New target for treating metabolism-associated fatty liver disease, regulatory molecule thereof, and use thereof

Abstract

The present invention provides a new target for treating metabolism-associated fatty liver disease, a regulatory molecule thereof, and use thereof. For the first time, the present invention discloses a novel target gene closely associated with the alleviation or treatment of metabolism-associated fatty liver disease and a signaling pathway associated with upstream and downstream regulation thereof. The present invention also discloses a new mechanism in which the signaling pathway is involved in regulating metabolism-associated fatty liver disease. The present invention further discloses a preferred medicament for the alleviation or treatment.

Description

New targets, regulatory molecules, and applications for the treatment of metabolic-related fatty liver disease

[0001] This application claims priority to patent application No. CN 202311790463.8, filed on December 22, 2023; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the field of biomedicine, and more specifically, relates to a new target for treating metabolism-related fatty liver disease, its regulatory molecules and applications. Background Art

[0003] Liver diseases can lead to alcohol-associated liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD), which can further induce liver tumors.

[0004] Fatty liver refers to a condition characterized by excessive fat accumulation within liver cells due to various factors. Fatty liver is often caused by impaired lipid metabolism in liver cells. Patients with this condition experience symptoms such as right upper abdominal distension, loss of appetite, general weakness, lethargy, nausea, and even jaundice in severe cases.

[0005] Nonalcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis, is the most common cause of chronic liver disease. It can lead to numerous chronic liver diseases, such as fibrosis, cirrhosis, and liver failure, and is also a risk factor for primary liver cancer. Liver tumors caused by nonalcoholic steatohepatitis (NASH), a type of NAFLD, are on the rise.

[0006] Non-alcoholic fatty liver disease is qualitatively different from simple fat accumulation in adipose tissue or fat accumulation in other organs / tissues. The liver is an important site for lipid synthesis and metabolism in the body, while adipose tissue is more inclined to store excess lipids. The liver can synthesize and decompose lipids from scratch in response to systemic lipid balance. The liver synthesizes excess free fatty acids into triglycerides, which are then transferred to other tissues such as adipose tissue for storage. Disturbances in the liver's lipid metabolism function are an important predisposing factor for the occurrence of metabolic diseases in the body (obesity, diabetes, hyperlipidemia, etc.).

[0007] Over the past few decades, changes in lifestyle and dietary habits have contributed to the prevalence of obesity and NAFLD. Currently, the number of NAFLD patients is increasing at an alarming rate. As liver diseases like NAFLD become a major health concern, further research and analysis are urgently needed to explore effective drug interventions. Uncovering the mechanisms underlying the development and progression of liver diseases like NAFLD and identifying potential therapeutic targets is a top priority. Summary of the Invention

[0008] The purpose of the present invention is to provide a new target for treating metabolic-associated fatty liver disease (MAFLD), its regulatory molecules and applications.

[0009] In a first aspect of the present invention, there is provided a use of TKT or a downregulator of the (insulin-)InsR-C / EBPα-TKT signaling pathway containing TKT for preparing a pharmaceutical composition for alleviating or treating metabolic-related fatty liver disease.

[0010] In another preferred embodiment, the InsR-C / EBPα-TKT signaling pathway includes: TKT gene / protein, C / EBPα gene / protein, InsR gene / protein; preferably also includes their upstream and downstream regulatory genes / proteins or chemical molecules.

[0011] In another preferred embodiment, the downregulator is selected from: TKT downregulator, C / EBPα-TKT interaction (mutual regulatory effect; preferably including C / EBP transcriptional regulation of TKT) downregulator, InsR-C / EBPα interaction (mutual regulatory effect) downregulator.

[0012] In another preferred embodiment, the TKT down-regulator includes (but is not limited to): an agent that silences, knocks down or knocks out the TKT gene, and an agent that inhibits the activity of the TKT protein; more preferably, it includes: an interfering molecule that specifically interferes with the expression of the TKT gene (such as siRNA, shRNA, miRNA, antisense nucleotides, etc.), a CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent targeting the TKT gene, and the reagent causes TKT to undergo a loss-of-function mutation.

[0013] In another preferred embodiment, the downregulators of the C / EBPα-TKT interaction include (but are not limited to): reagents that weaken the binding of C / EBPα to the TKT promoter, reagents that silence, knock down or knock out the C / EBPα gene, and reagents that inhibit the activity of the C / EBPα protein; more preferably, they include: reagents that reduce the phosphorylation level of the C / EBPα protein, interfering molecules that specifically interfere with the expression of the C / EBPα gene (such as siRNA, shRNA, miRNA, antisense nucleotides, etc.), CRISPR gene editing reagents, homologous recombination reagents or site-directed mutagenesis reagents for the C / EBPα gene, which cause C / EBPα to undergo a loss-of-function mutation.

[0014] In another preferred embodiment, the downregulators of the InsR-C / EBPα interaction include (but are not limited to): reagents that silence, knock down or knock out the InsR gene, reagents that inhibit the activity of the InsR protein, and reagents that weaken the binding of insulin to InsR; more preferably, they include: interfering molecules that specifically interfere with the expression of the InsR gene (such as siRNA, shRNA, miRNA, antisense nucleotides, etc.), CRISPR gene editing reagents, homologous recombination reagents or site-directed mutagenesis reagents targeting the InsR gene, which cause a loss-of-function mutation in InsR.

[0015] In another preferred embodiment, the mutual regulatory effect includes regulation at the transcriptional level.

[0016] In another preferred embodiment, the down-regulator is an interfering agent that silences the TKT gene; preferably, the interfering agent is siRNA; preferably, the siRNA is a siRNA having a nucleotide sequence (sense chain, the 3′ end of the corresponding antisense chain has a protected base) as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 50, SEQ ID NO: 53 to SEQ ID NO: 167; more preferably, the siRNA is a siRNA having a nucleotide sequence as shown in any one of SEQ ID NO: 37 (No. 1115), SEQ ID NO: 25 (No. 773), SEQ ID NO: 50 (siTKT16), SEQ ID NO: 49, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 88 or SEQ ID NO: 130.

[0017] In another preferred embodiment, the down-regulatory agent is a homologous recombination agent that knocks down or knocks out the TKT gene, which causes the gene encoding to be terminated.

[0018] In another preferred embodiment, the down-regulator is a homologous recombination agent that knocks down or knocks out the InsR gene, which causes the gene encoding to be terminated.

[0019] In another preferred embodiment, the siRNA forms an siRNA preparation for targeted delivery to hepatocytes; preferably, the siRNA is linked (covalently bound, forming a conjugate) to a targeting ligand that binds to the asialoglycoprotein receptor (ASGPR).

[0020] In another preferred embodiment, the targeting ligand includes: N-acetylgalactosamine (GalNAc), N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine.

[0021] In another preferred embodiment, the metabolism-related fatty liver disease includes: non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

[0022] In another preferred embodiment, the TKT or TKT-containing (insulin-)InsR-C / EBPα-TKT signaling pathway downregulator increases the level of inosine (inosine), enhances mitochondrial function, thereby improving liver lipid metabolism, alleviating or treating metabolism-related fatty liver disease; preferably, the downregulator: prevents R5P derived from inosine from entering glycolysis, promotes the synthesis of inosine from glucose-derived R5P, increases the intracellular level of inosine, thereby promoting the inosine-PKA-CREB pathway, enhances mitochondrial function, improves liver lipid metabolism, alleviating or treating metabolism-related fatty liver disease; preferably, after promoting the inosine-PKA-CREB pathway, it also includes: activating the PKA-CREB-ChoKβ axis, increasing phosphatidylcholine (PC) synthesis, thereby enhancing mitochondrial function; preferably, the increase in phosphatidylcholine (PC) synthesis is mediated through the CDP-choline pathway.

[0023] In another preferred embodiment, the metabolic-related fatty liver disease is a metabolic-related fatty liver disease in which TKT is highly expressed in the liver (cells).

[0024] In another preferred embodiment, the "high expression" is "high expression" in a statistical sense, for example, the average TKT expression of patients with "metabolic-related fatty liver disease with high expression of TKT" is significantly higher than that of healthy people (or a statistically sufficient number of people), by more than 10% or 20%, preferably by more than 30% or 50%, and more preferably by more than 80% or 100%.

[0025] In another aspect of the present invention, an siRNA or siRNA preparation for alleviating or treating metabolic-related fatty liver disease is provided, wherein the siRNA comprises an siRNA with a nucleotide sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 50, SEQ ID NO: 53 to SEQ ID NO: 167; preferably, the siRNA is an siRNA with a nucleotide sequence as shown in any one of SEQ ID NO: 37 (No. 1115), SEQ ID NO: 25 (No. 773), SEQ ID NO: 50 (siTKT16), SEQ ID NO: 49 (siTKT10), SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 88 or SEQ ID NO: 130.

[0026] In another preferred embodiment, the siRNA preparation is a stable siRNA preparation for targeted delivery to hepatocytes.

[0027] In another preferred embodiment, the siRNA is linked (covalently bound, forming a conjugate) to a targeting ligand that binds to the asialoglycoprotein receptor (ASGPR); the targeting ligand includes: N-acetylgalactosamine (GalNAc), N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine.

[0028] In the siRNA, the nucleosides in the sense strand and the antisense strand are modified by modifications selected from the group consisting of 2'-methoxy modification and 2'-fluoro modification; or phosphorothioate diester linkages between adjacent nucleosides or between nucleosides and targeting ligands.

[0029] In another preferred embodiment, in the sense strand of the siRNA, the first and second nucleosides from the 5' end and the second and third nucleosides are linked by phosphorothioate diester groups; in the corresponding antisense strand of the siRNA (with two protective bases at the 3' end), the first and second nucleosides from the 5' end, the first and second to last nucleosides from the 3' end, and the second and third nucleosides are linked by phosphorothioate diester groups.

[0030] In another preferred embodiment, in the sense strand of the siRNA, the nucleosides at positions 1, 2, 3, 4, 6, and 10-19 from the 5' end are 2'-methoxy modified; in the corresponding antisense strand of the siRNA (with two protected bases at the 3' end), the nucleosides at positions 1, 3, 4, 5, 7, 10-13, 15, and 17-21 from the 5' end are 2'-methoxy modified.

[0031] In another preferred embodiment, in the sense strand of the siRNA, the nucleosides at positions 5, 7, 8, and 9 from the 5' end are 2'-fluoro-modified, and in the corresponding antisense strand of the siRNA (with two protected bases at the 3' end), the nucleosides at positions 2, 6, 8, 9, 14, and 16 are 2'-fluoro-modified.

[0032] In another aspect of the present invention, a pharmaceutical composition or kit for alleviating or treating metabolic-related fatty liver disease is provided, comprising the siRNA or siRNA preparation for alleviating or treating metabolic-related fatty liver disease, and a pharmaceutically acceptable carrier or excipient.

[0033] In another aspect of the present invention, the use of the InsR-C / EBPα-TKT signaling pathway is provided for screening substances for alleviating or treating metabolism-related fatty liver disease.

[0034] In another preferred embodiment, the InsR-C / EBPα-TKT signaling pathway is a signaling pathway in hepatocytes.

[0035] In another aspect of the present invention, a method for screening substances for alleviating or treating metabolic-related fatty liver disease is provided, comprising: (1) contacting a candidate substance with a system containing the InsR-C / EBPα-TKT signaling pathway; (2) screening for substances that downregulate the InsR-C / EBPα-TKT signaling pathway, wherein the substances are substances (including potential substances) useful for alleviating or treating metabolic-related fatty liver disease; wherein the downregulation includes: downregulating TKT, downregulating C / EBPα-TKT interaction, and downregulating (insulin / )InsR-C / EBPα interaction.

[0036] In another preferred embodiment, step (1) comprises: adding a candidate substance to a system containing the InsR-C / EBPα-TKT signaling pathway.

[0037] In another preferred embodiment, step (2) comprises: detecting changes in each protein or its encoding gene in the InsR-C / EBPα-TKT signaling pathway, and comparing with a control group, wherein the control group is a system containing the InsR-C / EBPα-TKT signaling pathway without adding the candidate substance; if the candidate substance downregulates TKT, downregulates C / EBPα-TKT interaction, or downregulates (insulin / )InsR-C / EBPɑ interaction, then the candidate substance is a substance useful for alleviating or treating metabolic-related fatty liver disease.

[0038] In another preferred embodiment, the system containing the InsR-C / EBPα-TKT signaling pathway is selected from: a cell (culture) system, a subcellular (culture) system, a tissue (culture) system or an animal system.

[0039] In another preferred embodiment, the downregulation can also be referred to as inhibition, which is a statistical inhibition or downregulation, such as inhibition or downregulation of 10% or more than 20% compared with the control or substrate, preferably inhibition or downregulation of 40% or more than 50%, and more preferably inhibition or downregulation of 80% or more than 100%.

[0040] In another preferred embodiment, the candidate substances include (but are not limited to): regulatory molecules designed for the InsR-C / EBPα-TKT signaling pathway, or its pathway proteins, or its upstream or downstream proteins or genes (such as but not limited to upregulators, interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands)), CRISPR constructs, small molecule compounds, and compounds from a compound library.

[0041] In another aspect of the present invention, there is provided the use of liver TKT protein or its encoding gene in the preparation of a diagnostic reagent, wherein the diagnostic reagent is used to diagnose or prognose metabolism-related fatty liver disease; preferably, the diagnosis or prognosis includes: judging the occurrence or progression of metabolism-related fatty liver disease based on the expression of liver TKT protein, or judging whether it is suitable for a treatment regimen using "TKT or TKT-containing (insulin / )InsR-C / EBPα-TKT signaling pathway downregulators"; if TKT protein is highly expressed, this treatment regimen is applicable.

[0042] In another aspect of the present invention, the use of a reagent that specifically recognizes TKT protein or its encoding gene is provided for the preparation of a diagnostic reagent or diagnostic kit for diagnosing or prognosing metabolism-related fatty liver disease; preferably, the diagnosis or prognosis includes: judging the occurrence or progression of metabolism-related fatty liver disease based on the expression of TKT protein, or judging whether it is suitable for a treatment regimen using "TKT or TKT-containing (insulin / )InsR-C / EBPα-TKT signaling pathway downregulators"; if TKT protein is highly expressed, this treatment regimen is applicable.

[0043] In another preferred embodiment, the diagnostic reagent comprises a member selected from the group consisting of: primers that specifically amplify the gene encoding the TKT protein; probes that specifically recognize the gene encoding the TKT protein or its transcript; or antibodies specific to the TKT protein.

[0044] In another aspect of the present invention, a kit for diagnosing or prognosing metabolism-related fatty liver disease is provided, wherein the kit contains a diagnostic reagent for detecting the expression or expression level of TKT protein or its encoding gene.

[0045] In another preferred embodiment, the kit further comprises: nucleic acid extraction reagents, polymerase chain reaction reagents, protein immunoblotting reagents, and / or enzyme chain immunoassay reagents.

[0046] Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1. Analysis of TKT expression characteristics in liver tissue.

[0048] (A) Differential metabolic pathway enrichment map between healthy subjects and NAFLD patients.

[0049] (B) Volcano plot of changes in liver protein levels in healthy subjects and NAFLD patients.

[0050] (CE) Expression levels of non-oxidative pentose phosphate pathway (PPP) metabolic enzymes (C), TKT protein (D), and mRNA (E) in the livers of healthy controls and NAFLD patients (n = 4 in C and D, n = 8 in E).

[0051] (F) H&E, Oil Red O, and TKT immunohistochemical staining of healthy subjects and NAFLD patients (n=4).

[0052] (G-I) Expression levels of nonoxidative pentose phosphate pathway (PPP) metabolic enzymes (G), protein quantification (H), and mRNA (I) levels of TKT in the liver of wild-type mice fed a normal diet (NCD) or a high-fat diet (HFD) for 90 days (n = 5 for NCD, n = 6 for HFD).

[0053] (J) H&E, Oil Red O, and TKT immunohistochemical staining of liver tissues from wild-type mice fed a normal diet (NCD) or a high-fat diet (HFD) for 90 days (n = 5 in NCD, n = 6 in HFD).

[0054] (K) Expression levels of TKT protein in the liver of wild-type mice 10 weeks after injection of AAV-Flag-TKT adenovirus via the tail vein (n=4).

[0055] (L) Oil red O staining of liver tissues of wild-type mice after injection of AAV-Flag-TKT adenovirus via the tail vein and feeding of a high-fat diet for 10 weeks (n=4).

[0056] (M) Serum and liver TG levels in wild-type mice after injection of AAV-Flag-TKT adenovirus via the tail vein and feeding of a high-fat diet for 10 weeks (n=4).

[0057] For all data: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. Data are expressed as mean ± SEM. Two-tailed t-test was applied in D, E, H, I, and M.

[0058] Figure 2. Supplementary analysis of TKT expression characteristics in liver tissue.

[0059] (A) Clinical sample information corresponding to the liver tissue used in the experiment. HS (Hepatic Steatosis): Hepatic Steatosis.

[0060] (B) Fluorescent images of the liver of wild-type mice 10 weeks after injection of AAV-Flag-TKT adenovirus via the tail vein (n=3).

[0061] (C and D) H&E staining of liver tissue, serum and liver TC levels, and serum ALT and AST levels in wild-type mice injected with AAV-Flag-TKT adenovirus via the tail vein and fed a high-fat diet for 10 weeks (n = 4).

[0062] (E and F) H&E, Oil Red O, and Sirius Red staining of liver tissue, serum and liver TG and TC levels, and serum ALT and AST levels in wild-type mice injected with AAV-Flag-TKT adenovirus via the tail vein and fed an MCD diet for 4 weeks (n = 4).

[0063] For all data: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. Data are expressed as mean ± SEM. Two-tailed t-test was applied in D and F.

[0064] Fig. 3. Effect of hepatocyte-specific TKT knockout on disease progression.

[0065] (AI) Phenotypic analysis of wild-type and liver-specific TKT null mice fed a high-fat diet for 90 days, including (A) MRI of liver lipid content, (B) liver appearance, (C) H&E and Oil Red O staining, (D) serum TG levels, (E) serum TC levels, (F) serum ALT levels, (G) serum AST levels, (H) liver TG, and (I) liver TC levels (n = 5-8).

[0066] (JR) Phenotypic analysis of wild-type and liver-specific TKT null mice fed an MCD diet for 4 weeks, including (J) H&E staining, (K) Oil Red O staining, (L) mRNA levels of lipogenesis-related genes, (M) immunohistochemical staining of F4 / 80 and (N) CD45, (O) mRNA levels of inflammation-related genes, (P) Sirius red staining, (Q) immunohistochemical staining of α-smooth muscle actin (α-SMA), and (R) mRNA expression levels of TGFβ (n = 3-4).

[0067] (S) Strategy for constructing mice with liver-specific TKT gene knockout.

[0068] For all data: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. Data are expressed as mean ± SEM. Two-tailed t-test was applied in D, E, F, G, H, I, L, O, and R.

[0069] Figure 4. Effect of regulating TKT in the liver on disease progression.

[0070] (AD) (A) Body weight changes, (B) body shape images, (C) body weight, and (D) liver weight of wild-type and liver-specific TKT null mice fed a high-fat diet for 90 days (n=4).

[0071] (E and F) Liver pathological features of wild-type and liver-specific TKT null mice fed an MCD diet for 28 days, including (E) Sirius red staining and (F) TUNEL staining (n = 4).

[0072] (GN)TKT fl / fl Alb cre Phenotypic analysis of mice fed a high-fat diet for 10 weeks after tail vein injection of AAV-Flag-TKT adenovirus, including (G) H&E staining, (H) Oil Red O staining, (I) serum TG, (J) liver TG, (K) serum TC, (L) liver TC, (M) serum AST, and (N) serum ALT levels (n = 3 in the control group and n = 4 in the AAV-Flag-TKT group).

[0073] (OU)TKT fl / fl Alb cre Phenotypic analysis of mice fed an MCD diet for 3 weeks after tail vein injection of AAV-Flag-TKT adenovirus, including (O) H&E staining, Oil Red and Sirius Red staining, and (P) serum TC, (Q) liver TC, (R) serum TG, (S) liver TG, (T) serum AST, and (U) serum ALT levels (n = 4).

[0074] For all data: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. Data are expressed as mean ± SEM. Two-tailed t-test was applied in C, D, I, J, K, L, M, N, P, Q, R, S, T, U.

[0075] Figure 5. GalNAc-siRNA targeting liver TKT and its effects.

[0076] (A) GalNAc-siTKT injection strategy for NAFL treatment. Eight-week-old wild-type male mice were fed a high-fat diet for 90 days and then subcutaneously injected with 10 mg / kg of GalNAc-siTKT into the back. Injections were repeated five times at days 90+0, 3, 7, 14, and 21, and analysis was performed on day 120.

[0077] (B) TKT protein expression level in the liver of NAFL mice after GalNAc-siTKT treatment (n=5).

[0078] (CI) Phenotypic analysis of NAFL mice after GalNAc-siTKT treatment, including (C) H&E, Oil Red O staining, (D) serum TG, (E) liver TG, (F) serum TC, (G) liver TC, (H) serum ALT, and (I) AST levels (n = 5).

[0079] (J) GalNAc-siTKT injection strategy for the treatment of NASH. Eight-week-old wild-type male mice were subcutaneously injected with 10 mg / kg of GalNAc-siTKT at the beginning of the MCD diet. The injections were repeated five times on days 0, 3, 7, 14, and 21, and phenotypic analysis was performed on day 30.

[0080] (K) TKT protein expression level in the liver of NASH mice after GalNAc-siTKT intervention (n=2 in NC, n=4 in siTKT).

[0081] (LU) Phenotypic analysis of NASH mice after GalNAc-siTKT intervention, including (L) H&E, Oil Red O, and Sirius Red staining, as well as (M) serum TG, (N) liver TG, (O) serum TC, (P) liver TC, (Q) serum ALT, and (R) AST levels, (S) mRNA levels of lipid formation-related genes, (T) mRNA levels of inflammation-related genes, and (U) mRNA levels of fibrosis-related genes (n = 4).

[0082] For all data: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. Data are expressed as mean ± SEM. Two-tailed t-test was applied in D, E, F, G, H, I, M, N, O, P, Q, R, S, T, and U.

[0083] Figure 6. GalNAc-siRNA specifically targets and reduces liver TKT protein levels in mice.

[0084] (A and B) TKT (A) protein and (B) mRNA levels in mouse primary hepatocytes after treatment with non-liposome-coated GalNAc-siTKT.

[0085] (C-H) Protein expression levels of TKT in (C) liver, (D) lung, (E) heart, (F) kidney, (G) spleen, and (H) adipose tissue of wild-type mice 72 hours after subcutaneous injection of 10 mg / kg GalNAc-siTKT.

[0086] For all data: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. Data are expressed as mean ± SEM. Two-tailed t-test was applied in B.

[0087] Figure 7. Screening and effect analysis of human, mouse and monkey homologous sequences.

[0088] (A) Detection of the knockdown effect of 49 siRNAs targeting the human, mouse, and monkey homologous regions of the TKT gene on primary mouse hepatocytes.

[0089] (BC) Wild-type mice were subcutaneously injected with GalNAc-siTKT 773 and 1115, and the TKT knockdown effect in the liver was detected 7 days later. (B) mRNA level and (C) protein level.

[0090] (D) Changes in blood drug concentration after subcutaneous injection of GalNAc-siTKT1115 in wild-type mice.

[0091] Figure 8. Hyperinsulinemia causes elevated hepatic TKT levels.

[0092] (A) Serum insulin levels in wild-type mice fed a normal diet or a high-fat diet for 90 days (NCD group n = 5, HFD group n = 4).

[0093] (B-D) Relative quantification of (B) TKT mRNA levels, (C) TKT, pC / EBPa, and C / EBPa protein levels, and (D) TKT protein in primary hepatocytes from wild-type mice after treatment with 10 nM insulin (n=4).

[0094] (E) Binding ability of pC / EBPa and C / EBPa to TKT promoter after wild-type mouse primary hepatocytes were treated with 10 nM insulin (n=3).

[0095] (FH) Relative quantification of (F) TKT mRNA levels, (G) TKT, pC / EBPa, and C / EBPa protein levels, and (H) TKT protein in primary hepatocytes from liver-specific insulin receptor knockout mice after treatment with 10 nM insulin (n=3).

[0096] (IM) TKT protein (I) and (J) mRNA levels in the liver of mice with liver-specific insulin receptor knockout fed a normal diet or a high-fat diet for 90 days, H&E staining (K), Oil Red O staining (L), and Sirius Red staining (M) of liver tissue (n = 3-4).

[0097] Figure 9. TKT-deficient hepatocytes showed significant accumulation of R5P and inosine.

[0098] (A and B) (A) Principal component analysis (PCA) and (B) KEGG metabolic pathway enrichment analysis of differential metabolites in primary hepatocytes from wild-type and liver-specific TKT null mice (n=5).

[0099] (C) Heat map of differential metabolites in the purine metabolic pathway (n=5).

[0100] (D) 13 C-labeled inosine ( 13 C5-inosine) structure diagram.

[0101] (E) Primary hepatocytes from wild-type and liver-specific TKT-deficient mice were cultured in the presence of 5.6 mM 13 Metabolic flux analysis after 6 hours of culture in a medium containing C5-inosine (n=3).

[0102] (F) Isotope-labeled levels of R5P, G6P / F6P, and S7P in primary hepatocytes from wild-type and liver-specific TKT-deficient mice (n=3).

[0103] (G) Primary hepatocytes from wild-type and liver-specific TKT-deficient mice were cultured in the presence of 15 mM 1,2 13 Gluose and G6P / F6P labeling were analyzed after 30 minutes of incubation in the C2-glucose culture medium, Sed7P, R5P and R1P labeling were analyzed after 6 hours, and inosine labeling was analyzed after 48 hours (n=3).

[0104] (H and I) Labeling of R5P, G6P / F6P, and S7P in primary hepatocytes from wild-type and liver-specific TKT-deficient mice (n=3).

[0105] Figure 10. TKT deficiency enhances mitochondrial function through the inosine-PKA-CREB pathway.

[0106] (A) Expression levels of p-PKA substrates, p-CREB, CREB, UCP1, and TKT in primary hepatocytes from wild-type and liver-specific TKT-null mice (n=3).

[0107] (B, F) Analysis of mitochondrial morphology and quantity in liver tissue of wild-type and liver-specific TKT-deficient mice, including (B) low-magnification electron microscopy images, (C) high-magnification electron microscopy images, (D) mitochondrial density, (E) mitochondrial diameter, and (F) mitochondrial surface area (n = 3 in B and C, n = 10 in D, n = 120 in E and F).

[0108] (G, J) Analysis of mitochondrial function in primary hepatocytes from wild-type and liver-specific TKT-null mice, including (G) mitochondrial membrane potential, (H) ATP, (I) mitochondrial ROS (mtROS), and (J) OCR levels (n = 10-12 in J, K, and L, n = 3 in M).

[0109] (K and L) (K) Inosine and (L) OCR levels in primary hepatocytes of wild-type mice after treatment with 10 nM insulin (n=6).

[0110] (M) Inosine levels in primary hepatocytes from wild-type, liver-specific TKT-deficient, and liver-specific insulin receptor-deficient mice after treatment with 10 nM insulin (n=4-6).

[0111] Figure 11. TKT deficiency enhances mitochondrial function through the inosine-PKA-CREB pathway.

[0112] (A) Expression levels of p-PKA substrate, p-CREB, CREB, and UCP1 proteins in primary hepatocytes of wild-type mice after treatment with 56 μM inosine for 6 h (n=3).

[0113] (B-D) Mitochondrial DNA (mtDNA) content (B), protein expression (C), and mRNA expression (D) of mitochondrial genes in primary hepatocytes from wild-type and liver-specific TKT-deficient mice (n = 4 in B and D, n = 3 in C).

[0114] (E) Immunofluorescence staining of TOM20 protein in liver tissues of wild-type and liver-specific TKT-deficient mice (n=3).

[0115] (F) Western blot analysis of p-PKA substrate and TKT protein in primary hepatocytes from wild-type mice treated with 56 μM inosine and 10 μM H89 for 6 h.

[0116] (G and H) Analysis of (G) OCR, (H) ATP, and residual respiratory capacity in primary hepatocytes from wild-type and liver-specific TKT null mice after treatment with 56 μM inosine for 6 h (n=3).

[0117] (I) Changes in OCR of primary hepatocytes from wild-type mice after treatment with 56 μM inosine and 10 μM H89 for 6 h (n=3).

[0118] All data: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. Data are expressed as mean ± SEM. B and D were analyzed using a two-tailed t-test.

[0119] FIG12 . Inosine upregulates PC synthesis by activating the PKA-CREB-ChoKβ axis in hepatocytes.

[0120] (A) Heatmap of the correlation matrix of signature genes. Each row or column corresponds to a signature gene, including module number and TKT protein. Red and blue indicate positive and negative correlations, respectively. Modules 1, 3, and 7 showed Pearson correlation coefficients > 0.72, p < 0.01.

[0121] (B-D) Network diagrams of the top 10 lipid classes with the highest connectivity in (B) module 1, (C) module 3, and (D) module 7. Changes in module eigengenes (MEs) in the livers of NAFLD and healthy controls in (B) module 1, (C) module 3, and (D) module 7. The top and bottom lines in the box plots represent the maximum and minimum values ​​based on the Tukey test, and the middle line represents the median.

[0122] (E) Levels of six lipids in modules 1, 3, and 7 in livers of NAFLD and healthy subjects (n=24).

[0123] (F) Schematic diagram of the phosphatidylcholine synthesis pathway.

[0124] (G and H) Levels of (G) phosphatidylcholine (PC) and (H) CDP-choline pathway metabolites in primary hepatocytes from wild-type and liver-specific TKT-null mice (n = 6 in G, n = 4 in H).

[0125] (I and J) PC (I) and OCR (J) levels in primary hepatocytes after wild-type and liver-specific TKT null mice were fed a low-choline diet (choline content was 20% of that of the NCD diet) for 3 weeks (n = 4 in I, n = 3 in J).

[0126] (K) Relative levels of CDP-choline pathway metabolites in primary hepatocytes from wild-type mice after treatment with 56 μM inosine for 6 hours (n=3).

[0127] (L and M) ChoK mRNA and protein levels in primary hepatocytes from wild-type and liver-specific TKT null mice (L = 4, M = 2).

[0128] (N) Relative levels of CDP-choline pathway metabolites in wild-type mouse primary hepatocytes after treatment with 56 μM inosine and 10 μM H89 for 6 h (N=3).

[0129] FIG13 is a diagram showing target genes of the present invention and their upstream and downstream regulatory related signal pathways.

[0130] Figure 14. TKT deficiency enhances hepatocyte mitochondrial function by increasing phosphatidylcholine synthesis.

[0131] (A) Protein levels of PGC1α, NRF2, and DRP1 in TKT-deficient hepatocytes.

[0132] (B) Lipidomics results of mitochondria in TKT-deficient hepatocytes.

[0133] (CF) Levels of choline, phosphatidylcholine, CDP-choline, and PC in liver tissues of mice in the low-choline diet and normal diet groups.

[0134] (GJ) Mitochondrial membrane potential, ATP, mitochondrial ROS, and OCR levels in hepatocytes of mice in the low-choline diet group and the normal diet group.

[0135] (KP) Levels of (K) choline, (L) PC, and (M) CDP-choline, and hepatocyte (N) ATP, (O) mitochondrial membrane potential, and (P) mitochondrial ROS levels in primary hepatocytes from wild-type and liver-specific TKT-deficient mice fed a low-choline diet.

[0136] FIG15 shows the sense strand of siRNA according to Table 2, and the modified sense strand and antisense strand prepared by simultaneously considering the antisense strand thereof.

[0137] Figure 16. Modified groups; wherein Base represents a base. DETAILED DESCRIPTION

[0138] This invention, for the first time, reveals a novel target gene closely associated with the alleviation or treatment of metabolic-related fatty liver disease, and its upstream and downstream regulatory signaling pathways. It also discloses a novel mechanism for regulating metabolic-related fatty liver disease, involving this signaling pathway. It also discloses a preferred drug for alleviation / treatment.

[0139] Transketolase (TKT) or TKT-containing insulin / InsR-C / EBPα-TKT signaling pathway and its regulation

[0140] As used in the present invention, the "(signaling) pathway" and "(signaling) path" can be used interchangeably.

[0141] As used herein, the term "(signal) pathway" refers to a signaling system formed by the mutual constraints or interactions between a series of genes or proteins or their metabolites (synthetic or processed products). This also includes interactions between pathway proteins and other intracellular components or organelles, and sometimes also includes the participation of upstream and downstream genes or proteins, which generally lead to the occurrence of certain cellular events. The insulin / InsR-C / EBPα-TKT signaling pathway primarily includes the following elements: TKT gene / protein, C / EBPα gene / protein, and InsR gene / protein; preferably, it also includes their upstream and downstream regulatory genes / proteins or chemical molecules.

[0142] The nucleotide sequence of the TKT gene is shown in, for example, Gene ID: 7086 (human) and Gene ID: 21881 (mouse); the amino acid sequence of its protein is shown in, for example, NCBI Reference Sequence: NP_001055.1 (human) or NCBI Reference Sequence: NP_033414.1 (mouse).

[0143] The nucleotide sequence of the C / EBP gene is shown, for example, in Gene ID: 1050 (human) and Gene ID: 12606 (mouse); the amino acid sequence of its protein is shown, for example, in NCBI Reference Sequence: NP_004355.2 (human) and NCBI Reference Sequence: NP_031704.2 (mouse).

[0144] The nucleotide sequence of the InsR gene is shown, for example, in Gene ID: 3643 (human) and Gene ID: 16337 (mouse); the amino acid sequence of its protein is shown, for example, in NCBI Reference Sequence: NP_000199.2 (human) and NCBI Reference Sequence: NP_034698.2 (mouse).

[0145] In the present invention, unless otherwise stated, protein / gene information in some upstream and downstream signaling pathways related to the insulin / InsR-C / EBPa-TKT signaling pathway is known in the art.

[0146] Downstream of the insulin / InsR-C / EBPα-TKT signaling pathway in hepatocytes, a series of further regulatory pathways exist. Downregulating TKT or the TKT-containing insulin / InsR-C / EBPα-TKT signaling pathway can further reduce inosine levels, enhance mitochondrial function, and thereby improve hepatic lipid metabolism and alleviate or treat metabolic-related fatty liver disease. More specifically, after downregulating the TKT or TKT-containing insulin / InsR-C / EBPα-TKT signaling pathway, it is possible to prevent R5P derived from inosine from entering glycolysis, promote the synthesis of inosine from glucose-derived R5P, and increase the level of inosine in the cell; thereby promoting the inosine-PKA-CREB pathway, activating the PKA-CREB-ChoKβ axis, increasing phosphatidylcholine (PC) synthesis, enhancing mitochondrial function, improving liver lipid metabolism, and alleviating or treating metabolic-related fatty liver disease; preferably, the increase in phosphatidylcholine (PC) synthesis is mediated through the CDP-choline pathway.

[0147] As used herein, unless otherwise stated, the target genes / proteins discussed and the signaling pathways they participate in are those in the liver (cells).

[0148] When used as targets for artificial regulation or in artificially established screening systems, the above proteins or encoding genes can be naturally occurring, such as those purified and isolated from mammals; or they can be recombinantly produced, such as by producing recombinant proteins using conventional genetic recombination techniques. In addition, any modified forms that do not affect the biological activity of these proteins are also acceptable, such as derivatives or variants whose functions are not altered.

[0149] The above-mentioned protein (polypeptide) also includes its variant forms, including (but not limited to): deletion, insertion and / or substitution of several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8, 1-5) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. Any protein with high homology to the protein (such as 70% or higher homology to the polypeptide sequence; preferably 80% or higher homology; more preferably 90% or higher homology, such as 95%, 98% or 99% homology) and having the same function as the protein is also included in the present invention. The present invention also includes a mutant form of the protein or protein truncation, as long as the mutant protein or truncation substantially retains the function of the full-length protein.

[0150] The gene sequences mentioned above also include degenerate sequences thereof. The protein-encoding polynucleotides (genes) may be natural genes or their degenerate sequences.

[0151] As used in the present invention, the "down-regulator" and "inhibitor" can be used interchangeably, and also include: blocker, antagonist, etc.

[0152] The inventors have discovered that downregulating TKT, downregulating the C / EBPα-TKT interaction, and downregulating the insulin / InsR-C / EBPα interaction in the signaling pathway can alleviate or treat metabolic-related fatty liver disease. Therefore, this mode of action can be used to screen or design drugs suitable for targeted regulation.

[0153] It should be understood that once the function of the insulin / InsR-C / EBPα-TKT signaling pathway (preferably, including its upstream and downstream proteins or genes) is known, various methods well known to those skilled in the art can be used to regulate the insulin / InsR-C / EBPα-TKT signaling pathway. For example, various methods well known to those skilled in the art can be used to regulate the expression of pathway proteins or to abrogate their expression.

[0154] The present invention provides a method for down-regulating the insulin / InsR-C / EBPα-TKT signaling pathway, comprising targeted mutation, gene editing or gene recombination of TKT, C / EBPα or InsR genes in the insulin / InsR-C / EBPα-TKT signaling pathway, thereby achieving down-regulation.

[0155] As a more specific embodiment, a method for downregulating the expression of TKT, C / EBPɑ, or InsR genes is provided, comprising: introducing an interfering molecule that interferes with the expression of TKT, C / EBPα, or InsR genes into cells, or treating the cells through appropriate pathways to introduce the molecule into the cells, such as designing a transmembrane functional domain to impart transmembrane capabilities. RNA interference technology is a technique for silencing gene expression. The principle of RNA interference technology is that longer double-stranded RNA is cleaved and processed by the specific nuclease Dicer into 21-23nt small interfering RNAs consisting of sense and antisense strands. The small interfering RNA then forms an RNA-induced silencing complex (RISC) that unwinds into a single strand. The antisense strand guides the silencing complex to specifically bind to the target mRNA through base pairing, causing the mRNA to decompose.

[0156] As another embodiment of the present invention, the CRISPR / Cas system is used for gene editing to knock down, eliminate, or downregulate the target gene. A suitable sgRNA target site will lead to higher gene editing efficiency, so before commencing gene editing, a suitable target site should be designed and found. After designing a specific target site, in vitro cell activity screening is required to obtain an effective target site for subsequent experiments. This method can be used to convert the TKT, C / EBPα, or InsR gene into a truncated or mutant form that has lost its function.

[0157] Regulatory agents and pharmaceutical compositions

[0158] As a preferred embodiment of the present invention, a down-regulator is provided that down-regulates the expression or activity of TKT, down-regulates the C / EBPα-TKT interaction, or down-regulates the insulin / InsR-C / EBPα interaction. The down-regulator refers to any substance that can reduce the activity, stability, expression, activation level (such as phosphorylation level), effective action time, or inhibit the transcription and translation of TKT, C / EBPα or InsR. These substances can be used in the present invention as potentially useful substances for alleviating or treating metabolic-related fatty liver disease. They can be compounds, small chemical molecules, or biological molecules. The biological molecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

[0159] As used in the present invention, the "metabolic-related fatty liver disease" includes fatty liver.

[0160] As used in the present invention, the "metabolic-related fatty liver disease" includes two different indications: non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0161] In the present invention, the downregulator can be a nucleic acid inhibitor, protein inhibitor, antibody, ligand, compound, nuclease, nucleic acid binding molecule, etc., provided that it can downregulate the expression of TKT, C / EBPα, or InsR, inhibit its activity or function, or downregulate the C / EBPα-TKT interaction or downregulate the insulin / InsR-C / EBPα interaction. The nucleic acid inhibitor includes: shRNA, antisense nucleic acid, small interfering RNA, microRNA that targets the gene encoding TKT, C / EBPα, or InsR or its transcript, or a construct capable of expressing or forming such shRNA, antisense nucleic acid, small interfering RNA, or microRNA.

[0162] For example, the down-regulator is: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of TKT, C / EBPα or InsR or its upstream genes; or a homologous recombination, targeted mutation or gene editing agent that specifically targets TKT, C / EBPα or InsR or its upstream genes, etc.

[0163] As a preferred embodiment of the present invention, the down-regulator is an interfering molecule that specifically interferes with TKT expression. Small hairpin RNA (shRNA) is an RNA sequence that forms a sharp turn structure and can silence genes through RNA interference. The interfering molecule that specifically interferes with the expression of TKT, C / EBPα, or InsR genes can be an shRNA molecule targeting TKT, C / EBPα, or InsR genes, or it can be an siRNA molecule targeting TKT, C / EBPα, or InsR genes.

[0164] Focusing on the TKT target, the inventors conducted in-depth experimental demonstrations, including demonstrations at the cellular and animal levels. Based on this, the inventors optimized and obtained a series of highly effective siRNA reagents. The siRNAs are siRNAs with nucleotide sequences such as any one of SEQ ID NO: 1 to SEQ ID NO: 50, and SEQ ID NO: 53 to SEQ ID NO: 167; more preferably, the siRNAs are siRNAs with nucleotide sequences such as any one of SEQ ID NO: 37 (No. 1115), SEQ ID NO: 25 (No. 773), SEQ ID NO: 50 (siTKT16), SEQ ID NO: 49, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 88, or SEQ ID NO: 130 (siTKT10). These siRNAs can be used in multiple species, exhibit highly ideal targeted regulatory effects, lack nonspecific regulation, and possess high clinical application value.

[0165] Considering that the disease targeted by the present invention is liver disease, in a preferred embodiment, the siRNA is prepared as a stable siRNA formulation for targeted delivery to hepatocytes.

[0166] In a particularly preferred embodiment, the siRNA is covalently bound to N-acetylgalactosamine (GalNAc) to form a conjugate after being modified with fluorine and methoxy.

[0167] As another alternative embodiment of the present invention, the down-regulator is an inhibitor targeting mutation, gene editing, or gene recombination of TKT, C / EBPα, or InsR. As a more specific embodiment, any of the above methods is used to convert TKT, C / EBPα, or InsR into a mutant, thereby rendering it ineffective.

[0168] The present invention also provides a pharmaceutical composition for alleviating or treating metabolism-related fatty liver disease, comprising an effective amount of the down-regulator of the present invention.

[0169] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0170] As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents.

[0171] The present invention also provides a kit for alleviating or treating metabolic-related fatty liver disease, comprising an effective amount of the down-regulator of the present invention. More preferably, the kit also includes instructions for use to guide clinicians in the correct and appropriate use of the medication.

[0172] For ease of administration, the down-regulator is prepared in a unit dosage form and placed in a kit. "Unit dosage form" refers to a dosage form in which a drug is prepared for single administration for ease of administration, including but not limited to various liquids (such as injections), solids (such as tablets), capsules, and sustained-release formulations. In addition, the down-regulator can also be independently placed in different containers and mixed and applied as needed.

[0173] Although the specific embodiments of the present invention provide a dosage regimen for animals such as mice, it should be understood that it is easy for a person skilled in the art to convert the dosage for animals such as mice into a dosage suitable for humans, for example, it can be calculated according to the Meeh-Rubner formula: Meeh-Rubner formula: A = k × (W 2 / 3 ) / 10,000. Where A is the body surface area, expressed in m 2 Calculation: W is body weight in grams; K is a constant that varies by animal species, for example, but not limited to, 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans. It should be understood that dosage conversions may vary depending on the drug and clinical situation, based on the assessment of an experienced pharmacist.

[0174] Drug screening based on the InsR-C / EBPα-TKT signaling pathway

[0175] Based on the inventors' new discoveries, research on the (insulin / )InsR-C / EBPα-TKT signaling pathway has multiple applications, including screening for substances that modulate this signaling pathway for the relief or treatment of metabolic-related fatty liver disease. These modulators include downregulating TKT, downregulating the C / EBPα-TKT interaction, and downregulating the insulin / InsR-C / EBPα interaction.

[0176] The present invention provides a method for screening agents that downregulate the InsR-C / EBPα-TKT signaling pathway. The method involves adding a candidate substance to a system containing the InsR-C / EBPα-TKT signaling pathway and observing changes or interactions among proteins or genes in the pathway. If the candidate substance has the effects of downregulating TKT, downregulating the C / EBPα-TKT interaction, or downregulating the insulin / InsR-C / EBPα interaction, the candidate substance is considered useful for alleviating or treating metabolic-related fatty liver disease.

[0177] As used herein, the terms "inhibit" and "downregulate" refer to statistically significant "inhibition" or "downregulation." This refers to significant "inhibition" or "downregulation." For example, compared to a control group, the protein activity, protein expression, protein binding, or methylation level is significantly "inhibited" or "downregulated" by 10%, 20%, 30%, 40%, or 50% or more; more preferably, by 60%, 70%, or 80% or more.

[0178] The system containing the InsR-C / EBPα-TKT signaling pathway is selected from: a cell system (or cell culture system), a subcellular system (or subcellular culture system), a solution system, an animal system, or a tissue system (or tissue culture system). Preferably, the system containing the InsR-C / EBPα-TKT signaling pathway is a hepatocyte (or cell culture).

[0179] As a preferred embodiment of the present invention, the method further comprises: performing further cell experiments and / or animal experiments on the obtained potential substances to further select and determine substances useful for alleviating or treating metabolism-related fatty liver disease from the candidate substances.

[0180] When performing screening, various techniques well known in the art can be used to determine changes in proteins or their encoding genes and interactions.

[0181] A variety of conventional techniques can be used to identify the transcription or expression of genes in a system. These techniques include, but are not limited to, oligonucleotide hybridization techniques (e.g., probes), polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, and the like. Protein-protein interactions and the strength of these interactions can be detected using a variety of techniques well known to those skilled in the art, such as co-immunoprecipitation, GST precipitation, phage display, or yeast two-hybrid systems. Nuclear localization of proteins is also well known in the art.

[0182] The substances initially screened out by the above method can constitute a screening library, so that people can eventually screen out substances that are truly useful for alleviating or treating metabolic-related fatty liver disease.

[0183] The present invention also provides potential substances obtained by the screening method and can be used to alleviate or treat metabolism-related fatty liver disease.

[0184] The present invention also provides a method for preparing a drug for alleviating or treating metabolism-related fatty liver disease (especially alleviating or treating metabolism-related fatty liver disease), the method comprising: synthesizing and / or purifying a substance useful for alleviating or treating metabolism-related fatty liver disease obtained through the aforementioned screening, as a drug for alleviating or treating metabolism-related fatty liver disease.

[0185] The obtained substance useful for alleviating or treating metabolism-related fatty liver disease can be used to prepare a pharmaceutical composition, as described below in the present invention.

[0186] Methods for screening for substances that act on proteins or genes, or specific regions thereof, as targets are well known in the art and can be used in the present invention. The candidate substances can be selected from the group consisting of peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences. Depending on the type of substance to be screened, those skilled in the art will appreciate how to select an appropriate screening method.

[0187] Applications for diagnosis or prognosis

[0188] The present invention discovered that upregulation of TKT in the liver is a common feature of NAFLD in humans and mice. Upregulation of TKT promotes the progression of NAFLD in mice and promotes non-alcoholic steatohepatitis (NASH). TKT is also involved in a new signaling pathway: the insulin / InsR-C / EBPα-TKT signaling pathway. Therefore, TKT and the signaling pathways it participates in can be used as molecular markers to guide the diagnosis or prognosis (including medication guidance) of metabolic-related fatty liver disease: (i) disease classification and differential diagnosis; (ii) evaluation of therapeutic drugs, drug efficacy, prognosis, and selection of appropriate treatment methods for relevant populations. For example, populations with abnormal (increased) TKT gene expression can be isolated, allowing for more targeted treatment.

[0189] By determining the expression or activity of TKT or the pathway genes / proteins of the signaling pathways involved in the sample to be evaluated, the disease prognosis of the subject providing the sample to be evaluated can be predicted, and appropriate drugs can be selected for treatment. Generally, a threshold for TKT expression can be specified. When the expression of TKT is higher than the specified threshold, a TKT-inhibiting regimen is considered for treatment. The threshold is easy for those skilled in the art to determine. For example, the threshold for abnormal TKT expression can be obtained by comparing and analyzing the general TKT expression in patients with metabolic-related fatty liver disease or the expression in normal healthy people.

[0190] Therefore, the present invention provides the use of TKT or pathway genes / proteins of the signaling pathways in which it participates, for the preparation of reagents or kits for the prognosis assessment of metabolic-related fatty liver disease. Various techniques known in the art can be used to detect the presence or absence and expression of the corresponding genes or proteins, and these techniques are all included in the present invention. For example, existing techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR, etc. can be used, and these methods can be used in combination. The present invention also provides reagents for detecting the presence or absence and expression of genes or proteins in analytes. Preferably, when performing gene-level detection, specific amplification primers can be used; or specific recognition probes can be used to determine the presence or absence of the target gene; when performing protein-level detection, antibodies or ligands that specifically bind to the protein can be used to determine the expression of the target protein.

[0191] The kit may also include various reagents required for DNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution, color development solution, washing solution, etc. In addition, the kit may also include instructions for use and / or nucleic acid sequence analysis software.

[0192] The present invention will be further described below with reference to 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 procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0193] Example 1. Expression characteristics of TKT in liver tissue

[0194] Liver samples were obtained from patients with nonalcoholic fatty liver disease (NAFLD) or healthy liver transplant donors. The sample source information is shown in Figure 2A , and they were divided into NAFLD group (HS) and healthy control group (Control) according to clinical pathological diagnosis.

[0195] Metabolomic analysis revealed that the pentose phosphate pathway (PPP) was significantly altered in NAFLD livers compared with controls ( Figure 1A ).

[0196] According to further proteomic analysis of PPP and glycolytic enzymes, the non-oxidative PPP metabolic enzyme TKT showed the most significant difference between NAFLD livers and controls (Figure 1B).

[0197] Western blot and quantitative PCR analysis were performed to analyze the expression of the non-oxidative PPP metabolic enzyme TKT. The results showed that TKT was significantly upregulated in NAFLD livers (Figure 1C-E).

[0198] Immunohistochemical (IHC) analysis of a randomly selected set of human liver samples (n = 6) revealed that TKT was more deeply stained in the NAFLD group than in the healthy control group ( Figure 1F ).

[0199] Furthermore, NAFLD was induced by feeding wild-type mice a high-fat diet (HFD). The HFD group was fed a high-fat diet daily with free access to food, while the control NCD group was fed a normal diet daily with free access to food. This mimics the development and progression of NAFLD in humans. After 3 months, lipid accumulation and upregulation of TKT were observed in the livers of the mice (Figures 1G-J).

[0200] These findings therefore indicate that upregulation of TKT in the liver is a common feature of NAFLD in both humans and mice.

[0201] Example 2: Upregulation of TKT promotes the progression of NAFLD in mice

[0202] 1. Upregulation of TKT in the liver promotes non-alcoholic fatty liver disease (NAFL)

[0203] To investigate whether TKT upregulation contributes to the progression of NAFLD, we constructed an adenovirus (adeno-associated virus, AAV)-mediated gene overexpression system to overexpress TKT in mouse liver.

[0204] Tail vein injection of AAV-Flag-TKT into mice significantly increased TKT levels in the liver (Figure 1K and Figure 2B).

[0205] After mice were fed an HFD for 10 weeks, mice injected with AAV-Flag-TKT showed more severe hepatic steatosis ( Figure 1L and Figure 2C ), as well as higher liver and serum triglyceride levels ( Figure 1M ).

[0206] Overexpression of TKT in the liver also increased the levels of total cholesterol, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the mice ( Figure 2D ).

[0207] These results suggest that upregulation of TKT in the liver promotes high-fat diet-induced nonalcoholic fatty liver disease (NAFL).

[0208] 2. Upregulation of TKT in the liver promotes non-alcoholic steatohepatitis (NASH)

[0209] A methionine- and choline-deficient (MCD) diet is commonly used to induce NASH in mice, a progressive form of NAFLD. Mice were fed an MCD diet daily (ad libitum) and monitored for various parameters in animals injected with AAV-Flag-TKT and a control (AAV-ctrl).

[0210] The results showed that after 4 weeks of MCD diet, AAV-Flag-TKT-injected mice exhibited more severe hepatocyte ballooning degeneration, lipid accumulation, inflammation, and fibrosis than the control group (Figure 2E), accompanied by elevated hepatic triglyceride (TG) and total cholesterol (TC) levels, as well as serum aspartate aminotransferase (AST) levels (Figure 2F), indicating that AAV-Flag-TKT-injected animals had more significant NASH progression than controls.

[0211] Thus, upregulation of TKT in the liver significantly drives the progression of NAFL and NASH.

[0212] Example 3: Hepatocyte-specific TKT knockout improves NAFLD in mice

[0213] 1. Downregulation of TKT in the liver improves non-alcoholic fatty liver disease (NAFL)

[0214] To investigate whether TKT is essential for the development of NAFLD, a hepatocyte-specific TKT-deficient mouse strain (TKT) was constructed. flox / flox Albcre , abbreviated as TKT fl / fl Alb cre ).

[0215] TKT flox / flox Alb cre Establishment of mice: The mouse strain was C57BL / 6. LoxP sites were inserted between exons 1 and 2 and between exons 2 and 3 of the mouse TKT gene to obtain TKT. flox / flox mouse strain (Figure 3S), which was then hybridized with AlbCre mice to obtain TKT flox / flox Alb cre mouse.

[0216] TKT + / + Alb cre Establishment of mice: TKT was obtained by crossing wild-type C57BL / 6 mice with AlbCre mice. + / + Alb cre mouse.

[0217] Under normal diet (NCD) conditions, TKT flox / flox Alb cre Mice showed normal liver morphology and function. flox / flox Alb cre and TKT + / + Alb cre The mice were fed a high-fat diet (HFD) for 90 days. + / + Alb cre Compared with mice, TKT flox / flox Alb cre The weight gain rate of TKT mice was slower than that of control mice after 90 days of HFD feeding (Figure 4A). flox / flox Alb cre The body and liver weights of the mice were lower (Figure 4B-D).

[0218] TKT fed with HFD flox / flox Alb cre The mice showed milder hepatic steatosis than the control group, as confirmed by magnetic resonance imaging (MRI), liver appearance photographs, H&E, and Oil Red O staining results ( Figure 3A-C ).

[0219] And, TKT flox / flox Alb cre The levels of liver triglyceride (TG), serum triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the mice were lower than those in the control group ( Figure 3D-H ), while liver total cholesterol remained unchanged ( Figure 3I ).

[0220] Therefore, hepatocyte-specific TKT deletion in mice can significantly improve HFD-induced NAFL in mice.

[0221] 2. Downregulation of TKT in the liver alleviates non-alcoholic steatohepatitis (NASH)

[0222] We investigated whether hepatocyte-specific TKT knockout could alleviate NASH induced by MCD diet. flox / flox Alb cre ) and control mice (TKT + / + Alb cre ) of disease progression.

[0223] After 4 weeks of MCD diet, TKT-deficient mice (TKT flox / flox Alb cre ) showed significantly reduced hepatic lipid accumulation ( Figure 3J,K ), whereas the mRNA levels of lipid synthase genes were unchanged ( Figure 3L ).

[0224] TKT flox / flox Alb cre The infiltration of inflammatory cells in the livers of mice was much less than that in the control group ( Figure 3M,N ), accompanied by downregulation of inflammation-related genes ( Figure 3O ).

[0225] The MCD diet induced severe liver fibrosis in the control group, as confirmed by Sirius red staining and Masson staining, whereas TKT knockout in the liver alleviated this condition ( Figures 3P and 4E ).

[0226] Furthermore, TKT deletion reduced hepatocellular necrosis associated with NASH progression ( Figure 4F ).

[0227] Thus, TKT deficiency in hepatocytes can ameliorate NAFL and NASH in mice.

[0228] 3. Role of TKT in the development of NAFLD

[0229] To further demonstrate that TKT is a driver of NAFLD, an adenoviral overexpression system was used to express TKT. flox / flox Alb cre TKT was re-expressed in the liver of mice. flox / flox Alb cre) were used to establish HFD-induced NAFL and MCD-induced NASH models, and then adenovirus AAV-Flag-TKT and AAV-Ctrl were injected into the tail vein, respectively.

[0230] During HFD feeding, re-expression of TKT in the liver promoted hepatic lipid accumulation ( Figure 4G,H ), increased serum and hepatic triglyceride and total cholesterol levels, and serum ALT and AST levels ( Figure 4I–N ).

[0231] Consistent with this, re-expression of TKT in the liver restored MCD-induced hepatic lipid accumulation and fibrosis and upregulated hepatic triglyceride and cholesterol levels, as well as serum ALT and AST levels (Fig. 4P-U).

[0232] In conclusion, upregulation of TKT in the liver is both necessary and sufficient for the development of NAFLD in mice.

[0233] Example 4: Analysis of the regulatory effects of TKT in the liver and the regulatory pathways involved

[0234] 1. Insulin-InsR signaling drives TKT upregulation and lipid accumulation in NAFLD progression

[0235] The inventors studied the mechanism of TKT upregulation in NAFLD livers, which is often associated with hyperinsulinemia and insulin resistance. In mice fed an HFD, elevated serum insulin levels were observed ( FIG8A ). In insulin-treated primary hepatocytes, the increase in TKT mRNA and protein levels peaked at 40 minutes and then gradually declined ( FIG8B-D ), accompanied by fluctuations in the levels of the phosphorylated transcription factor CCAAT / enhancer binding protein α (C / EBPα) ( FIG8C ). Analysis suggested that C / EBPα is a candidate transcription factor for the TKT gene promoter.

[0236] Chromatin immunoprecipitation (ChIP) confirmed that insulin enhanced the binding of phosphorylated C / EBPα to the 643-795 bp region upstream of the TKT promoter through InsR ( Figure 8E ).

[0237] To further investigate the role of insulin receptor (InsR) in insulin-induced TKT expression, a mouse strain (InsR flox / flox Alb cre ), InsR is specifically deleted in hepatocytes.

[0238] InsR flox / flox Alb cre Mice were purchased from The Jackson laboratory.

[0239] InsR flox / flox Alb cre Primary hepatocytes did not show insulin-stimulated fluctuations in TKT and phosphorylated C / EBPα (Figure 8F-H), suggesting that insulin promotes TKT expression through InsR. flox / flox Alb cr e mice, and found that HFD neither increased the hepatic TKT mRNA and protein levels (Fig. 8I, J) nor induced InsR - / - The livers of mice with PD-L1 induced significant lipid accumulation and fibrosis (Figure 2K-M).

[0240] Thus, insulin-InsR signaling drives TKT upregulation and lipid accumulation during NAFLD progression.

[0241] 2. TKT-deficient hepatocytes significantly increased the accumulation of R5P and inosine

[0242] From TKT flox / flox Alb cre and TKT + / + Alb cre Primary hepatocytes were isolated from mice and subjected to metabolomics analysis. PCA results showed significant differences between the two groups (Figure 9A).

[0243] Metabolic pathway enrichment revealed that TKT deficiency significantly altered the purine metabolic pathway and also altered the PPP (Figure 9B). TKT deficiency upregulated the levels of nucleosides such as inosine, adenosine, and deoxyadenosine, especially inosine (Figure 9C). It has been reported that inosine not only provides ribose as an alternative carbon source for central carbon metabolism through the PPP, but also promotes adipose tissue mitochondrial function as an extracellular signal through the cAMP-PKA-UCP1 pathway.

[0244] Next, we investigated how TKT deficiency regulates hepatic inosine metabolism. 13 C5] Inosine-induced TKT flox / flox Alb cre and TKT + / + Alb cre Mouse primary hepatocytes, in which only ribose contains 13 C, for metabolic flux analysis.

[0245] The results confirmed that [ 13C5] labeled inosine can be taken up by primary hepatocytes without being interfered with by TKT deficiency (Figure 9D inosine). TKT deficiency prevented carbon transfer between R5P, S7P, and F6P, resulting in an increase in the relative abundance of inosine-derived R5P (Figure 9F), while the relative abundance of F6P and S7P decreased (Figure 9F). The increase in R5P levels may inhibit the decomposition rate of PNPase, resulting in a decrease in the isotope labeling efficiency of R1P and R5P (Figure 9E). These results indicate that TKT deficiency in hepatocytes impairs the entry of inosine-derived pentose into central carbon metabolism by blocking the non-oxidative PPP.

[0246] In hepatocytes, glucose is metabolized by PPP to generate R5P, which is used for the de novo synthesis of IMP, which is then dephosphorylated to generate inosine. Next, we investigated how TKT deficiency affects the synthesis of glucose-derived inosine. 13 C2] Glucose treatment TKT flox / flox Alb cre and TKT + / + Alb cre Glucose metabolic flux analysis was performed in primary hepatocytes. Considering the two reactions catalyzed by TKT, TKT deficiency not only hindered carbon transfer between F6P and X5P, resulting in increased relative abundance and isotope labeling efficiency of F6P (Figures 9G and 9H), but also reduced the relative abundance and isotope labeling efficiency of S7P (Figure 9I). In addition, the major isotope-labeled form of R5P shifted from M+2 to M+1 (Figure 9I), suggesting that compensatory oxidative PPP may produce a higher abundance of R5P (Figure 9H). Overall, the accumulation of R5P in TKT-deficient hepatocytes provides more pentose for inosine synthesis, leading to elevated inosine levels (Figure 9C).

[0247] In summary, TKT deficiency in hepatocytes not only prevents inosine-derived R5P from entering glycolysis, but also promotes the synthesis of inosine from glucose-derived R5P, ultimately leading to increased intracellular inosine levels.

[0248] 3. TKT deficiency enhances mitochondrial function by promoting the inosine-PKA-CREB pathway

[0249] It has been reported that inosine promotes energy expenditure in adipose tissue via the PKA-CREB-UCP1 pathway.

[0250] Next, the inventors studied the effect of inosine in hepatocytes. By treating wild-type primary hepatocytes with inosine, they found that inosine activated the PKA-CREB signaling pathway but did not increase the expression of UCP1 protein (Figure 11A). Interestingly, TKT was observed to flox / flox Alb cre The activity of the PKA-CREB axis in hepatocytes was increased, which was consistent with the accumulation of inosine caused by TKT deficiency (Figure 10A).The inventors further investigated whether TKT deficiency affects the mitochondrial morphology and function of hepatocytes.

[0251] TKT-deficient hepatocytes had higher mitochondrial DNA content (Figure 11B), and higher mRNA and protein levels of mitochondrial genes such as ATP6, Mt-Co1 / 2 / 3, Mt-Nd1 / 2 / 3 / 4 / 5 / 6, Cytb, Ndufa12, and Tom20 (Figures 11C-E). Transmission electron microscopy of liver tissue showed that TKT deficiency increased mitochondrial density, diameter, and surface area (Figures 10B-F). TKT-deficient hepatocytes exhibited higher mitochondrial membrane potential, ATP production capacity, and mitochondrial ROS levels (Figures 10G-I). Seahorse assay results showed that TKT-deficient hepatocytes had enhanced oxygen consumption rate (OCR) (Figure 10J).

[0252] The inventors found that inosine treatment promoted mitochondrial activity in hepatocytes, and the effect was more pronounced in TKT-deficient hepatocytes. After adding inosine, the OCR level, ATP production capacity, and spare respiratory capacity of TKT-knockout hepatocytes were significantly increased (Figure 11G, H). Therefore, TKT deficiency promotes hepatocyte mitochondrial activity and sensitivity to inosine stimulation, improving liver lipid metabolism.

[0253] To determine whether inosine enhances hepatic mitochondrial function through the PKA-CREB pathway, hepatocytes were treated with the PKA inhibitor H89. As expected, H89 effectively reduced TKT flox / flox Alb cre and TKT + / + Alb cre Phosphorylated PKA substrate levels in primary hepatocytes ( FIG. 11F ). Importantly, inosine failed to upregulate mitochondrial function in H89-treated primary hepatocytes ( FIG. 11I ).

[0254] It is reported that hyperinsulinemia can interfere with mitochondrial function. The inventors found that insulin reduced intracellular inosine levels (Figure 10K) and inhibited mitochondrial activity in WT primary hepatocytes (Figure 10L). To investigate whether InsR and TKT play an important role in insulin-induced inosine reduction, we examined the effect of InsR on the inosine level. flox / floxAlb cre 、TKT flox / flox Alb cre Primary hepatocytes were isolated from InsR- or TKT-deficient mice and control mice, and it was found that insulin did not affect the level of inosine in InsR- or TKT-deficient hepatocytes (Figure 10M).

[0255] 4. TKT deficiency promotes hepatocyte mitochondrial function by increasing phosphatidylcholine (PC) synthesis

[0256] To investigate the regulatory role of TKT deficiency in the alterations of mitochondrial morphology and function in hepatocytes, the inventors compared TKT flox / flox Alb cre and TKT + / + Alb cre The levels of PGC1α, NRF2, and DRP1 in primary hepatocytes, which regulate mitochondrial fission and fusion, were not altered by TKT deficiency (Figure 14A).

[0257] Alterations in cellular lipid composition have been reported to affect mitochondrial morphology and function. Therefore, lipidomic analysis of human liver samples (n = 48) was performed to analyze candidate lipids that are altered by TKT deficiency and may affect mitochondrial function. Approximately 700 lipids from 10 categories were identified. Weighted gene co-expression network analysis (WGCNA) was used to partition all lipids into 15 modules (Figure 12A), and these modules were correlated with TKT protein levels. Three lipid modules were negatively correlated with TKT protein levels, with Pearson correlation coefficients > 0.8 and p-values ​​< 0.01 (Figure 12A).

[0258] Analysis of the core lipids of these three modules identified six lipids: PC, sphingomyelin (SM), n-acylethanolamine (NAE), ceramide (Cer), TG, and diacylglycerol (DG) (Figure 12B-D). PC is the most abundant phospholipid in the mitochondrial membrane and the most significantly downregulated lipid in NAFLD patients (Figure 12e).

[0259] In addition, TKT deficiency in mouse hepatocytes leads to upregulation of PC (Figure 12G). Intracellular PC synthesis is through the phosphatidylethanolamine (PE) methylation pathway or the CDP-choline pathway (Figure 12F), and PC is essential for mitochondrial function. The inventors' lipidomics results showed that mitochondrial PC levels, but not PE levels, were upregulated in TKT-deficient hepatocytes (Figure 14B), indicating that the PE methylation pathway that depends on PE supply was not disturbed. As the main pathway for intracellular PC synthesis, the CDP-choline pathway produces 60-70% of the total intracellular PC. In addition to PC, TKT flox / flox Alb cre The levels of CDP-choline pathway metabolites, including choline, phosphatidylcholine, and CDP-choline, were upregulated in primary hepatocytes ( Figure 12H ), indicating that TKT deficiency increased mitochondrial PC levels by promoting the CDP-choline pathway.

[0260] To investigate whether intracellular PC levels are crucial for mitochondrial function in mouse hepatocytes, the inventors reduced dietary choline intake by 80% to limit PC biosynthesis. After 3 weeks of low choline diet, liver choline, phosphatidylcholine, CDP-choline, and PC levels decreased compared to the normal diet group (Figure 14C-F). Importantly, low choline intake impaired hepatocyte mitochondrial membrane potential, ATP production capacity, mitochondrial ROS, and OCR levels (Figure 14G-J). In addition, the choline-restricted diet eliminated TKT. flox / flox Alb cre and TKT + / + Alb cre Differences in CDP-choline pathway metabolite levels ( FIG. 12I and FIG. 14K-M ) and mitochondrial activity ( FIG. 12J and FIG. 14N-P ) between primary hepatocytes.

[0261] These results indicate that hepatic TKT deficiency enhances mitochondrial activity and improves hepatic lipid metabolism by increasing intracellular PC synthesis.

[0262] 5. Inosine stimulates PC synthesis by activating the PKA-CREB-ChoKβ axis in hepatocytes

[0263] Next, we investigated whether and how inosine regulates PC synthesis in hepatocytes. Inosine upregulated the levels of CDP-choline pathway metabolites in WT primary hepatocytes (Figure 12K). Choline kinase is a key enzyme in the CDP-choline pathway that catalyzes the first step of the CDP-choline pathway. Compared with the control group, TKT flox / flox Alb cre ChoKβ mRNA and protein levels were elevated in primary hepatocytes ( FIG. 12L , M ).

[0264] Using PROMO to predict potential transcription factors for ChoKβ, we found that CREB may regulate the transcriptional activity of ChoKβ. Importantly, the PKA inhibitor H89 abolished the inosine-induced CDP-choline pathway activity (Figure 12N).

[0265] These data indicate that in TKT-deficient mouse hepatocytes, inosine accumulation promotes intracellular PC synthesis and mitochondrial function by activating the PKA-CREB-ChoKβ pathway, thereby improving hepatic lipid metabolism.

[0266] In summary, a high-fat diet can induce hyperinsulinemia, promote TKT expression through the insulin receptor (InsR)-transcription factor CCAAT / enhancer binding protein α (C / EBPα) pathway, and reduce intracellular inosine levels in hepatocytes. TKT deficiency, on the other hand, promotes hepatic inosine levels. Inosine accumulation promotes intracellular PC synthesis and mitochondrial function by activating the PKA-CREB-ChoKβ pathway (Figure 13).

[0267] Example 5. Development and function of GalNAc-siRNA targeting liver TKT

[0268] 1. Development of GalNAc-siRNA and its regulation of TKT in hepatocytes

[0269] Targeted knockout approaches require altering the genome and processing at the pre-embryonic stage, making them impractical for clinical treatment. To explore the possibility of targeting liver TKT for the treatment of NAFLD, the inventors conducted screening and research analyses, resulting in the development of two siRNA reagents.

[0270] siTKT10 sequence:

[0271] sense: CCGUGGACAUUGCUAACAUTT (SEQ ID NO: 49);

[0272] antisense: AUGUUAGCAAUGUCCACGTT (SEQ ID NO: 51).

[0273] siTKT16 sequence:

[0274] sense: GCUGCAGAGAGUCUAAAGATT (SEQ ID NO: 50);

[0275] antisense: UCUUUAGACUCUCUGCAGCTT (SEQ ID NO: 52).

[0276] siNC sequence: irrelevant sequence.

[0277] The 3' end of the siRNA sense chain is covalently bound to N-acetylgalactosamine (GalNAc), and the preparation method adopts a CPG carrier solid-state binding synthesis method.

[0278] The GalNAc group is specifically recognized by the ASGPR receptor on the hepatocyte membrane, allowing the siRNA duplex to be endocytosed. Therefore, the covalently linked product enables targeted delivery of siRNA to hepatocytes. Two siTKT GalNAc conjugates, GalNAc siTKT10 and GalNAc siTKT16, were obtained, along with a GalNAc siNC control.

[0279] Two GalNAc conjugates of siTKT effectively knocked down TKT in mouse primary hepatocytes without lipid nanoparticle coating ( Figure 6A,B ).

[0280] The inventors conducted in vivo experiments in mice using GalNAc-siTKT16. Subcutaneous injection of GalNAc-siTKT16 reduced TKT protein levels in the liver but had no effect on other tissues (Figure 6C-H).

[0281] The structure of GalNAc-siTKT16 is as follows:

[0282] AS (antisense): UmCfUmUmUmAfGmAmCmUmCmUmCmUfGmCfAmGmCmAmGmCmCm;

[0283] SS(sense): CmUmGmCmUmGmCfAmGfAfGfAmGmUmCmUmAmAmAmGmAm-L96.

[0284] Therefore, GalNAc-siRNA targeting liver TKT has a significant downregulation effect on TKT and an ideal specific regulatory effect, thereby having the ability to effectively improve NAFL and NASH.

[0285] 2. Effect of GalNAc-siTKT on NAFL

[0286] To investigate the therapeutic effect of GalNAc-siTKT on NAFL, mice were fed a HFD for 90 days and then subcutaneously injected with GalNAc-siTKT on days 3, 7, 14, and 21 (Figure 5A). The mice were analyzed on day 30.

[0287] The results showed that GalNAc-siTKT significantly reduced TKT levels in the liver ( Figure 5B ), reduced hepatic lipid accumulation ( Figure 5C ), and decreased serum and hepatic triglyceride and total cholesterol levels ( Figure 5D–G ), as well as serum ALT and AST levels ( Figure 5H, I ).

[0288] 3. The therapeutic effect of GalNAc-siTKT on NASH

[0289] To further investigate the preventive potential of GalNAc-siTKT against MCD-induced NASH in mice, GalNAc-siTKT (10 mg / kg) was injected subcutaneously once at the onset of MCD and then once on days 3, 7, 14, and 21 ( Figure 5J ).

[0290] On day 30, GalNAc-siTKT was found to reduce TKT levels in the liver (Figure 5K), alleviate hepatic steatosis, and alleviate liver fibrosis (Figure 5L). GalNAc-siTKT intervention also reduced serum and liver triglyceride and total cholesterol levels (Figures 5M-P), as well as serum ALT and AST levels (Figures 5Q, R).

[0291] Furthermore, GalNAc-siTKT treatment reduced the levels of inflammation- and fibrosis-related genes but did not affect fatty acid synthesis genes ( Figure 5S–U ).

[0292] In conclusion, GalNAc-siTKT specifically targets TKT in hepatocytes and can significantly improve NAFL and NASH, providing a therapeutic strategy for the treatment of human NAFLD.

[0293] Example 6: Screening of human, mouse, and monkey homologous sequences and analysis of their effects

[0294] To identify siTKT sequences with optimal knockdown efficacy, the inventors conducted a comprehensive sequence screening and experimental analysis of the human, mouse, and monkey homologous regions of the TKT gene. They also considered other gene sequences throughout the genome to eliminate nonspecific knockdown. From this large pool of candidate siRNAs, they initially screened approximately 48 siRNAs (Table 1).

[0295] Table 1

[0296] By comparing the mRNA and protein knockdown effects in mouse primary hepatocytes and human LO2 cell lines, sequences 773 and 1115 were selected to have the best knockdown effects ( Figure 7A ).

[0297] GalNAc-modified sequences at 773 (SEQ ID NO: 25) and 1115 (SEQ ID NO: 37) to generate GalNAc-siTKT 773 and GalNAc-siTKT 1115 for in vivo mouse experiments. GalNAc-siTKT 773 and GalNAc-siTKT 1115 were dissolved in PBS and injected subcutaneously into the back of mice at a dose of 10 mg / kg.

[0298] After the mice were fed a normal diet for 7 days, their liver tissues were obtained by euthanasia to detect the expression level of TKT. It was found that the in vivo knockdown effect of sequence 1115 was better than that of sequence 773 (Figure 7B and C).

[0299] Subsequently, in vivo pharmacokinetic studies were conducted on 1115 in mice. The results showed that after subcutaneous injection of 5 mg / kg and 10 mg / kg of GalNAc-siTKT, the peak plasma concentration of the drug in mice was around 60 minutes, and the half-life of the drug in vivo was approximately 4 hours (Figure 7D).

[0300] Therefore, GalNAc-siTKT 773 and GalNAc-si1115 with human, mouse and monkey homologous sequences showed excellent knockdown effects in vivo and in vitro.

[0301] Example 7. Further development of siTKT sequences with knockdown effects

[0302] 1.siTKT

[0303] Based on an in-depth analysis of the TKT sequence, the inventors expanded the scale of sequence preparation and obtained a series of siRNAs, as shown in Table 2.

[0304] Table 2. Unmodified siRNA

[0305] According to the sense and antisense strands of siRNA in Table 2, the inventors prepared modified sense and antisense strands using the aforementioned method, as shown in FIG15 .

[0306] The modifications for each nucleoside are described as follows:

[0307] The letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside ( FIG. 16 );

[0308] The letter f indicates that the nucleoside adjacent to the left side of the letter f is a 2'-fluoro-modified nucleoside ( FIG. 16 );

[0309] The letter s indicates that the connection between the two nucleosides adjacent to the letter s or the connected nucleosides and the GalNAc is a phosphorothioate diester connection.

[0310] 2. Inhibitory effect of modified siTKT on target gene TKT

[0311] 24 hours before transfection, Hep3B cells were seeded at approximately 20,000 cells / well in a 96-well plate with 100 μL of culture medium per well. Transfection was performed using Lipofectamine RNAiMAX (Invitrogen, 13778150) according to the product instructions, with a single transfection concentration of 2.5 nM. After 24 hours of treatment, total RNA was extracted using a high-throughput cellular RNA extraction kit (FG0417-L / FG0418-XL, magnetic bead method). Human HQP mRNA levels were measured by RNA reverse transcription (ThermoFisher, AK16225) and real-time quantitative PCR (ThermoFisher, A25778). Human HQP mRNA levels were corrected for HPRT mRNA levels.

[0312] HEP3B cells were used to evaluate the inhibition of target gene TKT mRNA expression by modified sequences at a single concentration. As shown in Table 3, sequences with significantly better knockdown of TKT mRNA were screened based on the sequence characteristics of the target TKT mRNA. At a concentration of 2.5 nM, multiple sequences were able to reduce the target gene TKT mRNA expression to below 0.1, with the lowest being reduced to 0.03.

[0313] Combined with the inhibition efficiency of the single-point concentration of the sequences in Table 3, several sequences were selected for multi-point concentration evaluation of the inhibitory effect on the expression of the target gene TKTmRNA. 50 The value corresponds to the concentration of siRNA used when the remaining percentage of TKT mRNA expression is 50%, and the maximum inhibition ratio corresponds to the maximum value of the amount of TKT mRNA expression that can be inhibited by multiple concentrations.

[0314] Table 3

[0315] 24 hours before transfection, Hep3B cells were seeded at approximately 20,000 cells / well in a 96-well plate with 100 μL of culture medium per well. Transfections were performed using Lipofectamine RNAiMAX (Invitrogen, 13778150) according to the product instructions. The final concentrations for transfection were 5, 1, 0.2, 0.04, 0.008, 0.0016, and 0.00032 nM. After 24 hours of treatment, total RNA was extracted using a high-throughput cellular RNA extraction kit (FG0417-L / FG0418-XL, magnetic bead method). Human HQP mRNA levels were measured by RNA reverse transcription (ThermoFisher, AK16225) and real-time quantitative PCR (ThermoFisher, A25778). Human HQP mRNA levels were corrected for HPRT mRNA levels.

[0316] The IC values ​​of different sequences were calculated by nonlinear fitting based on the TKT mRNA residual values ​​corresponding to the above siRNA treatments at different concentrations. 50 value and maximum inhibition ratio, among which IC 50 The corresponding siRNA concentration is when the remaining percentage of TKT mRNA expression is 50%, and the maximum inhibition ratio corresponds to the maximum value of the multiple concentrations that can inhibit TKT mRNA expression. 50 The results of multi-concentration screening of the modified sequences to inhibit the expression of the target gene TKT mRNA are shown in Table 4.

[0317] The results of multi-concentration screening of modified sequences in HEP3B cells to inhibit the expression of target gene TKT mRNA are shown in Table 4.

[0318] Table 4

[0319] Based on the above, the inventors further found sequence regions suitable for designing siTKT with advantages, as well as advantageous inhibition analysis, including:

[0320] The double-stranded sequences are numbered PPP24074 to PPP24087, with PPP24078 being relatively more preferred; and their corresponding modified sequences and coupled molecules.

[0321] The double-stranded sequences are numbered PPP24002 to PPP24014, among which PPP24009 and PPP24011 are relatively more preferred; and their corresponding modified sequences and coupled molecules.

[0322] The double-stranded sequences are numbered PPP24032-PPP24039, with PPP24036 being relatively more preferred; and their corresponding modified sequences and coupled molecules.

[0323] The double-stranded sequences are numbered PPP24020-PPP24026, among which PPP24021, PPP24022, and PPP24026 are relatively more preferred; and their corresponding modified sequences and coupled molecules.

[0324] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Use of a down-regulator of transketolase (TKT) or insulin receptor (InsR)-CCAAT enhancer binding protein α (C / EBPα)-TKT signaling pathway containing TKT for preparing a pharmaceutical composition for alleviating or treating metabolic-related fatty liver disease.

2. The use according to claim 1, characterized in that The down-regulator includes a group consisting of: a TKT down-regulator, a C / EBPα-TKT interaction down-regulator, and an InsR-C / EBPɑ interaction down-regulator; Preferably, the TKT down-regulator includes: an agent for silencing, knocking down or knocking out the TKT gene, an agent for inhibiting the activity of the TKT protein; more preferably, it includes: an interfering molecule that specifically interferes with the expression of the TKT gene, a CRISPR gene editing agent, a homologous recombination agent or a site-directed mutagenesis agent for the TKT gene, wherein the agent causes TKT to undergo a loss-of-function mutation; Preferably, the down-regulator of the C / EBPα-TKT interaction includes: an agent that weakens the binding of C / EBPα to the TKT promoter, an agent that silences, knocks down or knocks out the C / EBPɑ gene, and an agent that inhibits the activity of the C / EBPα protein; more preferably, it includes: an agent that reduces the phosphorylation level of the C / EBPα protein, an interfering molecule that specifically interferes with the expression of the C / EBPα gene, a CRISPR gene editing agent, a homologous recombination agent or a site-directed mutagenesis agent for the C / EBPα gene, and the agent causes a loss-of-function mutation in C / EBPα; Preferably, the downregulator of the InsR-C / EBPα interaction includes: an agent for silencing, knocking down or knocking out the InsR gene, an agent for inhibiting the activity of the InsR protein, and an agent for weakening the binding of insulin to InsR; more preferably, it includes: an interfering molecule that specifically interferes with the expression of the InsR gene, a CRISPR gene editing agent, a homologous recombination agent or a site-directed mutagenesis agent for the InsR gene, wherein the agent causes a loss-of-function mutation in InsR.

3. The use according to claim 2, characterized in that The down-regulator is an interfering agent that silences the TKT gene; preferably, the interfering agent is siRNA; preferably, the siRNA is an siRNA with a nucleotide sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 50, SEQ ID NO: 53 to SEQ ID NO: 167; more preferably, the siRNA is an siRNA with a nucleotide sequence as shown in any one of SEQ ID NO: 37, SEQ ID NO: 25, SEQ ID NO: 50, SEQ ID NO: 49, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 88 or SEQ ID NO: 130; or The down-regulator is a homologous recombination agent that knocks down or knocks out the TKT gene, which causes the gene coding to be terminated; or The down-regulator is a homologous recombination agent that knocks down or knocks out the InsR gene, which causes the gene coding to be terminated.

4. The use according to claim 2, characterized in that The siRNA forms an siRNA preparation for targeted delivery to hepatocytes; preferably, the siRNA is connected to a targeting ligand that binds to the asialoglycoprotein receptor; the targeting ligand includes: N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine.

5. The use according to claim 1, characterized in that The metabolic-related fatty liver disease includes: non-alcoholic fatty liver disease, non-alcoholic steatohepatitis; or The TKT or TKT-containing InsR-C / EBPα-TKT signaling pathway downregulator increases the level of inosine, enhances mitochondrial function, thereby improving liver lipid metabolism, alleviating or treating metabolism-related fatty liver disease; preferably, the downregulator: prevents R5P derived from inosine from entering glycolysis, promotes the synthesis of inosine from R5P derived from glucose, increases the level of inosine in cells, enhances mitochondrial function, improves liver lipid metabolism, alleviating or treating metabolism-related fatty liver disease; preferably, the increase in phosphatidylcholine synthesis is mediated through the CDP-choline pathway.

6. An siRNA or siRNA preparation for alleviating or treating metabolic-related fatty liver disease, wherein the siRNA comprises an siRNA having a nucleotide sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 50, SEQ ID NO: 53 to SEQ ID NO: 167; preferably, the siRNA is an siRNA having a nucleotide sequence as shown in any one of SEQ ID NO: 37, SEQ ID NO: 25, SEQ ID NO: 50, SEQ ID NO: 49, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 88 or SEQ ID NO:

130.

7. The siRNA or siRNA preparation according to claim 6, characterized in that The siRNA preparation is a stable siRNA preparation for targeted delivery to hepatocytes; preferably, the siRNA is connected to a targeting ligand that binds to an asialoglycoprotein receptor; the targeting ligand includes: N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine.

8. The siRNA or siRNA preparation according to claim 6 or 7, characterized in that In the siRNA, the nucleosides in the sense strand and the antisense strand are modified by a modification selected from the following group: 2'-methoxy modification, 2'-fluoro modification; or, phosphorothioate diester linkages are included between adjacent nucleosides or between nucleosides and targeting ligands.

9. The siRNA or siRNA preparation according to claim 8, characterized in that In the sense strand of the siRNA, the first and second nucleosides from the 5' end and the second and third nucleosides are connected by phosphorothioate diester groups; in the corresponding antisense strand of the siRNA, the first and second nucleosides from the 5' end, the first and second to last nucleosides from the 3' end, and the second and third nucleosides are connected by phosphorothioate diester groups; Preferably, in the sense strand of the siRNA, the nucleosides at positions 1, 2, 3, 4, 6, 10-19 from the 5' end are 2'-methoxy modified; in the corresponding antisense strand of the siRNA, the nucleosides at positions 1, 3, 4, 5, 7, 10-13, 15, 17-21 from the 5' end are 2'-methoxy modified; Preferably, in the sense strand of the siRNA, the nucleosides at positions 5, 7, 8, and 9 from the 5' end are 2'-fluorinated, and in the corresponding antisense strand of the siRNA, the nucleosides at positions 2, 6, 8, 9, 14, and 16 are 2'-fluorinated.

10. A pharmaceutical composition or a medicine kit for alleviating or treating metabolic-related fatty liver disease, comprising the siRNA or siRNA preparation for alleviating or treating metabolic-related fatty liver disease according to any one of claims 6 to 8, and a pharmaceutically acceptable carrier or excipient.

11. Use of the InsR-C / EBPα-TKT signaling pathway for screening substances that alleviate or treat metabolic-related fatty liver disease.

12. A method for screening a substance for alleviating or treating metabolism-related fatty liver disease, comprising: (1) contacting the candidate substance with a system containing the InsR-C / EBPα-TKT signaling pathway; (2) Screening out substances that downregulate the InsR-C / EBPα-TKT signaling pathway, wherein the substances are useful for alleviating or treating metabolic-related fatty liver disease; wherein the downregulation includes: downregulating TKT, downregulating C / EBPα-TKT interaction, and downregulating insulin / InsR-C / EBPα interaction.

13. The method according to claim 12, characterized in that Step (1) comprises: adding a candidate substance to a system containing the InsR-C / EBPɑ-TKT signaling pathway; Step (2) comprises: detecting changes in each protein or its encoding gene in the InsR-C / EBPα-TKT signaling pathway, and comparing with a control group, wherein the control group is a system containing the InsR-C / EBPα-TKT signaling pathway without adding the candidate substance; if the candidate substance downregulates TKT, downregulates C / EBPα-TKT interaction, or downregulates insulin / InsR-C / EBPα interaction, then the candidate substance is a substance useful for alleviating or treating metabolic-related fatty liver disease.

14. Use of liver TKT protein or its encoding gene in the preparation of a diagnostic reagent for diagnosing or prognosing metabolic-related fatty liver disease; preferably, the diagnosis or prognosis comprises: Based on the expression of liver TKT protein, the occurrence or progression of metabolic-related fatty liver disease can be judged, or whether the patient is suitable for a treatment plan using "downregulators of TKT or TKT-containing InsR-C / EBPα-TKT signaling pathway" can be judged; if TKT protein is highly expressed, this treatment plan is applicable.

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