaH2PO4, 0.5 M NaCl, pH 7.4) for at least 5 column volumes. 3) The filtered culture medium was loaded onto the column using a pump. 4) The column was rinsed with wash buffer (20 mM NaH2PO4, 0.5 M NaCl, 30 mM imidazole, pH 7.4) for at least 20 column volumes until no protein flowed through. 5) A gradient elution was performed with elution buffer (20 mM NaH2PO4, 0.5 M NaCl, 500 mM imidazole, pH 7.4) to collect the protein peak. 6) The protein peak was concentrated to a total volume of less than 2 ml using a concentrator containing a 10 kDa NMWL filter. The protein peak was then filtered through a Superdex 200 Increase / Drop elution column. The sample was further separated and purified using a 10 / 300 GL molecular sieve based on the protein's size and shape. Mass spectrometry analysis of the purified protein revealed that, with the exception of lysine- and arginine-rich regions, the majority of the Cholesin sequence could be identified, indicating that Cholesin is secreted in its entirety.

[0300] These results indicate that Cholesin is secreted in its full-length form.

[0301] 1.3 Cholesin is a hormone that responds to eating

[0302] In this example, 10-12-week-old wild-type male mice with a C57BL / 6J genetic background were randomly divided into nine groups of eight. Group 1 consisted of mice starved overnight (fasting group); Groups 2 through 5 were starved overnight and then fed a regular diet (RD) for 1, 2, 4, and 12 hours (regular diet supplemented with 0.02% cholesterol); and Groups 6 through 9 were starved overnight and then fed a Western diet (WD) for 1, 2, 4, and 12 hours (Western diet supplemented with 1.25% cholesterol). Mice from these nine groups were sacrificed, and plasma was collected for cholesin levels using an enzyme-linked immunosorbent assay (ELISA).

[0303] The specific procedure is as follows: 3 μl of mouse plasma and 100 μl of coating solution (0.05 mol / L Na₂CO₃ / NaHCO₃, pH 8.0) were added to a polystyrene microplate. Plasma from a whole-body knockout mouse was used as a control for calibration in a 96-well microplate. A purified and quantified cholesin protein standard was used to establish a standard curve at concentrations of 0, 12.5, 25, 50, 100, and 200 pmol. After sample addition, the plate was incubated overnight at 4°C to allow protein adsorption to the well bottom. After overnight coating on a shaker at 4°C, the microplate was removed, the liquid in the wells was shaken dry, and 100 μl of 1× gelatin was added to each well for 2 hours at room temperature. The blocking solution was discarded, and the wells were washed four times with 200 μl of TBST solution per well, with the final shake to remove the liquid as much as possible. The primary antibody for detection was prepared in TBST, 100 μl of the primary antibody was added to each well, and the plate was incubated overnight on a shaker at 4°C. Discard the primary antibody and wash four times with 200 μl of TBST solution per well, shaking off as much liquid as possible the last time. Prepare the secondary antibody in TBST, add 100 μl to each well, and incubate on a shaker at room temperature for 1 hour. Discard the secondary antibody and wash four times with 200 μl of TBST solution per well, shaking off as much liquid as possible the last time. Add 100 μl of TMB substrate to each well and incubate on a shaker at room temperature for 10-30 minutes. Then, add 100 μl of stop solution (0.05 mol / L H2SO4) to each well. Read the absorbance at OD450nm using a microplate reader within 30 minutes. Calculate the cholesin content in the sample based on the absorbance of the standard curve.

[0304] The results showed that in mice fed after starvation, the level of cholesin in plasma increased rapidly and quickly reached a peak of approximately 1200 pM one hour after eating the Western diet. It then continued to decline and returned to the level of 0 hours after 12 hours after eating. The changes in cholesin in plasma after feeding with a normal diet also showed the same trend, but the changes were weaker than those in the Western diet group, with the peak level of cholesin being only around 700 pM one hour after eating (Figure 3A). These results indicate that eating a high-cholesterol diet promotes cholesin secretion, suggesting that cholesterol may be an inducer of cholesin secretion.

[0305] To investigate whether the same secretory changes as in mice occur in humans, we analyzed clinical data. We compared plasma cholesin levels in 13 healthy pairs of subjects after an overnight fast and after a one-hour meal. The results showed a significant increase in plasma cholesin levels after a meal (Figure 3B).

[0306] These results suggest that cholesin is a hormone that is responsive to feeding and that cholesterol is a possible inducer.

[0307] 1.4 Tissue Distribution of Cholesin

[0308] In this example, 5 wild-type (WT) and Cholesin knockout (KO) male mice of 10-12 weeks of age with a genetic background of C57BL / 6J were selected, and the brain, stomach, small intestine, colon, liver, kidney, brown adipose tissue (BAT), epididymal white adipose tissue (eWAT), and skeletal muscle of the mice were taken respectively. RIPA lysis buffer (25mM Tris, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.4) was prepared, and protease inhibitors and phosphatase inhibitors were added before use according to the volume of the lysis buffer to prepare tissue protein extracts. Appropriate amount of tissue was weighed, 500μl RIPA lysis buffer and magnetic beads were added, and the tissue grinder was cryogenically ground at 60Hz for 1 minute. After grinding, the tissue was centrifuged at 15000rpm in a 4°C pre-cooled centrifuge for 15 minutes. When the lipid content in the tissue was high, the supernatant of the first centrifugation was transferred to a new centrifuge tube and centrifuged again for 15 minutes under the same parameters. The supernatant was taken for protein quantification using the BCA method, 5× SDS loading buffer was added and mixed thoroughly, and then heated in a 100°C metal bath for 10 minutes to obtain a tissue protein sample. Then 25 μg of total protein was taken for SDS-PAGE electrophoresis to determine the tissue distribution of Cholesin at the protein level.

[0309] The results are shown in Figure 4 . The endogenous Cholesin protein is slightly smaller than 25 kDa in size, with the highest expression level in the small intestine, followed by the colon and stomach, and low expression levels in other tissues, indicating strong tissue specificity.

[0310] 1.5 Cholesin is an intestinal secretory hormone that responds to cholesterol absorption

[0311] Because Cholesin is most highly expressed in the intestine, and the small intestine is the primary organ for exogenous cholesterol absorption, it has been hypothesized that Cholesin may be secreted by the small intestine to regulate cholesterol homeostasis. To test this hypothesis, mice with intestinal-specific Cholesin knockout (Cholesin IKO) were generated as a research tool.

[0312] In this example, WT and IKO mice were randomly divided into two groups, each with 8 mice. The first group of mice was starved overnight, while the second group of mice was starved overnight and then fed a Western diet (WD) containing 1.25% cholesterol for 1 hour. Plasma and small intestine were then collected from the mice, and total cholesterol (TC) in the small intestine and cholesterol, TC, and triglyceride (TG) levels in the plasma were measured. The results are shown in Figures 5A and 5B.

[0313] To further confirm the role of cholesterol in promoting Cholesin secretion, WT and IKO mice were randomly divided into two groups, each with 7 mice. The mice were starved overnight. The first group of mice were gavaged with 150 μl corn oil (chol-) containing 20% ​​anhydrous ethanol, and the second group of mice were gavaged with 150 μl 200 mg kg -1 Body weight cholesterol (chol+, cholesterol dissolved in corn oil containing 20% ​​anhydrous ethanol) was measured. Plasma and small intestinal tissue were collected 1 hour after gavage. Small intestinal TC levels and plasma cholesin, TC, and TG levels were measured. The results are shown in Figures 5C and 5D.

[0314] The results in Figure 5A show that the cholesin content in the plasma of WT mice increased significantly after feeding WD, while the cholesin content in the plasma of IKO mice barely increased, and the cholesin content in the plasma of IKO mice was significantly lower than that of WT mice under starvation. These results suggest that the small intestine may be the main organ for cholesin secretion in response to the Western diet. The results in Figure 5A also show that the total cholesterol content in the small intestine increased after feeding WD, but there was no significant difference between the WT and IKO groups in both the starved and fed states. This result suggests that there is no difference in the cholesterol absorption capacity of the small intestine between WT and IKO mice, and that their response to the Western diet is not due to different small intestinal absorption capacities.

[0315] The results in Figure 5B show that there was no significant change in plasma TC in mice before and after eating, but the plasma TC of IKO mice was higher than that of the WT group before and after eating. Since plasma was collected 1 hour after eating, at which time exogenous cholesterol had not yet entered the blood circulation, plasma TC did not change with eating; the fact that the plasma TC in the IKO group was higher than that in the WT group suggests that Cholesin may negatively regulate plasma TC levels. Figure 5B also shows that the plasma TG content of mice decreased significantly before and after eating. There was no significant difference in plasma TG between WT and IKO mice under starvation conditions. After eating, the plasma TG of IKO mice was slightly higher than that of the WT group, indicating that eating reduces the total triglycerides circulating in the blood. The difference in plasma TG between the WT and IKO groups also suggests that Cholesin may have an effect on plasma TG.

[0316] Similar to the effects of WD feeding, the results in Figure 5C show that cholesin levels in the plasma of WT mice increased significantly after oral administration of cholesterol, while IKO mice showed little response to cholesterol stimulation, and cholesterol absorption in IKO mice was also unaffected. The results in Figure 5D show that there was no significant change in plasma TC levels in mice after oral administration of cholesterol, but plasma TC in IKO mice was higher than that in the WT group in both the control and cholesterol-treated groups. Total plasma TG levels increased after oral administration of cholesterol, but there was no significant difference in plasma TG between WT and IKO mice in either the experimental or control groups. This is because cholesterol is dissolved in corn oil, resulting in an increase in plasma TG levels after oral administration.

[0317] These results demonstrate that Cholesin is a hormone secreted by the small intestine in response to cholesterol absorption.

[0318] Example 2: NPC1L1-mediated cholesterol absorption promotes Cholesin secretion

[0319] 2.1 Cholesin expression in absorptive enterocytes

[0320] Cholesterol absorption is currently believed to be mediated by NPC1L1 on the apical surface of absorptive enterocytes in the intestine. Because cholesterol absorption promotes Cholesin secretion, we hypothesized that Cholesin might be primarily expressed in absorptive enterocytes. To test this hypothesis, we constructed double-knockout mice expressing Cholesin-GFP and Apoa1-mCherry (APOA1 is a marker protein for absorptive enterocytes) and used them for immunofluorescence experiments to identify the cell types expressing Cholesin. The specific experimental steps are as follows: 1) Collect the mouse small intestinal tissue, flush the intestinal feces with PBS and immediately place it in a 4% paraformaldehyde solution prepared in PBS and fix it overnight at 4°C; 2) Transfer the fixed tissue to a PBS solution containing 30% sucrose and dehydrate it at 4°C overnight; 3) After dehydration, the tissue should sink to the bottom of the sucrose solution. Remove the tissue from the sucrose solution, absorb the liquid on the surface of the tissue as much as possible, add OCT to the embedding box and place the tissue block, and freeze the embedded tissue in a -80°C refrigerator for 6 hours; 4) Before sectioning, place the tissue block in a -20°C refrigerator for equilibrium, cut slices with a thickness of 5-8 μm using a freezing microtome, and attach the tissue slices to a cationic anti-shedding slide; 5) Before staining, remove the slices and equilibrate at room temperature for 20 minutes, then place them in a 55°C oven for 10 minutes to prevent the tissue from falling off the slide, and circle the tissue on the slide with a histochemical pen; 6) Wash the tissue three times with TBST to wash off the OCT, and then use 0.2% Triton Permeabilize the tissue with Triton X-100 / PBS for 5-7 minutes; 7) Immediately remove the 0.2% Triton X-100 / PBS after penetration, rinse once with TBST, and then block with 5% BSA / TBST at room temperature for 30 minutes; 8) Dilute the primary antibody with 5% BSA / TBST, discard the blocking solution after blocking, add GFP primary antibody, 80-100 μl of primary antibody per tissue block, add double-distilled water to the humidified chamber, and incubate the sections at 4°C overnight; 9) Discard the primary antibody and wash the sections with TBST at room temperature three times, 5 minutes each; 10) Dilute the fluorescent secondary antibody and DAPI with 5% BSA / TBST and incubate at room temperature in the dark for 1 hour; 11) Discard the secondary antibody and DAPI, wash the sections with TBST at room temperature three times, 5 minutes each; 12) Mount the sections and image the Cholesin localization results using a laser confocal microscope.

[0321] As shown in Figure 6 , colocalization of Cholesin-GFP and Apoa1-mCherry was observed, indicating that Cholesin is expressed in absorptive enterocytes.

[0322] 2.2 Cholesin levels increased in a dose-dependent manner with increasing cholesterol concentration

[0323] To investigate whether cholesterol directly affects Cholesin secretion, HCT116 cells (human colorectal cancer cells) stably overexpressing Cholesin were constructed. The day before the experiment, HCT116 cells stably overexpressing Cholesin were plated in 24-well plates. The cells were in the logarithmic growth phase at the time of the experiment. Before treatment, the cells were washed once with prewarmed serum-free RPMI1640 medium. Exogenous cholesterol was diluted in serum-free RPMI1640 to final concentrations of 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM. 500 μl of serum-free RPMI1640 medium containing the final concentrations of cholesterol was added to each well of the 24-well plate and incubated in a 37°C incubator for 1 hour. After the incubation period, 400 μl of the medium was transferred to a 1.5 ml EP tube and centrifuged at maximum speed at 4°C for 5 minutes to remove cell debris. The culture medium collected from each well of cells was spotted into a microplate three times in parallel, with 100 μl in each well, and ELISA was performed to detect the secretion of Cholesin in the culture medium.

[0324] The total cholesterol level in the cells was also detected using the following method: 1) After the cells were aspirated to detect the amount of Cholesin secretion, the remaining cells were digested with Trypsin, resuspended in PBS, and the cells were counted; 2) The supernatant was discarded after high-speed centrifugation, 150 μl of isopropanol was added to each sample, and the cells were disrupted by ultrasonication on ice (40% intensity, ultrasonication for 2 seconds, interval of 3 seconds, total time of 3 minutes); 3) After the end of ultrasonication, the sample was centrifuged at 10,000 g and 4°C for 10 minutes. The supernatant was the total cholesterol extract of the cells; 4) The total cholesterol concentration in the extract was determined using the CHOD-PAP cholesterol determination kit (Sino-Biotech Holdings). The total cholesterol amount was then calculated based on the volume of the extract. Finally, the total cholesterol amount was divided by the total number of cells to convert it into the cholesterol content per 106 cells.

[0325] The results are shown in Figure 7. The results show that as cholesterol concentration increases, the level of cholesin in the culture medium increases in a dose-dependent manner. Furthermore, as the cholesterol treatment dose increases, the total amount of cholesterol in the cells also gradually increases.

[0326] NPC1L1 knockdown in HCT116 cells disrupts cholesterol absorption and cholesin secretion

[0327] To investigate the role of NPC1L1 in cholesterol-induced Cholesin secretion at the cellular level, NPC1L1 was knocked down (Npc1l1 KD) in the HCT116 stable cell line overexpressing Cholesin. Subsequently, immunoblotting confirmed that NPC1L1 knockdown had no effect on Cholesin expression (Figure 8A).

[0328] According to the method in 2.2, non-target knockdown control cells (NT) and NPC1L1 knockdown cells (Npc1l1 KD) were plated in 24-well plates, and then cultured with serum-free RPMI1640 containing 0μM and 10μM cholesterol for 1 hour. After the culture, the culture medium was collected and the secretion of Cholesin was detected by ELISA. After counting the cells, isopropanol was added to extract total cholesterol by ultrasonication. The total cholesterol concentration in the extract was determined using the CHOD-PAP cholesterol assay kit (Zhongsheng Beikong). The total cholesterol amount was calculated based on the volume of the extract, and finally the total cholesterol amount was divided by the total number of cells to convert it into 10 6 The results showed that knockdown of NPC1L1 in HCT116 cells impaired cholesterol absorption and Cholesin secretion (Figure 8B).

[0329] The results of Examples 2.2 and 2.3 together indicate that NPC1L1-mediated cholesterol absorption stimulates the secretion of Cholesin.

[0330] NPC1L1 inhibitor treatment and Npc1l1 gene knockout in mice effectively blocked small intestinal cholesterol absorption and cholesin secretion

[0331] To further verify whether Cholesin secretion in mice is dependent on NPC1L1-mediated cholesterol absorption, two methods were used: treating mice with the NPC1L1 inhibitor Ezetimibe and directly using Npc1l1 gene knockout mice.

[0332] NPC1L1 inhibitor treatment

[0333] Male mice aged 10-12 weeks were fasted overnight and then given 10 mg kg -1 One hour after gavage, mice were gavaged with 150 μl corn oil solvent control (chol-) or cholesterol (chol+, 200 mg kg -1 One hour after gavage, plasma was collected for determination of total cholesterol (TC) and cholesin levels. Small intestinal tissue was also collected to determine total cholesterol content. The results are shown in Figures 9A-9B.

[0334] The results showed that the plasma cholesin level of control mice (Chol-) fasted overnight was low, with a plasma content of approximately 500 pM. In mice not treated with ezetimibe, the plasma cholesin level increased significantly after oral administration of cholesterol. However, mice treated with ezetimibe no longer responded to the cholesterol administration stimulation, and the plasma cholesin level remained basically the same as in the starved state (Figure 9A). At the same time, the total cholesterol content in the small intestine indicated that ezetimibe treatment inhibited the absorption of cholesterol in the small intestine (Figure 9A). Determination of the plasma TC content in mice showed that short-term drug administration stimulation did not cause changes in plasma TC levels (Figure 9B).

[0335] Npc1l1 gene knockout

[0336] To further explore the role of NPC1L1 in cholesterol-induced Cholesin secretion, we constructed an Npc1l1 gene knockout (Npc1l1 - / - ) mice. Western blotting confirmed that the loss of Npc1l1 did not affect the expression of Cholesin (Figure 9C). + / + ) and Npc1l1 knockout (Npc1l1 - / - The mice were fasted overnight and then orally gavaged with 150 μl corn oil solvent control (chol-) or cholesterol (chol+, 200 mg kg -1 After 1 hour, whole blood was collected from the mice to separate the plasma for the detection of plasma cholesin and TC levels, and small intestinal tissue was collected to determine the TC content. The results are shown in Figures 9D-9E.

[0337] The results showed that Npc1l1 deficiency significantly inhibited cholesin secretion and cholesterol absorption in the small intestine of mice (Figure 9D). Measurement of plasma TC content in mice showed that short-term cholesterol treatment did not cause changes in plasma TC levels (Figure 9E).

[0338] The above in vitro and in vivo results of Example 2 indicate that NPC1L1-mediated cholesterol absorption promotes the secretion of Cholesin.

[0339] Example 3: Cholesin regulates cholesterol metabolism in humans

[0340] To investigate whether cholesin has a similar cholesterol-regulating effect in humans, plasma samples were collected from 600 individuals one hour after a meal and analyzed by ELISA. Levels of cholesin, total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were measured and correlated. The procedure was as follows: 3 μl of human plasma and 100 μl of coating buffer (0.05 mol / L Na₂CO₃ / NaHCO₃, pH 8.0) were added to a polystyrene microplate. Plasma from whole-body cholesin knockout mice was used as a control for calibration in a 96-well microplate. A standard curve was generated using purified and quantified cholesin protein standards at concentrations of 0, 12.5, 25, 50, 100, and 200 pmol. After sample addition, the plates were incubated overnight at 4°C to allow protein adsorption to the well bottom. After overnight coating at 4°C on a shaker, the microplates were removed, the liquid in the wells was dried, and 100 μl of 1× gelatin was added to each well and blocked for 2 hours at room temperature. Discard the blocking solution and add 200 μl of TBST to each well and wash four times, shaking thoroughly for the final time. Prepare the primary antibody in TBST and add 100 μl of primary antibody to each well. Incubate overnight on a shaker at 4°C. Discard the primary antibody and add 200 μl of TBST to each well and wash four times, shaking thoroughly for the final time. Prepare the secondary antibody in TBST and add 100 μl of secondary antibody to each well. Incubate for 1 hour on a shaker at room temperature. Discard the secondary antibody and add 200 μl of TBST to each well and wash four times, shaking thoroughly for the final time. Add 100 μl of TMB substrate to each well and incubate for 10-30 minutes on a shaker at room temperature. Then, add 100 μl of stop solution (0.05 mol / L H2SO4) to each well. Read the absorbance at OD450 nm using a microplate reader within 30 minutes. Calculate the cholesin content in the sample based on the absorbance of the standard curve. The data were analyzed by Pearson correlation analysis using Excel, and the p value and r were calculated. 2 .

[0341] Correlation analysis results showed that cholesin levels were negatively correlated with TC and LDL-C levels (Figures 10A and 10B), indicating that cholesin has a negative regulatory effect on cholesterol levels.

[0342] Example 4: Cholesin knockout increases hepatic cholesterol synthesis and VLDL secretion

[0343] 4.1 Determination of Plasma Total Cholesterol, HDL, LDL, VLDL, and Triglyceride Levels, as well as Liver Cholesterol and Triglyceride Levels in Cholesin IKO Mice

[0344] In this example, 12-14-week-old wild-type (WT) and intestinal cholesin knockout (cholesin IKO) male and female mice with a C57BL / 6J genetic background were fed a regular diet (RD) containing 0.02% cholesterol or a Western diet (WD) containing 0.2% cholesterol for 8 weeks. After overnight starvation and subsequent feeding for 6 hours, plasma was collected to measure total cholesterol (TC) and triglyceride (TG) levels. Liver tissue was also collected to measure TC and TG levels. The results are shown in Figures 11A and 11B.

[0345] Since cholesterol exists primarily in the form of lipoproteins (such as HDL, LDL, and VLDL) in blood, the content of each lipoprotein in plasma was analyzed using fast protein liquid chromatography. The experimental method was as follows: Plasma from all mice in each group was pooled, mixed, and placed on ice pending sample loading. A Superose 6 Increase 10 / 300 GL column was connected to an AKTA purification system and equilibrated with PBS. 1 ml of total plasma was accurately injected using a sample loop. After complete injection, eluted fractions were collected without waiting for peak elution. 400 μl of each fraction was collected, and sampling was stopped when the salt peak appeared. The total cholesterol content of each fraction was determined enzymatically, and FPLC curves were plotted. The relative levels of very low-density lipoprotein (VLDL), intermediate / low-density lipoprotein cholesterol (IDL / LDL), and high-density lipoprotein cholesterol (HDL) were calculated based on the area under the FPLC curve. The results are shown in Figure 11C.

[0346] Figure 11A shows that when wild-type mice were fed a standard diet, plasma TC levels in male mice were approximately 100 mg / dL, while those in female mice were slightly lower. However, when fed a WD diet, plasma TC levels in male mice reached approximately 170 mg / dL, while those in female mice reached 140 mg / dL. Plasma TC levels in IKO male and female mice fed a standard diet or a Western diet were approximately 20% higher than those in wild-type mice. Regarding plasma triglyceride levels in mice, IKO mice showed slight increases in plasma TG compared to wild-type mice when fed either a RD or WD diet.

[0347] Figure 11B shows that in both male and female mice fed either RD or WD, liver TC content in IKO mice was higher than in WT mice. While there was no significant difference in liver TC content between males and females, the difference between WT and IKO liver TC was amplified when fed WD. Regarding liver TG, there was no significant difference between WT and IKO mice under RD, but under WD, liver TG in IKO mice was slightly higher than that in WT mice.

[0348] Figure 11C shows that high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and very-low-density lipoprotein cholesterol (VLDL-C) were increased in Cholesin IKO mice.

[0349] These results indicate that cholesin deficiency leads to elevated plasma cholesterol and triglyceride levels.

[0350] 4.2 Elevated plasma cholesterol levels in Cholesin IKO mice are not caused by enhanced intestinal cholesterol absorption

[0351] According to previous reports, elevated plasma cholesterol levels may be caused by enhanced intestinal cholesterol absorption, increased hepatic cholesterol synthesis and VLDL secretion, or decreased cholesterol excretion. To investigate whether elevated plasma cholesterol levels in IKO mice were due to enhanced intestinal cholesterol absorption, body weight, body fat percentage, food and water intake, small intestinal total cholesterol and triglyceride levels, and chylomicron breakdown rate were measured in the mice described in Example 4.1.

[0352] Mice were randomly fed, and their body weight and body fat were measured using an EchoMRI awake animal body composition analyzer. Each mouse was measured three times. Body fat percentage was calculated using the following formula: Body fat percentage = mouse body fat / mouse body weight × 100%. The results are shown in Figures 12A and 12B.

[0353] Food intake was measured in individually housed mice using the PhenoMaster system from TSE Systems. Mice were allowed at least 24 hours to acclimate before measurement. Food and water were provided ad libitum in an appropriate device, and data were collected continuously for 48 hours using a built-in automated instrument. The results are shown in Figures 12C and 12D.

[0354] To determine the total cholesterol and triglyceride content in the small intestine of mice, the small intestine tissues of the mice starved overnight and then fed for 6 hours in Example 4.1 were collected. The measurement results are shown in FIG12E .

[0355] A lipid tolerance test was conducted by orally administering a dose of olive oil to mice to assess the rate of chylomicron breakdown in the small intestine. The mice described in Example 4.1 were fasted for 6 hours to synchronize their intestinal status. Subsequently, 200 μl of olive oil was orally administered. Blood was collected from the tail vein before and at various time points (0, 1, 2, 3, and 4 hours) after the oral administration. Plasma triglyceride levels were then separated and measured, and the triglyceride secretion rate (ΔTG) was calculated (plasma TG at 1, 2, 3, and 4 hours minus the plasma TG at time 0 for each mouse) to assess the rate of chylomicron breakdown. The results are shown in Figure 12F.

[0356] Figure 12A shows that there was no significant difference in body weight between WT and IKO groups of male and female mice under the two dietary treatments. Figure 12B shows that there was no significant difference in body fat percentage between WT and IKO groups of male and female mice under the two dietary treatments. Figures 12C and 12D show that there was no significant difference in food intake and water intake between WT and IKO groups of male and female mice under the two dietary treatments. Figure 12E shows that there was no significant difference in total cholesterol and triglyceride content in the small intestine between IKO mice and WT mice in male and female mice fed a normal diet or a Western diet. Figure 12F shows that the chylomicron decomposition rate of IKO mice was not affected in male and female mice fed a normal diet or a Western diet.

[0357] These results indicate that intestinal absorption is similar in IKO and WT mice, and therefore the elevated plasma cholesterol levels in IKO mice are not due to enhanced cholesterol absorption in the small intestine.

[0358] 4.3 Elevated plasma cholesterol levels in Cholesin IKO mice are not caused by decreased cholesterol excretion

[0359] To investigate whether the elevated plasma cholesterol levels in IKO mice were caused by decreased cholesterol excretion, the bile cholesterol and bile acid levels, as well as the cholesterol level in feces, of the mice of Example 4.1 were measured.

[0360] Cholesterol in mice is generally excreted through bile acid or feces. The cholesterol content in bile acid and feces was measured to explore whether the plasma TC level of IKO mice was affected by changes in cholesterol excretion. Mouse bile is excreted from the gallbladder during eating and is mainly stored in the gallbladder when hungry. The mice were starved overnight, and the bile in the gallbladder was collected with a syringe. The volume of the gallbladder was then weighed, and the concentrations of total bile cholesterol (cholesterol determination kit (CHOD-PAP method, Zhongsheng Beikong) and bile acid (total bile acid (TBA) kit, Nanjing Jiancheng) were measured using a kit. The results are shown in Figure 13A.

[0361] To measure the cholesterol content in feces, mouse feces were collected and total cholesterol and triglycerides were extracted from the feces using the following method: 40 mg of feces were weighed and dried in a 55°C oven. The dried, crushed feces were then suspended in 1200 μl of a 2:1 chloroform-methanol (v / v) mixture. The suspension was then mixed with 300 μl of water and vortexed vigorously for 60 seconds. The mixture was then centrifuged at 4000 rpm for 10 minutes at 4°C, and the organic layer was carefully transferred to a new test tube for drying. The dried lipids were then resuspended in 100 μl of a 9:1 isopropanol-Triton X-100 (v / v) solution, and the resulting fecal extract was used for enzymatic assays to detect total cholesterol and triglyceride levels. The results are shown in Figure 13B.

[0362] Figure 13A shows that there were no significant differences in total bile volume, bile cholesterol levels, and bile acid levels between WT and IKO mice treated with either diet. Figure 13B shows that there were no significant differences in fecal total cholesterol and triglyceride levels between WT and IKO mice treated with either diet.

[0363] These results indicate that the elevated plasma cholesterol levels in IKO mice are not caused by decreased cholesterol excretion.

[0364] 4.4 The elevated plasma cholesterol levels in Cholesin IKO mice are due to increased cholesterol synthesis in the liver.

[0365] To investigate whether the elevated plasma cholesterol levels in IKO mice were caused by enhanced hepatic cholesterol synthesis, the expression of cholesterol synthesis genes in the liver was examined at the mRNA and protein levels.

[0366] qPCR was used to measure mRNA levels of genes involved in cholesterol synthesis in the liver. The specific experimental procedures were as follows: 1) Approximately 20 mg of liver tissue was weighed and total RNA was extracted using the HP Total RNA Kit (Omega); 2) 1 μg of RNA was reverse transcribed using the REVERTAID 1ST CDNA SYNTH KIT (thermo); 3) the reverse-transcribed cDNA was subjected to qPCR. The results are shown in Figure 14A.

[0367] Immunoblotting was used to examine the protein expression levels of cholesterol biosynthesis genes in the liver. The specific procedures were as follows: 1) Weigh 20 mg of liver tissue, add 500 μl of RIPA lysis buffer and magnetic beads, and cryo-grind at 60 Hz using a tissue grinder for 1 minute; 2) Centrifuge at maximum speed at 4°C for 10 minutes; 3) Transfer the supernatant to a fresh EP tube, taking care not to aspirate the fat layer; add 5× loading buffer, mix thoroughly, and boil the sample at 100°C for 10 minutes; 4) Quantify protein using the BCA assay; 5) Analyze the expression levels of SREBP2 (including pSREBP2 and mSREBP2), HMGCR, HMGCS1, and LDLR in the tissue by immunoblotting. The results are shown in Figure 14B.

[0368] Figures 14A and 14B show that compared to wild-type mice, the expression levels of cholesterol synthesis-related genes Hmgcs1, Hmgcr, Mvd, Mvk, Pmvk, and sqle in the liver of IKO mice fed a normal diet and a Western diet were significantly increased, as well as the transcription factor SREBP2 that regulates the expression of these cholesterol synthesis-related genes and its downstream regulated LDLR. Notably, the expression levels of SREBP2 and cholesterol synthesis-related genes were lower in mice fed a Western diet, indicating that intestinal cholesterol absorption inhibits hepatic cholesterol synthesis.

[0369] 4.5 The elevated plasma cholesterol levels in Cholesin IKO mice are caused by enhanced VLDL secretion in the liver.

[0370] To investigate whether the elevated plasma cholesterol levels in IKO mice were caused by enhanced VLDL secretion from the liver, we injected the mice with poloxamer 407, an inhibitor of lipoprotein lipase and VLDL metabolism, and monitored the plasma cholesterol and triglyceride levels of the mice to assess VLDL secretion from the liver.

[0371] Mice were fasted for 4 hours and then intraperitoneally injected with 1 g kg-1 of Poloxamer 407. Blood samples were collected from the tail vein before, 1 hour after, and 2 hours after the Poloxamer 407 injection. Plasma total cholesterol (TC) and triglyceride (TG) levels were measured at each time point, and TC and TG secretion rates were calculated (calculated as the TC or TG level at 1 or 2 hours for each mouse minus the TC or TG level at 0 hours for that mouse). The results are shown in Figure 15.

[0372] The results in Figure 15 showed that the secretion rates of TC and TG in male and female IKO mice fed a normal diet and a Western diet were significantly increased compared with those in WT mice, indicating that Cholesin deficiency increases the secretion of VLDL in the liver, thereby leading to increased plasma cholesterol levels.

[0373] The above results of Example 4 show that knockout of Cholesin increases cholesterol synthesis and VLDL secretion in the liver, thereby increasing plasma cholesterol and triglyceride levels. This suggests that administration of Cholesin can reduce cholesterol synthesis and VLDL secretion in the liver, thereby reducing plasma cholesterol and triglyceride levels.

[0374] Example 5: GPR146 is a receptor for Cholesin

[0375] 5.1 Target tissues of cholesin

[0376] In order to identify the target tissues of Cholesin, 8-10 week old wild type (Gpr146 + / + ) mice, and purified GST-Cholesin was incubated with different mouse tissues and then subjected to in vitro binding experiments on frozen tissue sections. The specific process is as follows: 1) Collect mouse tissues: According to the experimental design, the mouse adipose tissue (epididymal white adipose tissue (eWAT) and brown adipose tissue (BAT)), muscle, liver, pancreas, small intestine and kidney tissues were collected and immediately placed in 4% paraformaldehyde solution prepared in PBS for fixation overnight at 4°C; 2) Tissue dehydration and embedding: Different dehydration treatments were performed according to the different OCT and paraffin embedding agents. Except for white adipose tissue for paraffin embedding, other tissues were subsequently embedded in OCT; 3) Tissue sectioning: The thickness of the sections was 10 μm using a freezing microtome, and the thickness of the paraffin sections was 5 μm; 4) The frozen sections were placed in an immunofluorescence humidified box and thawed at room temperature. The paraffin sections required antigen retrieval. The pretreated sections were marked with a closed circle on the periphery of the tissue with an immunohistochemistry pen and washed twice with PBS; 5) Protein binding: The binding protein (GST-Cholesin 200 nM; GST 200 nM), incubate at room temperature for 30 minutes; 6) After the protein incubation, wash twice with PBS and block with 5% BSA blocking solution prepared in PBS at room temperature for 1 hour; 7) Discard the blocking solution and incubate with primary antibody diluted in blocking solution at 4°C overnight; 8) Discard the primary antibody, wash three times with TBST, and incubate with secondary antibody diluted in blocking solution and DAPI at a final concentration of 0.5 mg / ml at room temperature for 1 hour; 10) Discard the secondary antibody and DAPI, wash three times with TBST; 11) Try to dry the solution on the tissue surface, add an appropriate amount of mounting medium to the surface of the slide according to the size of the tissue, carefully cover with a coverslip to avoid bubbles, and place at room temperature in the dark until the mounting medium solidifies. After placing at room temperature in the dark, perform imaging observation.

[0377] The immunofluorescence results showed that GST-Cholesin binding signals were present in muscle, liver, kidney, and adipose tissue, and the binding signals in muscle, liver, kidney, and adipose tissue were relatively stronger (Figure 16A). This indicates that muscle, liver, kidney, and adipose tissue are target tissues for Cholesin.

[0378] 5.2 Identification of Cholesin Receptors

[0379] To identify receptors for cholesin, a genome-wide screen was performed using a synthetic viral library containing 65,383 sgRNAs targeting 19,050 human protein-coding genes, 21,864 sgRNAs targeting miRNAs, and 3,000 non-targeting control sgRNAs. Based on the screening results, GPR146 was identified as a candidate cholesin receptor. GPR146 is a conserved orphan G protein-coupled receptor involved in the regulation of cholesterol synthesis.

[0380] To investigate whether GPR146 is a receptor for Cholesin, 8-10 week-old Gpr146 knockout mice (Gpr146) with a C57BL / 6J genetic background were selected. - / - ) mice, and an in vitro binding assay was performed as described in Example 5.1. The immunofluorescence results are shown in FIG16A .

[0381] In order to prove that GPR146 is a specific receptor for Cholesin, GPR146 mutants (R90A, H101A, H169A, R179A, D187A, E265A) were designed based on the conservation and acid-base properties of the amino acids in the extracellular domain of GPR146. Since the liver is the main target tissue of Cholesin, the wild type (Gpr146 + / + ) and Gpr146 knockout (Gpr146 - / - ) mouse primary hepatocytes were stained for binding. - / - Mouse primary hepatocytes were transfected with plasmids to overexpress wild-type GPR146 (WT) and mutant GPR146 (Mut). The specific experimental method is as follows: 1) According to the experimental needs, an appropriate number of 12mm diameter cell slides were spread in a 6-well plate in advance, and Gpr146 was isolated at the same time. + / + and Gpr146 - / - Primary mouse hepatocytes were diluted to an appropriate density and plated in a 6-well plate; 2) Gpr146 - / -Six hours after mouse cells adhered, plasmids were transfected to overexpress wild-type GPR146 (WT) and mutant GPR146 (Mut); 3) After 36 to 48 hours of cell culture, the cells in the 6-well plate were transferred to a 24-well plate and washed once with PBS buffer. GST and GST-Cholesin were diluted to 200 nM in fresh culture medium, and Cholesin-His was diluted to 20 μM. WT cells were divided into three groups: GST negative control group, GST-Cholesin binding group, and GST-Cholesin and Cholesin-His co-incubation group. Gpr146 - / - The cells were incubated with 200nM GST-Cholesin only. 200μl of ligand-containing culture medium was added to each well and the cells were incubated in a 37°C constant temperature incubator for 30min; 4) After incubation, the culture medium was aspirated and the cells were washed three times with PBS buffer; 5) 250μl of 4% paraformaldehyde / PBS solution was added to each well and the cells were fixed on a horizontal shaker at room temperature for 30min; 6) The paraformaldehyde solution was aspirated and the cells were washed once with PBS and then 250μl of 5% BSA / PBS solution was added to the cells and blocked at room temperature for 30min on a horizontal shaker; 7) The primary antibody was diluted in an appropriate ratio with 5% BSA / PBS solution, the blocking solution was aspirated and 200μl of the primary antibody solution was added to the cells and incubated at room temperature for 1h; 8) The primary antibody was recovered and stored at 4°C and washed three times with PBS solution for 10min each time; 9) The cells were fixed with 5% Dilute the fluorescent secondary antibody and DAPI in BSA / PBS, add 200 μl of the secondary antibody and DAPI solution to each well, and incubate on a horizontal shaker at room temperature in the dark for 1 hour. 10) Aspirate the secondary antibody and DAPI solution and wash three times with PBS for 10 minutes each. 11) Wipe the slide with dust-free paper and mark the experimental conditions. Add an appropriate amount of mounting medium to the slide. Gradually blot the remaining liquid along the edge of the slide with absorbent paper. Gently lower the mounting medium with the cell side facing the mounting medium, avoiding bubbles. Incubate at room temperature in the dark overnight. Wait for the mounting medium to solidify before imaging. The experimental results are shown in Figure 16B.

[0382] At the same time, 8-10 week old wild-type mice with C57BL / 6J genetic background and Gpr146 - / - Saturation binding experiments were performed on primary mouse hepatocytes. The specific process is as follows: 1) Isolation of WT and Gpr146 - / - Mouse primary hepatocytes were plated in 12-well plates and four groups were set up: WT cells, Gpr146 - / - Cells, Gpr146 - / - Cells complemented with wild-type GPR146, and Gpr146 - / - Complement the mutant GPR146 cells. - / -1) Adenovirus overexpressing wild-type and mutant GPR146 was added to the cells; 2) Purified Cholesin was labeled with a biotin labeling kit, and the concentration of biotin-labeled Cholesin was determined; 3) The culture medium of the cells infected for 24 hours was discarded, 5% BSA (prepared with M199 culture medium) was added, and the cells were incubated for 30 minutes for blocking; 4) The blocking solution was discarded, and different gradients of Cholesin-biotin protein were added to 4 groups of primary hepatocytes, with 3 replicates for each experimental group, and incubated at room temperature for 30 minutes; 5) After incubation, the culture medium was discarded, the cells were washed three times with PBS, and high-sensitivity-HRP-streptavidin antibody (1:8000) prepared with 1% BSA in PBS buffer was added and incubated at room temperature for 1 hour; 6) The culture medium was discarded, the cells were washed three times with PBS buffer, and 200 μL of cell lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1% Cells were lysed with Triton X-100 (Triton X-100), and 10 μl of the sample was used for protein quantification. 30 μl of the sample was transferred to a 96-well plate, and 100 μl of a soluble single-component TMB substrate solution was added. The sample was incubated at room temperature in the dark for color development. After the desired color development reached, 100 μl of 0.1 mol / L sulfuric acid solution was added to terminate the reaction. The absorbance at 450 nm was measured in a microplate reader. The ratio of the absorbance to the protein level represents the binding of a specific concentration of Cholesin to the cells. Graphpad software was used to analyze the relationship between the binding of different concentrations of Cholesin to primary hepatocytes, thereby generating a saturation binding curve. The experimental results are shown in Figure 16C.

[0383] The results in Figure 16A show that GST-Cholesin is expressed in Gpr146 - / - The binding signals in the muscle, liver, kidney and adipose tissue of mice were significantly weakened compared with those of wild-type mice. As shown in the immunofluorescence results in Figure 16B, GST-Cholesin can bind to the cell membrane of WT hepatocytes, and this binding can be competed by Cholesin-His; Gpr146 - / - The binding of cells to GST-Cholesin was significantly weakened. Cells supplemented with wild-type GPR146 restored their binding to GST-Cholesin, while cells supplemented with mutant GPR146 still could not bind to GST-Cholesin. As shown in the saturation curve of Figure 16C, Gpr146 - / - The saturation binding level of the cells decreased significantly. Cells supplemented with wild-type GPR146 restored their binding to Cholesin, while cells supplemented with mutant GPR146 still could not bind to Cholesin.

[0384] These in vitro and in vivo binding assays indicate that GPR146 is a receptor for Cholesin.

[0385] 5.3 Determination of the binding affinity of cholesin to GPR146

[0386] Microthermophoresis is a technique that quantifies the interactions between biomacromolecules based on the thermophoretic properties of molecules. In this example, this technique was used to measure the binding affinity of Cholesin to wild-type (WT) and mutant (Mut) GPR146. The specific experimental steps are as follows: 1) Receptor fluorescent labeling: We purified wild-type and mutant GPR146 proteins from Hi5 cells and concentrated them to about 100ul, with a concentration of about 0.4ug / ul. 90ul of protein sample was fluorescently labeled using the RED-NHS protein amino labeling kit. After labeling, the concentration was 1 / 5 of the pre-labeling concentration. The volume was expanded to 450ul, and the labeled sample was subjected to fluorescence signal detection. Based on the fluorescence signal intensity, the final receptor concentration used in the MST experiment was determined to be 50nM; 2) Cholesin protein was labeled using a Superdex 200 Incresae A 5 / 15GL molecular sieve chromatography column was replaced with a HEPES-NaCl buffer containing DDM and CHS. Cholesin was diluted to 6 μM with the buffer and then diluted 1:1 with the buffer to form 16 concentration gradients. 10 μl of 50 nM labeled wild-type and mutant GPR146 were mixed thoroughly with 10 μl of different concentrations of Cholesin, respectively, and allowed to stand at room temperature for 20 minutes. 3) Samples were aspirated using a Monolith NT.115 capillary tube and placed on a sample stage in descending order of Cholesin concentration. The samples were then placed in a microthermophoresis instrument for measurement using measurement parameters set to 10% LED power and 40% MST power. The experimental results were analyzed and calculated using NanoTemper analysis software to determine the equilibrium dissociation constants (kd) between Cholesin and wild-type and mutant GPR146 proteins.

[0387] The results are shown in FIG17 , which show that Cholesin has a high binding affinity to wild-type GPR146, with a Kd value of 21.34±0.98 nM, while the binding of Cholesin to mutant GPR146 is weakened, with a Kd value of 971.0±91.5 nM.

[0388] Example 6: Cholesin inhibits cAMP production through the GPR146-coupled Gαi signaling pathway

[0389] 6.1 Cholesin inhibits the interaction between GPR146 and GNAI1

[0390] After confirming the interaction between cholesin and GPR146, the downstream signaling of GPR146 was further investigated in primary mouse hepatocytes. GPR146 is a GPCR. The G protein associated with this GPCR is a heterotrimer composed of three distinct subunits: α, β, and γ. Ligand binding to the GPCR triggers a conformational change in the receptor, which in turn binds and activates the G protein. The active form of the G protein is then released from the receptor surface and dissociates into its α and β / γ subunits. Both subunits then activate their specific effectors, releasing a second messenger. This messenger is recognized by protein kinases, leading to their activation and triggering a signaling cascade that leads to cellular events. The Gαs and Gαi isoforms, respectively, activate or inactivate adenylate cyclase, an enzyme that converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP), releasing inorganic pyrophosphate in the process. Guanine nucleotide-binding protein G(i) subunit α1 (GNAI1) is an inhibitory subunit of G proteins. Dissociation of Gαi inhibits downstream cAMP signaling. It is hypothesized that cholesin binding to GPR146 promotes Gαi dissociation, thereby inhibiting downstream cholesterol synthesis by inhibiting cAMP. To verify this hypothesis, co-immunoprecipitation experiments were performed.

[0391] Primary hepatocytes from wild-type mice were isolated and allowed to adhere to the culture medium. Adenovirus overexpressing HA-tagged GNAI1 was then added. The cells were then divided into three groups: empty adenovirus, Flag-tagged wild-type GPR146 (WT), and Flag-tagged mutant GPR146 (Mut). Twenty-four hours after adenoviral infection, each group was treated with 0 nM and 200 nM Cholesin for 10 minutes, respectively. Immediately afterward, cells were lysed using RIPA buffer. A 40 μl sample of the lysate was added to 5× SDS loading buffer and boiled at 100°C for 10 minutes to serve as the input sample. Flag-tagged beads were then added to the cell lysate, and the cells were incubated overnight at 4°C on a rotating mixer. After binding, the beads were centrifuged at 5000 rpm for 3 minutes at 4°C. The supernatant was discarded and 600 μl of pre-chilled RIPA lysis buffer was added and vortexed to remove residual liquid sample and proteins nonspecifically bound to the beads. The beads were centrifuged and the supernatant was discarded. Washing was repeated six times to thoroughly remove residual liquid. The samples were then boiled at 100°C for 10 minutes in 40 μl of 1× SDS loading buffer for SDS-PAGE and immunoblotting. The immunoprecipitation results are shown in Figure 18.

[0392] The results showed that Cholesin treatment disrupted the interaction between wild-type GPR146 and GNAI1, while mutant GPR146 had no such effect, indicating that Cholesin disrupts the interaction between GPR146 and GNAI1.

[0393] 6.2 Cholesin treatment reduces cAMP levels in wild-type primary hepatocytes

[0394] Isolation of wild type (Gpr146 + / + ) and Gpr146 knockout (Gpr146 - / - ) mouse primary hepatocytes and placed in 6-well plates. Set up 4 groups: Gpr146 + / + Cells, Gpr146 - / - Complemented empty vector (Vec) cells, Gpr146 - / - Complementation of wild-type GPR146 (WT) cells and Gpr146 - / - Complemented mutant GPR146 (Mut) cells. After primary hepatocytes adhered, adenoviruses expressing wild-type and mutant GPR146 complementation were added. Twenty-four hours after infection, cells in each group were treated with 0 nM or 200 nM Cholesin for 10 minutes. The cell culture medium was then immediately aspirated and cAMP levels in the cells were measured using the Cayman Cyclic AMP ELISA Kit. The results are shown in Figure 19.

[0395] The results showed that cAMP levels in primary hepatocytes of wild-type mice decreased after Cholesin treatment, while Gpr146 - / - Primary hepatocytes no longer respond to Cholesin treatment, and after wild-type GPR146 is restored, Gpr146 - / - While the response to Cholesin was restored, complementation of mutant GPR146 was still unresponsive to Cholesin's inhibitory effect on cAMP, indicating that Cholesin treatment reduces cAMP levels in primary hepatocytes.

[0396] The above results of Example 6 indicate that Cholesin inhibits the production of cAMP through the GPR146-coupled Gαi signaling pathway.

[0397] Example 7: Intraperitoneal injection of Cholesin reduces Hmgcr mRNA levels and plasma and liver cholesterol levels

[0398] 7.1 Dose-dependent inhibition of Hmgcr mRNA levels in mouse liver by intraperitoneal injection of Cholesin

[0399] In this example, 10-12 week-old wild-type male mice with a C57BL / 6J genetic background were randomly divided into four groups of 10 mice each. The mice were fasted overnight and injected with 0 mg / kg, 1 mg / kg, 5 mg / kg, and 10 mg / kg of eukaryotic purified Cholesin-his protein the following day. Liver tissue was collected 6 hours after the mice ate. Total RNA from mouse liver tissue was extracted and qPCR was performed to measure Hmgcr expression levels. Statistical analysis was performed using Rpl32 as an internal reference.

[0400] The results are shown in FIG20 . Injection of exogenous Cholesin can inhibit the expression of Hmgcr in the liver, and this inhibitory effect is dose-dependent.

[0401] 7.2 Effects of intraperitoneal injection of Cholesin on Hmgcr mRNA levels in mouse liver and TC levels in plasma and liver

[0402] Eight-week-old wild-type male mice with a genetic background of C57BL / 6J were selected and divided into two groups. They were injected with 0 mg / kg and 5 mg / kg of eukaryotic purified Cholesin-his protein, respectively. The injections were given twice a week for a total of two weeks. When the mice were 10 weeks old, the mice were starved and then fed for 6 hours, and then liver tissue and whole blood were collected. The operation process is shown in Figure 21A. For mouse liver, total RNA of mouse liver tissue was extracted for qPCR to detect the expression level of Hmgcr; at the same time, total cholesterol (TC) of the liver was also extracted to detect the level of TC in the extract. For mouse whole blood, plasma was separated by low-speed centrifugation, and the level of TC in plasma was detected. The Hmgcr mRNA level in mouse liver, the TC level in mouse plasma and liver are shown in Figures 21B, 21C and 21D, respectively.

[0403] The results showed that Cholesin injection could inhibit the expression of Hmgcr in mouse liver ( FIG. 21B ). After Cholesin injection, the plasma TC level ( FIG. 21C ) and liver TC level ( FIG. 21D ) of mice decreased.

[0404] Example 8: Cholesin inhibits cholesterol synthesis via GPR146

[0405] To investigate whether Cholesin inhibits cholesterol synthesis in mice through GPR146, 10-week-old wild-type (Gpr146) mice with a C57BL / 6J genetic background were selected. fl / flExperiments were conducted on Gpr146 knockout (LKO) mice and Gpr146 knockout (LKO) mice. At 10-12 weeks of age, Gpr146 LKO mice were divided into three groups of 12 mice each. One group received tail vein injections of adenovirus control (Vec), one group received tail vein injections of adenovirus expressing wild-type GPR146 (WT), and the other group received tail vein injections of adenovirus expressing mutant GPR146 (Mut). Each mouse received a total of 5×10^8 adenovirus via tail vein injection. Each group was then further divided into two subgroups of 6 mice each, each receiving two weekly injections of 0 mg / kg or 5 mg / kg of Cholesin for four consecutive injections. At 14.5 weeks of age, the mice were starved and then fed for 6 hours before being harvested for liver tissue and whole blood. The procedure is shown in Figure 22A.

[0406] For mouse liver, total liver RNA was extracted and then subjected to qPCR to detect the expression levels of cholesterol synthesis-related genes Hmgcs1, Hmgcr, Mvd, Mvk, and Pmvk. The results are shown in Figure 22B. At the same time, total liver protein was extracted and immunoblotted with antibodies to SREBP2, HMGCR, HMGCS1, LDLR, pKA Sub, and HSP90, with HSP90 as an internal reference. The results are shown in Figure 22C. Total cholesterol (TC) in the liver was also extracted to detect the level of TC in the extract. The results are shown in Figure 22D. For mouse whole blood, low-speed centrifugation was performed to separate plasma, and the level of TC in the plasma was detected. The results are shown in Figure 22E.

[0407] The results in Figures 22B and 22C show that cholesin inhibits the expression of PKA and SREBP2 in wild-type mice, thereby reducing the expression of genes involved in cholesterol synthesis and lowering liver and plasma TC levels. However, this effect of cholesin was abolished in GPR146 knockout mice. Addition of wild-type GPR146 restored the effect of cholesin, but addition of mutant GPR146 did not restore the effect of cholesin.

[0408] These results suggest that Cholesin regulates cholesterol homeostasis by inhibiting hepatic SREBP2 expression through GPR146.

[0409] Example 9: Cholesin can prevent hypercholesterolemia and atherosclerosis

[0410] Ldlr - / - Ldlr gene knockout mice are currently a commonly used disease model for studying atherosclerosis. - / - The plasma cholesterol of mice was significantly higher than that of wild-type mice, and atherosclerotic plaques were formed under the induction of Western diet (WD). - / -The mice were fed a Western diet for 4 weeks and then divided into four groups, with 6-7 mice in each group. The first group received Cholesin alone (5 mg kg -1 , twice a week); the second group received rosuvastatin (Ros) alone in drinking water (10 mg kg -1 sky -1 ); the third group was Cholesin (5 mg kg -1 , twice a week) and rosuvastatin (10 mg kg -1 sky -1 ) combined with (Cholesin+Ros) administration; the fourth group was the vehicle control group (Veh), and the specific operation is shown in Figure 23. During the period, the tail vein blood of the mice was collected every two weeks to detect the plasma total cholesterol (TC) and triglyceride (TG) levels, and the mouse body weight and food intake were detected. At the same time, the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the mouse plasma were also detected after 8 weeks of administration. After 8 weeks of continuous administration, the mice were killed, and the liver tissues were collected to detect the liver TC and TG levels, the expression levels of cholesterol synthesis-related genes in the liver, and the liver tissues were stained with HE to observe the lipid accumulation in the mouse liver. In addition, the whole aorta of the mice after 8 weeks of administration was stained with oil red to observe the changes in atherosclerotic plaques.

[0411] Plasma and liver TC and TG levels in mice

[0412] The changes in plasma TC levels in mice are shown in Figure 24A. Rosuvastatin or Cholesin alone significantly reduced Ldlr levels in mice. - / - TC levels in mouse plasma were observed. A slight increase in rosuvastatin plasma TC levels was observed after four weeks of rosuvastatin administration at the same dose, suggesting the potential for drug resistance in the later stages of rosuvastatin administration. The combination of rosuvastatin and cholesin demonstrated the greatest effect in reducing plasma TC, surpassing either rosuvastatin or cholesin alone. The combined treatment achieved a strong inhibitory effect early in the treatment phase and maintained this inhibitory effect for a prolonged period. The area under the curve (AUC) for changes in plasma TC over an eight-week period was calculated to provide a more intuitive comparison of the effects of the three treatment regimens. Both cholesin and rosuvastatin alone significantly reduced plasma TC levels, and the combined treatment exhibited a more pronounced inhibitory effect, achieving a synergistic effect.

[0413] Figure 24B shows TC levels in mouse livers after 8 weeks of administration. TC levels in the livers of mice treated with either rosuvastatin or cholesin were significantly reduced. Combined administration of rosuvastatin and cholesin was most effective in reducing liver TC, surpassing the effects of either rosuvastatin or cholesin alone, achieving a synergistic effect.

[0414] The TG levels in mouse plasma and liver are shown in Figures 24C and 24D. Both Cholesin and rosuvastatin administered alone reduced TG levels in plasma or liver, and their combined use had the most significant inhibitory effect on TG levels in plasma or liver, indicating a synergistic effect.

[0415] Expression levels of cholesterol synthesis-related genes in mouse liver

[0416] The reduction in total cholesterol may be due to the inhibition of cholesterol synthesis. Therefore, the expression levels of cholesterol synthesis-related genes Hmgcs1, Hmgcr, Mvd, Mvk, Pmvk, and Sqle in mouse livers, as well as the transcription factor SREBP2 that regulates the expression of these genes, were measured by qPCR and immunoblotting. The results are shown in Figures 25A and 25B.

[0417] The results showed that after 8 weeks of administration, the expression of cholesterol synthesis-related genes and transcription factor SREBP2 in the livers of mice treated with rosuvastatin were significantly upregulated, while the injection of Cholesin could significantly inhibit the expression of cholesterol synthesis-related genes and transcription factor SREBP2. When Cholesin and rosuvastatin were used in combination, the upregulation of cholesterol synthesis genes caused by statins was significantly alleviated, and the expression level of cholesterol synthesis-related genes was suppressed to a lower level.

[0418] Oil red staining of whole mouse aorta

[0419] To visualize the overall pathological changes in the mouse arteries, the entire aorta was stained with Oil Red O. Oil Red O is a strong lipid staining agent that specifically stains neutral triglycerides, lipids, and lipoproteins red. Because atherosclerotic plaques are rich in lipids, the plaques are partially stained red. The proportion of the stained plaque area to the entire aorta was calculated to assess the severity of the disease.

[0420] The specific experimental steps are as follows: 1) Anesthetize the mouse and fix it on a foam board in a supine position. Open the chest cavity to expose the heart. Draw 20ml of PBS into the syringe. Insert the syringe needle into the left ventricle from the apex of the heart. Make a small cut in the right atrium. Manually push PBS into the whole body for perfusion. The liver color changes from red to yellow after perfusion. Leave the needle in place and draw 10ml of PBS into the syringe. 1) Continue perfusion with 4% paraformaldehyde / PBS. After the fixative is injected, the mouse tail can be seen swinging slightly, indicating that the perfusion fixation effect is good; 2) Remove the mouse's internal organs, expose the spine, and carefully dissect the heart and the entire aorta under a stereo microscope. Cut the aorta below the bifurcation of the iliac artery, and cut the ascending aorta near the heart. After the entire aorta is dissected, the fatty tissue on the adventitia of the blood vessels must be carefully removed. Blunt dissection is mainly used to avoid damaging the blood vessels. The processed entire aorta is stored in 4% paraformaldehyde / PBS and awaits staining; 3) Remove the fully fixed aorta, and use Vannas scissors to extend into the blood vessels from the fracture of the ascending aorta under a stereo microscope. Cut the aortic arch longitudinally along the side of the greater curvature of the aortic arch to the distal end, leaving three branches intact. For processing, unfold the dissected aorta with the intima facing upward and fix it to a black-bottomed plastic plate using the smallest insect pins. 4) Prepare Oil Red O staining solution: Weigh 0.5 g of Oil Red O powder and add 100 ml of isopropyl alcohol. Incubate at 90°C in a water bath for 1 hour. Filter to obtain a saturated Oil Red O solution. For use, mix the saturated solution with double-distilled water in a ratio of 3:2, incubate at 42°C in a water bath for 10 minutes, and then filter again to obtain the Oil Red working solution. Both the saturated solution and the working solution must be stored at low temperature and protected from light. 5) For staining, pretreat the specimen in 60% isopropyl alcohol for 10 minutes, then stain in Oil Red working solution for 15 minutes. Decolorize in 60% isopropyl alcohol for 5 minutes to reduce background. The stained specimen can be stored in the fixative for a long time. 6) Subsequently, image the stained aorta using a Zeiss Stemi 508 stereomicroscope. Quantify the lesion area in the thoracic aorta using Image J software. Plaque area and total aortic intima area were measured using ImageJ. The relative percentage of plaque was calculated by dividing the plaque area by the total intima area × 100%. Dividing by the intima area was primarily to eliminate the effects of differences in mouse body size.

[0421] The experimental results are shown in FIG26 . Obvious plaques were observed in the vehicle control group. Rosuvastatin and Cholesin alone could effectively inhibit arterial lesions, while their combined use could achieve better therapeutic effects and a synergistic effect.

[0422] Mouse body weight and food intake

[0423] Changes in mouse body weight and food intake during drug administration are shown in Figures 27A and 27B, respectively. The results showed no significant differences in body weight between the rosuvastatin and control groups, while both Cholesin alone and combined with rosuvastatin treatment inhibited the extent of weight gain in mice (Figure 27A). Furthermore, food intake was unaffected during drug administration, with no significant differences between the four groups (Figure 27B), indicating that changes in mouse body weight were not due to differences in food intake.

[0424] HE staining of mouse liver tissue

[0425] The liver lipid accumulation was observed by HE staining of liver tissue. The specific operation was as follows: 1) a piece of mouse liver tissue was removed and fixed in 4% paraformaldehyde overnight; 2) alcohol was used as a dehydrating agent from low to high concentrations to gradually remove the water in the tissue block, and then the tissue block was placed in xylene to make it transparent, and the alcohol in the tissue block was replaced with xylene, and then immersed in wax and embedded; 3) the transparent tissue block was placed in the melted paraffin, and placed in a wax melting box for insulation, and embedded after the paraffin was completely immersed in the tissue block; 4) the embedded wax block was fixed on a microtome and cut into thin slices, generally 5 μm, and placed in a 45°C constant temperature box for drying after the slices were mounted; 5) the slices were placed in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 5 min, and anhydrous ethanol II in sequence. 5 min - 95% alcohol 5 min - 90% alcohol 5 min - 80% alcohol 5 min - 70% alcohol 5 min - distilled water wash; 6) Stain the sections with Harris hematoxylin for 3-8 min, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, blue with 0.6% ammonia solution, and rinse with running water; 7) Stain the sections with eosin solution for 1-3 min; 8) Dehydrate and transparentize the sections in 95% alcohol I for 5 min - 95% alcohol II for 5 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - xylene I for 5 min - xylene II for 5 min, remove the sections from the xylene, air dry slightly, and mount with neutral gum; 9) Examine under a microscope and collect and analyze images.

[0426] The HE staining results are shown in Figure 28. The white area in the cells is lipid accumulation. It can be observed that the control group has the most white lipid droplets, while the experimental groups have significantly weakened them, indicating that lipid accumulation in the liver of mice is significantly reduced after Cholesin administration.

[0427] ALT and AST levels in mouse plasma

[0428] To evaluate the liver damage in mice after administration, the AST and ALT levels in the plasma of the four groups of mice were detected using an alanine aminotransferase (alanine aminotransferase / ALT / GPT) kit (Nanjing Jiancheng) and an aspartate aminotransferase (aspartate aminotransferase / AST / GOT) kit (Nanjing Jiancheng). The results are shown in Figures 29A and 29B.

[0429] The results showed that AST and ALT levels were significantly reduced after Cholesin was administered alone or in combination with rosuvastatin, indicating that Cholesin has an alleviating effect on liver damage.

[0430] The above results of Example 9 indicate that Cholesin, whether used alone or in combination with rosuvastatin, has a significant protective effect against hypercholesterolemia and atherosclerosis.

[0431] This application makes reference to various issued patents, published patent applications, journal articles and other publications, all of which are incorporated herein by reference. If any incorporated reference conflicts with this specification, this specification shall prevail. In addition, any specific embodiment of the present invention that falls within the scope of the prior art may be explicitly excluded from any one or more claims. Because the embodiments are considered to be known to those skilled in the art, they may be excluded, even if the exclusion is not explicitly listed in this application. Any specific embodiment of the present invention may be excluded from any claim for any reason, regardless of whether it is related to the existence of the prior art.

[0432] Although the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition, method, or process step to the objective, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims

1. A method for reducing plasma cholesterol level and / or triglyceride level in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

2. The method of claim 1, wherein the plasma cholesterol is selected from the group consisting of plasma total cholesterol and plasma LDL-cholesterol.

3. A method for inhibiting hepatic cholesterol synthesis in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

4. The method according to claim 3, wherein the method inhibits the expression of a gene related to cholesterol synthesis or uptake, and the gene is selected from the group consisting of Hmgcr, Hmgcs1, Mvd, Mvk, Pmvk, Sqle and Ldlr. 5 . The method according to claim 3 or 4 , wherein the method inhibits the expression of a transcription factor that promotes the expression of a gene involved in cholesterol synthesis or uptake, and the transcription factor is SREBP2.

6. A method for inhibiting hepatic VLDL-cholesterol secretion in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

7. A method for treating or preventing a disease associated with elevated plasma cholesterol levels in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

8. The method according to any one of claims 1 to 7, wherein the Cholesin protein is human Cholesin protein.

9. The method according to any one of claims 1 to 8, wherein the nucleic acid is DNA or RNA.

10. The method of any one of claims 1-9, wherein the expression vector is selected from a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

11. The method according to any one of claims 7 to 10, wherein the disease associated with elevated plasma cholesterol levels is selected from hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, nephrotic syndrome and gallstones.

12. The method according to claim 11, wherein the disease associated with elevated plasma cholesterol levels is hypercholesterolemia.

13. The method of claim 11, wherein the disease associated with elevated plasma cholesterol levels is atherosclerosis.

14. The method according to any one of claims 1 to 13, wherein the method further comprises the step of administering a second therapeutic agent, preferably, the second therapeutic agent is selected from proteins or peptides, nucleic acids and small molecule drugs.

15. The method of claim 14, wherein the second therapeutic agent is a cholesterol-lowering drug selected from the group consisting of an HMGCR inhibitor, a PCSK9 inhibitor, and a cholesterol absorption inhibitor.

16. The method of claim 15, wherein the HMGCR inhibitor is a statin.

17. The method according to claim 16, wherein the statin is selected from the group consisting of rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin, preferably rosuvastatin.

18. The method of claim 15, wherein the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran and Tafolecimab.

19. The method according to claim 15, wherein the cholesterol absorption inhibitor is an NPC1L1 inhibitor, preferably ezetimibe or hebomibe.

20. The method of any one of claims 14-19, wherein the second therapeutic agent is administered before, after, or simultaneously with the Cholesin protein, the nucleic acid, or the expression vector.

21. The method of any one of claims 1-20, wherein the subject is a human.

22. The method of any one of claims 1-14 and 20-21, wherein the subject is unresponsive or intolerant to one or more of a cholesterol absorption inhibitor, an HMGCR inhibitor, and a PCSK9 inhibitor, e.g., the subject has an LDL receptor (LDLR) defect.

23. A composition comprising a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent for enhancing the expression or activity of human endogenous Cholesin protein, and a pharmaceutically acceptable carrier or excipient.

24. The composition according to claim 23, wherein the composition is used for one or more of the following: 1) reducing plasma cholesterol levels in a subject; 2) reducing plasma total cholesterol or plasma LDL-cholesterol levels in a subject; 3) reducing plasma triglyceride levels in a subject; 4) inhibiting hepatic cholesterol synthesis in a subject; 5) inhibiting hepatic VLDL-cholesterol secretion in a subject; and 6) Treating or preventing a disease associated with elevated plasma cholesterol levels in a subject.

25. The composition according to claim 23 or 24, wherein the Cholesin protein is human Cholesin protein.

26. The composition according to any one of claims 23-25, wherein the composition further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from proteins or peptides, nucleic acids and small molecule drugs.

27. The composition of claim 26, wherein the second therapeutic agent is selected from cholesterol-lowering drugs.

28. The composition of claim 27, wherein the cholesterol-lowering drug is selected from the group consisting of HMGCR inhibitors, PCSK9 inhibitors, and cholesterol absorption inhibitors.

29. The composition of claim 28, wherein the HMGCR inhibitor is a statin.

30. The composition according to claim 29, wherein the statin is selected from the group consisting of rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin, preferably rosuvastatin.

31. The composition of claim 28, wherein the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran and Tafolecimab.

32. The composition according to claim 28, wherein the cholesterol absorption inhibitor is an NPC1L1 inhibitor, preferably ezetimibe or hebomibe.

33. The composition according to any one of claims 23-32, wherein the composition is formulated as an injectable preparation or an oral preparation.

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