Use of targeting nr3c1 in the treatment of liver fibrosis

Abstract

This invention relates to the application of NR3C1 targeting in the treatment of liver fibrosis. Specifically, this invention provides the use of an NR3C1 agonist for preparing a composition or formulation for the prevention and / or treatment of liver fibrosis. The composition or formulation of this invention is effective in treating liver fibrosis caused by parasitic diseases.

Description

Technical Field

[0001] This invention relates to the field of medical technology, specifically to the application of targeting NR3C1 in the treatment of liver fibrosis. Background Technology

[0002] Chronic liver disease is a significant global public health issue, affecting 800 million people worldwide each year and causing 2 million deaths. Liver fibrosis is the common pathological basis for many chronic liver diseases, including viral hepatitis, schistosomiasis, alcohol abuse, non-alcoholic fatty liver disease, and cholestatic liver injury. Liver fibrosis can further progress to cirrhosis, leading to portal hypertension and related complications (upper gastrointestinal bleeding, ascites, hepatic coma, etc.). Patients with liver fibrosis also have a significantly increased risk of developing liver cancer.

[0003] Hepatic stellate cells (HSCs) are the main effector cells in various types of liver fibrosis, and 90% of the myofibroblasts in the liver are composed of activated HSCs. In a normal liver, HSCs are in a non-proliferative, quiescent state, playing roles such as storing vitamin A and regulating intrahepatic blood flow. When liver injury occurs, quiescent HSCs can be activated and transdifferentiated into myofibroblasts. These cells have enhanced proliferation, migration, and contractile abilities, and secrete large amounts of collagen that deposit in the liver tissue, leading to liver fibrosis.

[0004] Studies have shown that liver fibrosis is a reversible process, and activated HSCs can undergo senescence, apoptosis, or return to a quiescent state. In recent years, although several drugs for treating liver fibrosis have been developed based on its etiology, stage, and progression, such as PPARγ agonists, LPAR1 inhibitors, and NOX inhibitors that inhibit HSC activation, or drugs that block the function of effector cytokines in liver fibrosis, such as IL-4 / IL-13 antibodies or TGF-β blockers, a universally recognized and effective anti-liver fibrosis drug is still lacking.

[0005] Therefore, there is an urgent need in this field to explore new methods to inhibit hepatic stellate cell activation or to effectively treat liver fibrosis. Summary of the Invention

[0006] This invention provides a method for effectively inhibiting the activation of hepatic stellate cells and an effective method for treating liver fibrosis.

[0007] In a first aspect of the invention, there is provided the use of an active ingredient for preparing a formulation or composition for preventing and / or treating liver fibrosis caused by parasitic diseases; wherein the active ingredient is selected from the group consisting of:

[0008] (a) Glucocorticoid receptor (NR3C1) agonists;

[0009] (b) Per1 gene expression promoters;

[0010] (c) A combination of (a) and (b) above.

[0011] In another preferred embodiment, the Per1 gene expression promoter promotes or significantly upregulates Per1 gene expression.

[0012] In another preferred embodiment, the significant upregulation refers to the ratio of the expression level C1 of Per1 in the detected liver fibrotic cells or tissues to the expression level C0 of Per1 in the normal liver fibrotic cells or tissues (i.e., C1 / C0) ≥ 1.5, preferably ≥ 2, and more preferably ≥ 3.

[0013] In another preferred embodiment, the liver fibrotic cells or tissue are hepatic stellate cells (HSCs).

[0014] In another preferred embodiment, the prevention and / or treatment includes:

[0015] (i) Activation of glucocorticoid receptors;

[0016] (ii) Promotes significant upregulation of Per1 gene expression;

[0017] (iii) Promotes significant upregulation of Per2 gene expression; and

[0018] (iv) Promotes significant upregulation of Usep2 gene expression.

[0019] In another preferred embodiment, the liver fibrosis caused by the parasitic disease is selected from the group consisting of: liver fibrosis caused by schistosomiasis infection, liver fibrosis caused by liver fluke infection, liver fibrosis caused by echinococcosis infection, or a combination thereof.

[0020] In another preferred embodiment, the liver fibrosis is not compound-induced liver fibrosis.

[0021] In another preferred embodiment, the liver fibrosis is not caused by a viral infection.

[0022] In another preferred embodiment, the liver fibrosis caused by the parasitic disease is liver fibrosis caused by schistosomiasis infection.

[0023] In another preferred embodiment, the liver fibrosis is NR3C1-negative liver fibrosis.

[0024] In another preferred embodiment, the NR3C1 negative finger indicates that the expression and / or activity of NR3C1 is significantly reduced compared to normal control cells.

[0025] In another preferred embodiment, the significant decrease refers to the ratio of NR3C1 expression level E1 in liver fibrotic cells or tissues to NR3C1 expression level E0 in normal liver cells or tissues (i.e., E1 / E0) ≤ 1 / 2, preferably ≤ 1 / 3, more preferably ≤ 1 / 5; and / or the ratio of NR3C1 activity A1 in liver fibrotic cells or tissues to NR3C1 activity A0 in normal lung cells or tissues (i.e., A1 / A0) ≤ 1 / 2, preferably ≤ 1 / 3, more preferably ≤ 1 / 5.

[0026] In another preferred embodiment, the liver fibrosis is NR3C1-deficient liver fibrosis.

[0027] In another preferred embodiment, the composition or formulation is applied to mammals.

[0028] In another preferred embodiment, the mammal includes humans or non-human mammals.

[0029] In another preferred embodiment, the non-human mammals include rodents (such as rats and mice) and primates (such as monkeys).

[0030] In another preferred embodiment, the composition is a pharmaceutical composition.

[0031] In another preferred embodiment, the NR3C1 agonist is selected from the group consisting of small molecule compounds, miRNAs, antisense nucleic acids (such as antisense RNA), mRNAs, antibodies, gene editing reagents, or combinations thereof.

[0032] In another preferred embodiment, the composition or formulation is used to inhibit liver fibrosis.

[0033] In another preferred embodiment, the NR3C1 agonist is used to inhibit hepatic stellate cell (HSC) activation.

[0034] In another preferred embodiment, the NR3C1 agonist includes dexamethasone, prednisolone, budesonide, or a combination thereof.

[0035] In a second aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0036] (a1) NR3C1 agonist;

[0037] (a2) Optional other medications for the prevention and / or treatment of liver fibrosis; and

[0038] (b) Pharmaceutically acceptable carriers.

[0039] In another preferred embodiment, the component (a1) accounts for 0.1-99.9 wt% of the total weight of the pharmaceutical composition, more preferably 10-99.9 wt%, and even more preferably 70%-99.9 wt%.

[0040] In another preferred embodiment, the NR3C1 agonist is selected from the group consisting of small molecule compounds, miRNAs, antisense nucleic acids (such as antisense RNA), mRNAs, antibodies, gene editing reagents, or combinations thereof.

[0041] In another preferred embodiment, the NR3C1 agonist is selected from the group consisting of dexamethasone, prednisolone, budesonide, or combinations thereof.

[0042] In another preferred embodiment, the NR3C1 agonist is used to upregulate (or increase) the quantity and / or activity of PLK3.

[0043] In another preferred embodiment, the pharmaceutical composition is liquid, solid, or semi-solid.

[0044] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral dosage form, an injection, or a topical dosage form.

[0045] In another preferred embodiment, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral liquids, or injections.

[0046] In another preferred embodiment, the composition is a liquid composition.

[0047] In another preferred embodiment, the composition is an oral formulation.

[0048] In another preferred embodiment, the carrier is selected from the group consisting of infusion carriers and / or injection carriers. Preferably, the carrier is one or more carriers selected from the group consisting of physiological saline, glucose saline, or combinations thereof.

[0049] In another preferred embodiment, the composition or formulation may be used alone or in combination for the prevention and / or treatment of liver fibrosis.

[0050] In another preferred embodiment, the combined use includes: combined use with other drugs for the prevention and / or treatment of liver fibrosis.

[0051] In a third aspect of the invention, the use of the pharmaceutical composition described in the second aspect of the invention is provided for preparing a pharmaceutical product for the prevention and / or treatment of liver fibrosis.

[0052] In a fourth aspect of the invention, a method for inhibiting hepatic stellate cell activation in vitro is provided, comprising the step of co-culturing hepatic stellate cells with a medically effective amount of a formulation or composition containing an NR3C1 agonist.

[0053] In another preferred embodiment, the cells are derived from mammals.

[0054] In another preferred embodiment, the mammal includes (but is not limited to) mice and humans.

[0055] In another preferred embodiment, the cells are cells cultured in vitro.

[0056] In another preferred embodiment, the cells are liver fibrosis cells.

[0057] In another preferred embodiment, the method is an in vitro method.

[0058] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0059] In another preferred embodiment, the NR3C1 agonist is selected from the group consisting of dexamethasone, prednisolone, budesonide, or combinations thereof.

[0060] In another preferred embodiment, the cells are derived from mammals.

[0061] In a fifth aspect of the invention, a method for slowing down liver fibrosis is provided, comprising the step of contacting liver fibrotic cells with a medically effective amount of a formulation or composition containing an NR3C1 agonist.

[0062] In another preferred embodiment, the cells are derived from mammals.

[0063] In another preferred embodiment, the mammal includes (but is not limited to) mice and humans.

[0064] In another preferred embodiment, the cells are cells cultured in vitro.

[0065] In another preferred embodiment, the cells are liver fibrosis cells.

[0066] In another preferred embodiment, the method is an in vitro method.

[0067] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0068] In another preferred embodiment, the NR3C1 agonist is selected from the group consisting of dexamethasone, prednisolone, budesonide, or combinations thereof.

[0069] In another preferred embodiment, the cells are derived from mammals.

[0070] In a sixth aspect of the invention, a method for treating liver fibrosis is provided, comprising the step of administering a safe and effective amount of an NR3C1 agonist to a subject in need.

[0071] In another preferred embodiment, the liver fibrosis includes: liver fibrosis caused by schistosomiasis infection, liver fibrosis caused by liver fluke infection, liver fibrosis caused by echinococcosis infection, liver fibrosis caused by hepatitis B virus infection, alcohol-induced liver fibrosis, non-alcoholic fatty liver fibrosis, or a combination thereof.

[0072] In another preferred embodiment, the object is a human or a non-human mammal.

[0073] In another preferred embodiment, the subject is a patient with liver fibrosis.

[0074] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0075] Figure 1 This diagram illustrates the nuclear transport of glucocorticoid receptors in different hepatic stellate cells induced by dexamethasone in Example 1 of the present invention.

[0076] Figure 2 This diagram illustrates the inhibition of gene transcription levels in primary mouse hepatic stellate cells by dexamethasone in Example 2 of the present invention.

[0077] Figure 3 This diagram illustrates the inhibition of gene transcription levels in the mouse hepatic stellate cell line JS-1 by dexamethasone in Example 2 of the present invention.

[0078] Figure 4 This diagram illustrates the inhibition of gene transcription levels in the human hepatic stellate cell line LX-2 by dexamethasone in Example 2 of the present invention.

[0079] Figure 5 This diagram illustrates the inhibition of α-SMA protein expression in the mouse hepatic stellate cell line JS-1 by dexamethasone in Example 2 of the present invention.

[0080] Figure 6 This diagram illustrates the inhibition of hydroxyproline content in the liver of schistosomiasis-infected mice by dexamethasone in Example 3 of the present invention.

[0081] Figure 7 This diagram illustrates the degree and size of fibrosis of schistosomiasis egg granulomas in the liver of mice infected with Schistosomiasis in Example 3 of the present invention.

[0082] Figure 8 This diagram illustrates the inhibition of gene transcription levels related to stellate cell activation in the liver of schistosomiasis-infected mice by dexamethasone in Example 3 of the present invention.

[0083] Figure 9 This diagram illustrates the inhibition of profibrotic cytokine gene transcription levels in the livers of schistosomiasis-infected mice by dexamethasone in Example 3 of the present invention.

[0084] Figure 10 This diagram illustrates that dexamethasone did not affect ALT and AST levels in mouse serum in Example 3 of the present invention.

[0085] Figure 11 This is a schematic diagram illustrating the successful knockout of NR3C1 in the JS-1 cell line in Example 4 of this invention.

[0086] Figure 12 This is a schematic diagram showing that after knocking out NR3C1 in Example 4 of the present invention, dexamethasone cannot inhibit the expression of JS-1 activation-related genes.

[0087] Figure 13 This diagram illustrates how dexamethasone failed to inhibit the protein expression levels of JS-1 activation-related genes after NR3C1 knockout in Example 4 of the present invention.

[0088] Figure 14 This is a schematic diagram illustrating the reduced NR3C1 expression in the whole liver of C57 / BL6 mice in Example 5 of the present invention.

[0089] Figure 15 This is a schematic diagram illustrating how dexamethasone failed to inhibit liver fibrosis and hepatic stellate cell activation in mice after NR3C1 was knocked out in mouse hepatic stellate cells in Example 5 of this invention.

[0090] Figure 16 This is a schematic diagram of the CUT & Tag sequencing results in Example 6 of the present invention.

[0091] Figure 17 This is a schematic diagram of the transcriptional sequencing results in Example 6 of the present invention.

[0092] Figure 18 This is a schematic diagram of the clustering analysis results of NR3C1 target genes in Example 6 of the present invention.

[0093] Figure 19 This diagram illustrates the expression of rhythm genes in primary HSCs of mice induced by dexamethasone in Example 7 of the present invention.

[0094] Figure 20 This diagram illustrates the increased expression of activation-related genes in primary mouse HSCs after Per1 knockdown in Example 7 of the present invention.

[0095] Figure 21 This diagram illustrates how knocking down Per1 weakens the inhibitory effect of dexamethasone on HSC activation in Example 7 of the present invention. Detailed Implementation

[0096] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that glucocorticoid receptor agonists (e.g., dexamethasone) can activate the glucocorticoid receptor (NR3C1) and, by upregulating the expression of the downstream target gene Per1, inhibit the activation of hepatic stellate cells (HSCs), thereby alleviating / treating liver fibrosis. Specifically, in a mouse model of liver fibrosis, the inventors found that administration of NR3C1 agonists (e.g., dexamethasone) significantly reduced the degree of liver fibrosis in mice, and significantly decreased the mRNA levels of hepatic stellate cell activation-related genes Col1α1, α-SMA, and Timp1 in the mouse liver.

[0097] Furthermore, experiments of this invention have shown that knocking out NR3C1 eliminates the effects of NR3C1 agonists (e.g., dexamethasone) on hepatic stellate cell activation-related genes. Therefore, NR3C1 agonists inhibit liver fibrosis by targeting NR3C1. Moreover, knocking out the downstream rhythm gene Per1 of NR3C1 significantly weakens the inhibitory effect of dexamethasone on HSC activation. This invention is based on these findings.

[0098] liver fibrosis

[0099] Liver fibrosis is a chronic pathological process of injury repair, characterized by the excessive accumulation of extracellular matrix (ECM), which is mainly secreted by myofibroblasts. Hepatic stellate cells (HSCs) are the main effector cells in various types of liver fibrosis, and 90% of the myofibroblasts in the liver are composed of activated HSCs. HSCs are located between the sinusoidal endothelial cells and hepatocytes, accounting for 10% of the total number of intrinsic liver cells. In a normal liver, HSCs are in a non-proliferative, quiescent state, playing roles such as storing vitamin A and regulating intrahepatic blood flow. When liver injury occurs, quiescent HSCs can be activated and transdifferentiated into myofibroblasts, with enhanced cell proliferation, migration, and contraction capabilities, secreting large amounts of collagen that deposit in the liver tissue, leading to liver fibrosis.

[0100] Glucocorticoid receptor (NR3C1)

[0101] Glucocorticoid receptors (GR), also known as NR3C1, are genes whose expression levels are significantly downregulated in the progression of liver fibrosis caused by various diseases, but their relationship with liver fibrosis and hepatic stellate cell activation is currently unclear. Synthetic glucocorticoid drugs (such as dexamethasone) can bind to NR3C1, promoting its nuclear translocation and exerting a gene expression regulatory function. These drugs are widely used clinically, readily available, and inexpensive; however, no research has yet demonstrated that NR3C1 is a target for liver fibrosis treatment.

[0102] Active ingredients

[0103] As used herein, "active ingredient of the invention" refers to NR3C1 agonists and / or Per1 gene expression promoters, which can be used to increase or upregulate the activity or quantity of NR3C1 and / or promote the expression of rhythmic genes (such as Per1, Per2, and Usp2 genes). It should be understood that this term includes a single compound, a combination of two or more compounds, or an extract. Furthermore, this term also includes any agent that can upregulate the activity or quantity of NR3C1, including gene editing agents or RNA interference agents. For example, Cas9-based gene editing agents can be used to inactivate the NR3C1 gene.

[0104] Pharmaceutical Composition

[0105] The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and effective amounts of the following active ingredients: an agonist targeting NR3C1 and optionally a Per1 gene expression promoter.

[0106] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0107] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0108] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration; the dosage forms of the pharmaceutical compositions of the present invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. They are prepared, for example, using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

[0109] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Generally, satisfactory results are obtained when the active ingredient of this invention is administered daily at a dose of approximately 0.00001 mg to 50 mg / kg animal body weight (preferably 0.0001 mg to 10 mg / kg animal body weight). For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0110] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0111] The main advantages of this invention include:

[0112] (a) Activation of NR3C1 can significantly inhibit hepatic stellate cell activation and reduce the degree of liver fibrosis.

[0113] (b) Synthetic glucocorticoids (such as dexamethasone) are NR3C1 agonists, which are widely used, readily available and inexpensive in clinical practice.

[0114] (c) The liver fibrosis treatment method of the present invention brings hope for the treatment of liver fibrosis and breaks through the bottleneck of liver fibrosis treatment, and is expected to generate great social and economic benefits.

[0115] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0116] Example 1

[0117] Dexamethasone can induce nuclear transport of glucocorticoid receptors in hepatic stellate cells.

[0118] Experimental methods:

[0119] Primary mouse hepatic stellate cells, mouse hepatic stellate cell line JS-1, and human hepatic stellate cell line LX-2 were cultured in DMEM medium containing 10% fetal bovine serum, penicillin, and streptomycin at 37°C and 5% CO2. Cells were cultured at a density of 3 x 10⁻⁶ cells per well. 5 Cells were seeded onto cell slides in 12-well cell culture plates. The experimental group was treated with dexamethasone (800 ng / mL), while the control group was treated with an equal volume of solvent. After 4 hours, the cell slides were fixed with 10% paraformaldehyde. The primary antibody was rabbit anti-mouse glucocorticoid receptor IgG, and the secondary antibody was goat anti-rabbit IgG. The cell nuclei were stained with DAPI, and immunofluorescence was performed.

[0120] The results are as follows Figure 1 As shown, after dexamethasone stimulation, glucocorticoid receptors in all three types of hepatic stellate cells were transported from the cytoplasm to the nucleus.

[0121] Example 2

[0122] Dexamethasone inhibits the transcriptional levels of genes involved in activation in hepatic stellate cells.

[0123] Experimental methods:

[0124] Primary mouse hepatic stellate cells, mouse hepatic stellate cell line JS-1, and human hepatic stellate cell line LX-2 were cultured in DMEM medium containing 10% fetal bovine serum, penicillin, and streptomycin at 37°C and 5% CO2.

[0125] According to 3*10 per hole 5Cells were seeded in 12-well cell culture plates. The experimental group received dexamethasone (800 ng / mL), while the control group received an equal volume of solvent. After 24 hours, cells were collected, and total RNA was extracted using TRizol. The total RNA was reverse transcribed into cDNA according to the Novizan reverse transcription kit instructions. The amount of cDNA was calculated from 1 μg of reverse transcribed RNA. The obtained cDNA was mixed with sterile water, Roche FastStart Universal Probe Master (Rox) (0.2 μL), and Col1α1, α-SMA, and Timp1 specific primers to prepare a 10 μL reaction mixture.

[0126] The above reaction system was tested using an ABIQ6 PCR instrument. The reaction conditions were: 95℃ for 10 min; 95℃ for 15 s, 56℃ for 15 s, 30 cycles; 60℃ for 30 s.

[0127] According to 6*10 per hole 5 JS-1 cells were seeded in 6-well cell culture plates. The experimental group was treated with dexamethasone (800 ng / mL), while the control group was treated with an equal volume of solvent. After 48 h, the cells were collected and total protein was extracted using RIPA lysis buffer. Western blotting was used to detect changes in α-SMA protein levels.

[0128] Experimental results:

[0129] The test showed that dexamethasone significantly inhibited (NR3C1 inhibits by regulating downstream genes) mouse primary hepatic stellate cells. Figure 2 ), mouse hepatic stellate cells JS-1 ( Figure 3 ) and human hepatic stellate cells LX-2 ( Figure 4 It activates the expression levels of related genes such as type I collagen (Col1α1), α-smooth muscle actin (α-SMA), and matrix metalloproteinase inhibitor 1 (Timp1) mRNA. Simultaneously, it can also inhibit the expression levels of these gene proteins. Figure 5 ).

[0130] Example 3

[0131] Dexamethasone inhibits liver fibrosis in schistosomiasis-infected mice

[0132] Twenty C57 / BL6 mice were randomly divided into four groups:

[0133] (1) PBS group (n=4);

[0134] (2) DEX group (n=4);

[0135] (3) Schistosomiasis infection + PBS group (n=6);

[0136] (4) Schistosomiasis infection + DEX group (n=6).

[0137] Mice were fixed in place, their abdominal hair was shaved off, and 16 cercariae of Schistosoma japonicum were infected through the abdominal skin to establish a mouse model of fibrosis caused by schistosomiasis japonicus.

[0138] Starting from week 3 post-infection, mice were administered dexamethasone (1 mg / kg) or an equal volume of PBS via intraperitoneal injection three times a week for three consecutive weeks. At week 6 post-infection, mice were anesthetized and dissected. Liver tissue was collected from each group to assess the effect of dexamethasone on the degree of liver fibrosis. Serum was collected from each group to assess alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

[0139] First, the hydroxyproline content in liver tissue was determined using the alkaline hydrolysis method described in the instructions for the Nanjing Jiancheng hydroxyproline reagent kit. The results are as follows: Figure 6 As shown. Hydroxyproline is a raw material for synthesizing collagen fibers, and its content can reflect the degree of fibrosis. From... Figure 6 It is known that dexamethasone can significantly reduce the hydroxyproline content in the liver of mice infected with Schistosoma.

[0140] In addition, immunohistochemical techniques, including hematoxylin-eosin (H&E) staining and Masson staining, were used to assess the size and degree of fibrosis of hepatic parasite egg granulomas.

[0141] The method for measuring parasite egg granulomas is as follows: Under a microscope, measure the maximum and minimum diameters of all parasite eggs in a single H&E section, and count the number of parasite egg granulomas. The area of ​​a single parasite egg granuloma = maximum diameter of the egg × minimum diameter. The total area of ​​liver parasite egg granulomas is the sum of the areas of each parasite egg granuloma. The average area of ​​a single sample of parasite egg granulomas = total area of ​​liver parasite egg granulomas / number of granulomas.

[0142] The method for assessing the degree of fibrosis is as follows: collagen fibers in Masson sections show blue staining, and the intensity of the color and the size of the area can reflect the degree of liver fibrosis. Microscopic observation was performed, and the color and area of ​​collagen fibers in each sample were scored. Both color and area were divided into four levels, ranging from 1 to 4 points. The final fibrosis degree for each sample was calculated as: color score × area score.

[0143] The results are as follows Figure 7 As shown, the H&E results indicated that, compared with the control group, the size of parasite egg granulomas in the mouse liver was significantly reduced after injection of dexamethasone; the Masson results indicated that, compared with the control group, the degree of liver fibrosis in mice was significantly reduced after injection of dexamethasone.

[0144] Finally, qPCR was used to detect the mRNA levels of stellate cell activation-related genes (including type I collagen (Col1α1), α-smooth muscle actin (α-SMA) and matrix metalloproteinase inhibitor 1 (Timp1)) and profibrotic cytokines (IL-4, IL-13, TGF-β1) in mouse liver.

[0145] The results are as follows Figure 8 As shown, compared with the control group, after injection of dexamethasone, the mRNA levels of hepatic stellate cell activation-related genes Col1α1, α-SMA, and Timp1 in the livers of schistosomiasis-infected mice were significantly reduced. Figure 9 As shown, the mRNA levels of profibrotic cytokines IL-4, IL-13, and TGF-β1 were also significantly reduced.

[0146] Therefore, NR3C1 can serve as a target for synthetic glucocorticoids (such as dexamethasone) in the treatment of liver fibrosis and can be used in the treatment of liver fibrosis.

[0147] In addition, serum ALT and AST levels were measured according to the methods described in the instructions for the Nanjing Jiancheng Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) test kits. Results are as follows: Figure 10 As shown, dexamethasone did not affect the levels of ALT and AST in mouse serum.

[0148] Example 4

[0149] Dexamethasone inhibits hepatic stellate cell activation via NR3C1.

[0150] JS-1 cells stably expressing Cas9 in vitro were cultured in DMEM medium containing 10% fetal bovine serum, penicillin, and streptomycin at 37°C and 5% CO2. Cells were cultured at a density of 6 x 10⁶ cells per well. 5 Cells were seeded in 6-well cell culture plates, and 2 μg of NR3C1-targeting sgRNA overexpression plasmid and 2 μL of transfection reagent jetOPTIMUS were added. After 48 h, single clones were picked and cultured for further to obtain NR3C1 knockout JS-1 cells.

[0151] Western blot analysis was performed on proteins extracted from NR3C1 knockout JS-1 cells, and the results are as follows: Figure 11 The results showed that, compared with wild-type JS-1 cells, NR3C1 knockout JS-1 cells did not express NR3C1.

[0152] Wild-type JS-1 cells and NR3C1 knockout JS-1 cells were mixed at a ratio of 3*10 cells per well. 5Cells were seeded in 12-well cell culture plates. The experimental group received dexamethasone (800 ng / mL), while the control group received an equal volume of solvent. After 24 hours, cells were collected, and total RNA was extracted using TRizol. qPCR was used to detect whether the inhibitory effect of dexamethasone on JS-1 activation disappeared after NR3C1 knockout.

[0153] The results are as follows Figure 12 As shown, after knocking out NR3C1, dexamethasone was no longer able to suppress the expression level of mRNA in JS-1 activation-related genes.

[0154] Wild-type JS-1 cells and NR3C1 knockout JS-1 cells were mixed at a ratio of 6*10 cells per well. 5 Cells were seeded in 6-well cell culture plates. The experimental group was treated with dexamethasone (800 ng / mL), while the control group was treated with an equal volume of solvent. After 48 h, cells were collected and total protein was extracted using RIPA lysis buffer. Western blotting was used to determine whether the inhibitory effect of dexamethasone on JS-1 activation disappeared after NR3C1 knockout.

[0155] The results are as follows Figure 13 As shown, after knocking out NR3C1, dexamethasone was no longer able to suppress the expression of protein levels in JS-1 activation-related genes.

[0156] The above results indicate that dexamethasone inhibits the activation of JS-1 cells by targeting NR3C1.

[0157] Example 5

[0158] In vivo, dexamethasone inhibits hepatic stellate cell activation via NR3C1.

[0159] In C57 / BL6 mice, the Cre expression cassette was knocked into the stop codon site of the Lrat gene, enabling LRAT-expressing cells in these mice to specifically express the Cre enzyme. Lrat is specifically expressed in hepatic stellate cells in the liver. The Cre enzyme recognizes a specific DNA sequence, the LoxP site, and deletes the sequence between two LoxP sites. These mice were then crossed with mice that had LoxP sites inserted at both ends of the third exon of the NR3C1 gene to obtain mice with specific NR3C1 knockout in hepatic stellate cells.

[0160] Western blot analysis was performed on proteins extracted from the liver of this mouse, and the results are as follows: Figure 14 As shown, the expression level of NR3C1 protein in the liver of this mouse was significantly reduced.

[0161] Eight C57 / BL6 mice were randomly divided into two groups, and twelve NR3C1-specific knockout mice were randomly divided into two groups:

[0162] (1) Schistosomiasis infection + PBS group (n=4);

[0163] (2) Schistosomiasis infection + DEX group (n=4);

[0164] (3) NR3C1 knockout + schistosomiasis infection + PBS group (n=6);

[0165] (4) NR3C1 knockout + schistosomiasis infection + DEX group (n=6).

[0166] Mice were immobilized, their abdominal hair was shaved, and 16 cercariae of Schistosoma japonicum were infected via the abdominal skin to establish a mouse model of schistosomiasis fibrosis. Starting from week 3 post-infection, mice were intraperitoneally injected with dexamethasone (1 mg / kg) or an equal volume of PBS three times a week for three consecutive weeks. At week 6 post-infection, mice were anesthetized and dissected, and liver tissue was collected from each group of mice to detect relevant indicators.

[0167] The results are as follows Figure 15 As shown in the figure, the hydroxyproline detection results indicated that after knocking out NR3C1, the effect of dexamethasone in inhibiting liver fibrosis in mice disappeared.

[0168] H&E results showed that knocking out NR3C1 did not reduce the size of parasite egg granulomas in the mouse liver.

[0169] Masson results showed that knocking out NR3C1 did not reduce the degree of liver fibrosis in mice.

[0170] qPCR results showed that after knocking out NR3C1, dexamethasone did not significantly reduce the mRNA levels of Col1α1, α-SMA, Col1α3, and the profibrotic cytokine TGF-β1 in the liver of schistosomiasis-infected mice.

[0171] Example 6

[0172] Dexamethasone induces NR3C1 nuclear transport regulation of downstream target gene expression

[0173] Experiment 1:

[0174] JS-1 cells were cultured in DMEM medium containing 10% fetal bovine serum, penicillin, and streptomycin at 37°C and 5% CO2. Cells were cultured at a rate of 3 x 10⁻⁶ cells per well. 5Cells were seeded in 12-well cell culture plates. The experimental group received dexamethasone (800 ng / mL), while the control group received an equal volume of solvent. After 4 hours, CUT&Tag experiments were performed according to the Novizan TD903 CUT&Tag kit instructions. The target DNA sequence was recognized using NR3C1 rabbit monoclonal antibody and secondary antibody, and the target DNA sequence was captured and fragmented using pA-Tn5. Subsequently, the purified product was amplified by PCR to construct a CUT&Tag library, which was then sequenced using Illumina NovaSeq.

[0175] Sequencing data were aligned using BWA alignment software, and peak calling analysis was performed using MAC2. The locations of signal peaks were annotated using ChIPseeker. The selection criteria for differentially expressed signal peaks between groups were a fold change greater than 2 and a p-value less than 0.05.

[0176] The results are as follows Figure 16 As shown, after dexamethasone stimulation, the binding of NR3C1 to gene transcription start sites increased. Compared with the control group, after dexamethasone stimulation, 8705 signal peaks were enhanced and 137 signal peaks were weakened.

[0177] Experiment 2:

[0178] JS-1 cells were cultured in DMEM medium containing 10% fetal bovine serum, penicillin, and streptomycin at 37°C and 5% CO2. Cells were cultured at a rate of 3 x 10⁻⁶ cells per well. 5 Cells were seeded in 12-well cell culture plates. The experimental group was treated with dexamethasone (800 ng / mL), while the control group was treated with an equal volume of solvent. After 24 h, RNA was extracted from the cells using Trizol, and the RNA was constructed using the NEBNextUltra™ RNA Library Prep Kit. Sequencing was performed using Novaseq 6000.

[0179] The sequencing results were compared and analyzed using STAR software. The screening criteria for differentially expressed genes between groups were a fold change greater than 1.5 and a p-value less than 0.05.

[0180] The results are as follows Figure 17 As shown, after dexamethasone stimulation, the expression of 178 genes was upregulated and the expression of 263 genes was downregulated.

[0181] The BETA software was used to perform a combined analysis of the differential signal peaks between groups in the CUT&Tag sequencing results and the differentially expressed genes in the RNA sequencing results.

[0182] The results are as follows Figure 18As shown, NR3C1 is predicted to promote the expression of downstream genes, with a total of 117 potential target genes identified. Cluster analysis of these 117 potential target genes using Metascape revealed that NR3C1 mainly regulates the activation of hepatic stellate cells by modulating signaling pathways such as circadian rhythm genes and lipid metabolism.

[0183] Example 7

[0184] Dexamethasone induces NR3C1 upregulation of the target gene Per1, inhibiting hepatic stellate cell activation.

[0185] Experiment 1:

[0186] Primary mouse HSCs were cultured in DMEM medium containing 10% fetal bovine serum, penicillin, and streptomycin at 37°C and 5% CO2. The culture was performed at a rate of 3 x 10⁻⁶ cells per well. 5 Cells were seeded in 12-well cell culture plates. The experimental group was treated with dexamethasone (800 ng / mL), while the control group was treated with an equal volume of solvent. After 24 h, the mRNA levels of the circadian rhythm genes Per1, Per2, and Usp2 were detected by qPCR.

[0187] The results are as follows Figure 19 As shown, dexamethasone induction increased the expression of circadian rhythm genes Per1, Per2, and Usp2 in HSCs. This indicates that dexamethasone can induce the expression of these genes in HSCs.

[0188] Experiment 2:

[0189] Primary mouse HSCs were cultured in DMEM medium containing 10% fetal bovine serum, penicillin, and streptomycin at 37°C and 5% CO2. The culture was performed at a rate of 3 x 10⁻⁶ cells per well. 5 Cells were seeded in 12-well cell culture plates. The experimental group was given siRNA targeting the rhythm genes Per1, Per2 and Usp2 and transfection reagent Lipofectamine 3000. The control group was given control siRNA and transfection reagent Lipofectamine 3000. After 48 hours of transfection, the mRNA level of activation-related genes in hepatic stellate cells was detected by qPCR.

[0190] result Figure 20 As shown, knocking down Per1 with siRNA increased the expression of activation-related genes in primary mouse HSCs, indicating that Per1 can inhibit HSC activation.

[0191] Experiment 3:

[0192] Primary mouse HSCs were cultured in DMEM medium containing 10% fetal bovine serum, penicillin, and streptomycin at 37°C and 5% CO2. The culture was performed at a rate of 3 x 10⁻⁶ cells per well. 5 Cells were seeded in 12-well cell culture plates. The experimental group was treated with siRNA targeting the rhythm genes Per1, Per2 and Usp2 and transfection reagent Lipofectamine 3000. The control group was treated with control siRNA and transfection reagent Lipofectamine 3000. 24 h after transfection, dexamethasone (800 ng / mL) was added to both the experimental and control groups. 48 h after transfection, qPCR was used to detect the mRNA levels of activation-related genes in hepatic stellate cells.

[0193] The results are as follows Figure 21 As shown, knocking down Per1 significantly weakens the inhibitory effect of dexamethasone on HSC activation, indicating that after dexamethasone activates NR3C1, it exerts its inhibitory effect on HSC activation by upregulating the expression of Per1, a downstream target gene of NR3C1.

[0194] Therefore, both NR3C1 agonists and Per1 gene expression promoters can inhibit HSC activation, thereby suppressing liver fibrosis.

[0195] discuss

[0196] Liver fibrosis is the common pathological basis of many chronic liver diseases, and currently there are no universally recognized effective drugs for its treatment. NR3C1 expression is significantly downregulated in the progression of liver fibrosis caused by various diseases, but its relationship with liver fibrosis and hepatic stellate cell activation remains unclear. This invention provides the use of NR3C1 as a drug target for the treatment of liver fibrosis.

[0197] The experiments of this invention show that using the NR3C1 agonist dexamethasone to activate NR3C1 can inhibit HSC activation both in vivo and in vitro, and can also inhibit the process of liver fibrosis in vivo.

[0198] In vitro knockout of NR3C1 eliminated the inhibitory effect of dexamethasone on HSC activation; in vivo specific knockout of NR3C1 in HSCs eliminated the inhibitory effect of dexamethasone on liver fibrosis. These results indicate that dexamethasone targets NR3C1, thereby inhibiting HSC activation and the progression of liver fibrosis.

[0199] Immunofluorescence results showed that dexamethasone activated NR3C1, causing NR3C1 to be transported into the nucleus.

[0200] Combined CUT & Tag sequencing and transcriptome analysis showed that NR3C1 mainly functions to upregulate the expression of downstream genes in the nucleus. Cluster analysis of its target genes showed that NR3C1 can regulate the activation of HSCs by regulating the expression of genes in pathways such as circadian rhythm genes and lipid metabolism.

[0201] Further verification revealed that dexamethasone activation of NR3C1 significantly upregulated the expression of the downstream rhythmic gene Per1; knocking down Per1 with siRNA significantly weakened the inhibitory effect of dexamethasone on HSC activation. This indicates that dexamethasone activation of NR3C1 inhibits HSC activation by upregulating the expression of its downstream rhythmic gene Per1.

[0202] Therefore, NR3C1 and / or Per1 can be used as targets for the treatment of liver fibrosis, and can be used to prepare anti-liver fibrosis drugs, especially targeted drugs that activate NR3C1 and / or promote Per1 gene expression.

[0203] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of an active ingredient, characterized in that, This preparation is used to develop a formulation or composition for the prevention and / or treatment of liver fibrosis caused by parasitic diseases; wherein the active ingredient is selected from the group consisting of: (a) Glucocorticoid receptor (NR3C1) agonists; (b) Per1 gene expression promoters; (c) A combination of (a) and (b) above.

2. The use as described in claim 1, characterized in that the liver fibrosis caused by the parasitic disease is selected from the group consisting of: liver fibrosis caused by schistosomiasis infection, liver fibrosis caused by liver fluke infection, liver fibrosis caused by echinococcosis infection, or a combination thereof.

3. The use as described in claim 1, characterized in that, The liver fibrosis mentioned is NR3C1 negative liver fibrosis, and NR3C1 negative means that the expression and / or activity of NR3C1 is significantly reduced compared with normal control cells.

4. The use as described in claim 3, characterized in that, The significant decrease refers to the ratio of NR3C1 expression level E1 in liver fibrotic cells or tissues to NR3C1 expression level E0 in normal liver cells or tissues (i.e., E1 / E0) ≤ 1 / 2, preferably ≤ 1 / 3, more preferably ≤ 1 / 5; and / or the ratio of NR3C1 activity A1 in liver fibrotic cells or tissues to NR3C1 activity A0 in normal lung cells or tissues (i.e., A1 / A0) ≤ 1 / 2, preferably ≤ 1 / 3, more preferably ≤ 1 / 5.

5. The use as described in claim 1, characterized in that, The NR3C1 agonist is selected from the group consisting of dexamethasone, prednisolone, budesonide, or combinations thereof.

6. The use as described in claim 1, characterized in that, The active ingredient is used to inhibit the activation of hepatic stellate cells (HSCs).

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a1) NR3C1 agonist; (a2) Optional Per1 gene expression promoter; (a3) Optional other medications for the prevention and / or treatment of liver fibrosis caused by parasitic diseases; and (b) Pharmaceutically acceptable carriers.

8. Use of the pharmaceutical composition according to claim 7, characterized in that, This product is used to prepare a pharmaceutical product for the prevention and / or treatment of liver fibrosis caused by parasitic diseases.

9. A method for inhibiting hepatic stellate cell activation in vitro, characterized in that, The procedure includes: co-culturing hepatic stellate cells with a medically effective amount of a formulation or composition containing an NR3C1 agonist and / or a Per1 gene expression promoter.

10. The method as described in claim 9, characterized in that, The cells are derived from mammals.

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