New target for treating metabolism-related fatty liver disease, and regulation molecule and application thereof

Abstract

The invention provides a new target for treating metabolism-related fatty liver diseases, a regulation molecule and application thereof. The invention discloses a novel target gene closely related to relieving or treating the metabolism-related fatty liver disease and a signal channel related to upstream and downstream regulation and control of the target gene for the first time. The invention discloses a new mechanism participated by the signal channel and used for regulating and controlling the metabolism-related fatty liver disease. Preferred remission / therapeutic agents are also disclosed.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. More specifically, the present invention relates to a new target for treating metabolic associated fatty liver disease, its regulatory molecules and applications. Background Art

[0002] Liver diseases can lead to alcohol-associated liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), etc. These liver diseases can further induce liver tumors.

[0003] Fatty liver refers to a pathological condition in which there is excessive fat accumulation in liver cells due to various reasons. Fatty liver is generally more common in patients with lipid metabolism disorders in liver cells caused by various reasons, presenting symptoms such as abdominal distension in the upper right abdomen, loss of appetite, general weakness, listlessness, nausea, etc. In severe cases, patients may even develop jaundice.

[0004] Non-alcoholic fatty liver disease (NAFLD) is mainly characterized by hepatic steatosis and is the most common cause of chronic liver diseases. It can cause many chronic liver diseases such as liver fibrosis, cirrhosis and liver failure, and is also a risk factor for primary liver cancer. As a type of NAFLD, liver tumors caused by non-alcoholic steatohepatitis (NASH) are on the rise.

[0005] There are qualitative differences between non-alcoholic fatty liver disease and 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 respond to the body's lipid balance and carry out de novo synthesis and decomposition of lipids. The liver synthesizes excessive free fatty acids into triglycerides and then transfers them to other tissues such as adipose tissue for storage. The disorder of the liver's lipid metabolism function is an important inducement for the occurrence of metabolic diseases in the body (such as obesity, diabetes, hyperlipidemia, etc.).

[0006] In the past few decades, changes in lifestyle and eating habits have promoted the prevalence of obesity and NAFLD. Currently, the growth rate of NAFLD patients is astonishing. As NAFLD and other liver diseases become major health problems, there is an urgent need for more in-depth research and analysis in this field to explore effective drug intervention methods. Revealing the mechanisms of the occurrence and development of liver diseases such as NAFLD and discovering potential therapeutic targets are the top priorities. Summary of the Invention

[0007] The object of the present invention is to provide a new target for treating metabolic associated fatty liver disease (MAFLD), its regulatory molecule and application.

[0008] In the first aspect of the present invention, there is provided the use of TKT or a down-regulator of the (insulin-) InsR-C / EBPα-TKT signaling pathway containing TKT for preparing a pharmaceutical composition for relieving or treating metabolic associated fatty liver disease.

[0009] In another preferred example, the InsR-C / EBPα-TKT signaling pathway includes: the TKT gene / protein, the C / EBPα gene / protein, the InsR gene / protein; preferably also includes their upstream and downstream regulatory genes / proteins or chemical molecules.

[0010] In another preferred example, the down-regulator includes those selected from: a TKT down-regulator, a down-regulator of the C / EBPα-TKT interaction (mutual regulatory effect; preferably including the transcriptional regulation of TKT by C / EBP), and a down-regulator of the InsR-C / EBPα interaction (mutual regulatory effect).

[0011] In another preferred example, the TKT down-regulator includes (but is not limited to): a reagent for silencing, knocking down or knocking out the TKT gene, a reagent for inhibiting the activity of the TKT protein; more preferably, it includes: an interfering molecule (such as siRNA, shRNA, miRNA, antisense nucleotide, etc.) specifically interfering with the expression of the TKT gene, a CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent for the TKT gene, and the reagent causes a loss-of-function mutation of TKT.

[0012] In another preferred example, the down-regulator of the C / EBPα-TKT interaction includes (but is not limited to): a reagent for weakening the binding of C / EBPα to the TKT promoter, a reagent for silencing, knocking down or knocking out the C / EBPα gene, a reagent for inhibiting the activity of the C / EBPα protein; more preferably, it includes: a reagent for reducing the phosphorylation level of the C / EBPα protein, an interfering molecule (such as siRNA, shRNA, miRNA, antisense nucleotide, etc.) specifically interfering with the expression of the C / EBPα gene, a CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent for the C / EBPα gene, and the reagent causes a loss-of-function mutation of C / EBPα.

[0013] In another preferred example, the downregulators of the InsR-C / EBPα interaction include (but are not limited to): reagents for silencing, knocking down or knocking out the InsR gene, reagents for inhibiting the activity of the InsR protein, and reagents for weakening 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 for the InsR gene, and the reagents cause loss-of-function mutations of InsR.

[0014] In another preferred example, the mutual regulation includes regulation at the transcriptional level.

[0015] In another preferred example, the downregulator is an interfering reagent for silencing the TKT gene; preferably, the interfering reagent is siRNA; preferably, the siRNA is an siRNA with a nucleotide sequence as shown in SEQ ID NO:1 to SEQ ID NO:50; more preferably, the siRNA is an siRNA with a nucleotide sequence as shown in SEQ ID NO:37 (No. 1115), SEQ ID NO:25 (No. 773), SEQ ID NO:50 (siTKT16) or SEQ ID NO:49 (siTKT10).

[0016] In another preferred example, the downregulator is a homologous recombination reagent for knocking down or knocking out the TKT gene, which terminates the gene encoding.

[0017] In another preferred example, the downregulator is a homologous recombination reagent for knocking down or knocking out the InsR gene, which terminates the gene encoding.

[0018] In another preferred example, the siRNA forms an siRNA preparation for hepatocyte-targeted delivery; preferably, the siRNA is covalently bound to N-acetylgalactosamine (GalNAc) to form a conjugate.

[0019] In another preferred example, the metabolic associated fatty liver disease includes: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH).

[0020] In another preferred embodiment, the downregulator of the TKT or the (insulin-)InsR-C / EBPα-TKT signaling pathway increases the inosine level, enhances mitochondrial function, thereby improving hepatic lipid metabolism and alleviating or treating metabolic associated fatty liver disease; preferably, the downregulator: blocks the entry of R5P derived from inosine into glycolysis, promotes the synthesis of inosine from R5P derived from glucose, increases the intracellular inosine level, thereby promoting the inosine-PKA-CREB pathway, enhancing mitochondrial function, improving hepatic lipid metabolism and alleviating or treating metabolic associated fatty liver disease; preferably, after promoting the inosine-PKA-CREB pathway, it further includes: activating the PKA-CREB-ChoKβ axis to increase the synthesis of phosphatidylcholine (PC), thereby enhancing mitochondrial function; preferably, the increase in the synthesis of phosphatidylcholine (PC) is mediated by the CDP-choline pathway.

[0021] In another preferred embodiment, the metabolic associated fatty liver disease is metabolic associated fatty liver disease with high expression of TKT in the liver (cells).

[0022] In another preferred embodiment, the "high expression" is "high expression" in a statistical sense. For example, compared with the average expression of TKT in healthy people (or a statistically sufficient number of people), the expression of TKT in patients with "metabolic associated fatty liver disease with high expression of TKT" is significantly higher by 10% or more, preferably 30% or more, more preferably 80% or more, or 100% or more.

[0023] In another aspect of the present invention, there is provided an siRNA or an siRNA preparation for alleviating or treating metabolic associated fatty liver disease, wherein the siRNA includes siRNAs having nucleotide sequences as shown in SEQ ID NOs: 1 to 50; preferably, the siRNA is an siRNA having a nucleotide sequence as shown in SEQ ID NO: 37 (No. 1115), SEQ ID NO: 25 (No. 773), SEQ ID NO: 50 (siTKT16) or SEQ ID NO: 49 (siTKT10); the siRNA preparation is a stable siRNA preparation for hepatocyte-targeted delivery; preferably, the siRNA is covalently conjugated with N-acetylgalactosamine (GalNAc) after being fluorinated and methoxylated modification.

[0024] In another aspect of the present invention, there is provided a pharmaceutical composition or a kit for alleviating or treating metabolic associated fatty liver disease, which includes the siRNA or the siRNA preparation for alleviating or treating metabolic associated fatty liver disease, and a pharmaceutically acceptable carrier or excipient.

[0025] In another aspect of the present invention, there is provided the use of the InsR-C / EBPα-TKT signaling pathway for screening substances for alleviating or treating metabolic associated fatty liver disease.

[0026] In another preferred embodiment, the InsR-C / EBPα-TKT signaling pathway is the signaling pathway in hepatocytes.

[0027] In another aspect of the present invention, there is provided a method for screening substances for alleviating or treating metabolic associated fatty liver disease, comprising: (1) contacting a candidate substance with a system containing the InsR-C / EBPα-TKT signaling pathway; (2) screening for substances that down-regulate the InsR-C / EBPα-TKT signaling pathway, and such substances are substances (including potential substances) useful for alleviating or treating metabolic associated fatty liver disease; wherein the down-regulation includes: down-regulating TKT, down-regulating the C / EBPα-TKT interaction, and down-regulating the (insulin / )InsR-C / EBPα interaction.

[0028] In another preferred embodiment, step (1) includes: adding a candidate substance to a system containing the InsR-C / EBPα-TKT signaling pathway.

[0029] In another preferred embodiment, step (2) includes: 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 down-regulates TKT, down-regulates the C / EBPα-TKT interaction or down-regulates the (insulin / )InsR-C / EBPα interaction, then the candidate substance is a substance useful for alleviating or treating metabolic associated fatty liver disease.

[0030] 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.

[0031] In another preferred embodiment, the down-regulation can also be referred to as inhibition, which is a statistical inhibition or down-regulation, such as a 10% or more, preferably 40% or more, more preferably 80% or more or 100% or more inhibition or down-regulation compared to a control or a basal level.

[0032] In another preferred example, the candidate substances include (but are not limited to): regulatory molecules (such as but not limited to upregulators, interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands)) designed against the InsR-C / EBPα-TKT signaling pathway, or its pathway proteins, or its upstream or downstream proteins or genes, CRISPR constructs, small molecule compounds, compounds from compound libraries.

[0033] 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 for diagnosing or prognosticating metabolic associated fatty liver disease; preferably, the diagnosis or prognosis includes: judging the occurrence or progression of metabolic associated fatty liver disease according to the expression level of liver TKT protein, or judging whether it is suitable for a treatment regimen using a "downregulator of TKT or the (insulin / )InsR-C / EBPα-TKT signaling pathway containing TKT"; if the TKT protein is highly expressed, then this treatment regimen is applicable.

[0034] In another aspect of the present invention, there is provided the use of a reagent that specifically recognizes TKT protein or its encoding gene for preparing a diagnostic reagent or diagnostic kit for diagnosing or prognosticating metabolic associated fatty liver disease; preferably, the diagnosis or prognosis includes: judging the occurrence or progression of metabolic associated fatty liver disease according to the expression level of TKT protein, or judging whether it is suitable for a treatment regimen using a "downregulator of TKT or the (insulin / )InsR-C / EBPα-TKT signaling pathway containing TKT"; if the TKT protein is highly expressed, then this treatment regimen is applicable.

[0035] In another preferred example, the diagnostic reagent includes those selected from: primers that specifically amplify the encoding gene of TKT protein; probes that specifically recognize the encoding gene of TKT protein or its transcript; or antibodies that specifically recognize TKT protein.

[0036] In another aspect of the present invention, there is provided a kit for diagnosing or prognosticating metabolic associated fatty liver disease, which contains a diagnostic reagent for detecting the expression level or expression amount of TKT protein or its encoding gene.

[0037] In another preferred example, the kit further includes: nucleic acid extraction reagents, polymerase chain reaction reagents, protein immunoblotting reagents, and / or enzyme-linked immunosorbent assay reagents.

[0038] Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Analysis of the expression characteristics of TKT in liver tissues.

[0040] (A) Enrichment map of differential metabolic pathways in healthy individuals and NAFLD patients.

[0041] (B) Volcano plot of changes in liver protein levels in healthy individuals and NAFLD patients.

[0042] (C-E) Expression levels of non-oxidative pentose phosphate pathway (PPP) metabolic enzymes (C), TKT protein (D), and mRNA (E) in the livers of healthy individuals and NAFLD patients (n = 4 in C and D, n = 8 in E).

[0043] (F) H&E, Oil Red O, and TKT immunohistochemical staining in the livers of healthy individuals and NAFLD patients (n = 4).

[0044] (G-I) Expression levels of non-oxidative pentose phosphate pathway (PPP) metabolic enzymes (G), protein quantification of TKT (H), and mRNA (I) levels in the livers of wild-type mice fed a normal chow diet (NCD) or a high-fat diet (HFD) for 90 days (n = 5 in NCD, n = 6 in HFD).

[0045] (J) H&E, Oil Red O, and TKT immunohistochemical staining of liver tissues in wild-type mice fed a normal chow diet (NCD) or a high-fat diet (HFD) for 90 days (n = 5 in NCD, n = 6 in HFD).

[0046] (K) Expression level of liver TKT protein 10 weeks after intravenous injection of AAV-Flag-TKT adenovirus into wild-type mice (n = 4).

[0047] (L) Oil Red O staining of liver tissues 10 weeks after intravenous injection of AAV-Flag-TKT adenovirus into wild-type mice and feeding them a high-fat diet (n = 4).

[0048] (M) Serum and liver TG levels 10 weeks after intravenous injection of AAV-Flag-TKT adenovirus into wild-type mice and feeding them a high-fat diet (n = 4).

[0049] For all data: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. Data are presented as mean ± SEM. A two-tailed t-test was applied in D, E, H, I, and M.

[0050] Figure 2 Supplementary analysis of the expression characteristics of TKT in liver tissues.

[0051] (A) Clinical sample information corresponding to the liver tissue used for experiments. HS (hepatic steatosis): Hepatic Steatosis.

[0052] (B) Fluorescence images of the liver in wild-type mice injected with AAV-Flag-TKT adenovirus via the tail vein for 10 weeks (n = 3).

[0053] (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).

[0054] (E and F) H&E, Oil Red O and Sirius Red staining of liver tissue, serum and liver TG and TC levels, serum ALT and AST levels in wild-type mice injected with AAV-Flag-TKT adenovirus via the tail vein and fed a MCD diet for 4 weeks (n = 4).

[0055] For all data: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. Data are presented as mean ± SEM. A two-tailed t-test was applied in D and F.

[0056] Figure 3 The effect of hepatocyte-specific TKT knockout on disease progression.

[0057] (A-I) Phenotypic analysis of wild-type and liver-specific TKT-deficient mice after 90 days of feeding a high-fat diet, including (A) nuclear magnetic resonance imaging of liver lipid content, (B) liver appearance, (C) H&E and Oil Red O staining, (D) serum TG level, (E) serum TC level, (F) serum ALT level, (G) serum AST level, (H) liver TG and (I) liver TC levels (n = 5 - 8).

[0058] (J-R) Phenotypic analysis of wild-type and liver-specific TKT-deficient mice after 4 weeks of feeding a MCD diet, including (J) H&E staining, (K) Oil Red O staining, (L) mRNA levels of lipidogenesis-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 level of TGFβ (n = 3 - 4).

[0059] (S) Mouse construction strategy for liver-specific knockout of the TKT gene.

[0060] For all data: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. Data are presented as mean ± SEM. A two-tailed t-test was applied in D, E, F, G, H, I, L, O, R.

[0061] Figure 4 The effect of regulating TKT in the liver on disease progression.

[0062] (A-D) Changes in body weight (A), body shape images (B), body weight (C), and liver weight (D) of wild-type and liver-specific TKT-deficient mice after 90 days of high-fat diet feeding (n = 4).

[0063] (E and F) Liver pathological features of wild-type and liver-specific TKT-deficient mice after 28 days of MCD diet feeding, including Sirius red staining (E) and TUNEL staining (F) (n = 4).

[0064] (G-N) Phenotypic analysis of TKT fl / fl Alb cre Phenotypic analysis of Alb mice after 10 weeks of high-fat diet feeding following 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, n = 4 in the AAV-Flag-TKT group).

[0065] (O-U) TKT fl / fl Alb cre Phenotypic analysis of Alb mice after 3 weeks of MCD diet feeding following 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).

[0066] For all data: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. Data are presented as mean ± SEM. A two-tailed t-test was applied in C, D, I, J, K, L, M, N, P, Q, R, S, T, U.

[0067] Figure 5 GalNAc-siRNA targeting liver TKT and its effects.

[0068] (A) Injection strategy of GalNAc-siTKT for the treatment of NAFL. Wild-type male mice at 8 weeks of age were fed a high-fat diet for 90 days and then injected subcutaneously in the back with GalNAc-siTKT at a dose of 10 mg / kg. Five repeated injections were given at 90+0, 3, 7, 14, and 21 days respectively, and the analysis was performed on the 120th day.

[0069] (B) Expression level of TKT protein in the liver of NAFL mice after GalNAc-siTKT treatment (n = 5).

[0070] (C-I) 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).

[0071] (J) Injection strategy of GalNAc-siTKT for the treatment of NASH. Wild-type male mice at 8 weeks of age were injected subcutaneously in the back with GalNAc-siTKT at a dose of 10 mg / kg at the start of the MCD diet. Five repeated injections were given at 0, 3, 7, 14, and 21 days respectively, and the phenotypic analysis was performed on the 30th day.

[0072] (K) Expression level of TKT protein in the liver of NASH mice after GalNAc-siTKT intervention (n = 2 in NC, n = 4 in siTKT).

[0073] (L-U) Phenotypic analysis of NASH mice after GalNAc-siTKT intervention, including (L) H&E, Oil Red O, Sirius red staining, and (M) serum TG, (N) liver TG, (O) serum TC, (P) liver TC, (Q) serum ALT, and (R) AST levels, (S) mRNA levels of lipidogenesis-related genes, (T) mRNA levels of inflammation-related genes, and (U) mRNA levels of fibrosis-related genes (n = 4).

[0074] For all data: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. Data are represented as mean ± SEM. A two-tailed t-test was applied in D, E, F, G, H, I, M, N, O, P, Q, R, S, T, U.

[0075] Figure 6 GalNAc-siRNA specifically targets and reduces the level of TKT protein in the liver in mice.

[0076] (A and B) Protein and (B) mRNA levels of TKT in primary mouse hepatocytes after treatment with GalNAc-siTKT without liposome coating.

[0077] (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.

[0078] For all data: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. Data are represented as mean ± SEM. A two-tailed t-test was applied in B.

[0079] Figure 7 Screening and efficacy analysis of human-mouse-monkey homologous sequences.

[0080] (A) Detection of the knockdown effect of a total of 49 siRNAs targeting the human, mouse, and monkey homologous regions of the TKT gene on primary mouse hepatocytes.

[0081] (B-C) Detection of the TKT knockdown effect in the liver of wild-type mice 7 days after subcutaneous injection of GalNAc-siTKT 773 and 1115, (B) mRNA level and (C) protein level.

[0082] (D) Changes in blood drug concentration of wild-type mice after subcutaneous injection of GalNAc-siTKT1115.

[0083] Figure 8 Hyperinsulinemia causes an increase in liver TKT levels.

[0084] (A) Serum insulin levels in wild-type mice fed a normal diet or a high-fat diet for 90 days (n = 5 in the NCD group, n = 4 in the HFD group).

[0085] (B-D) TKT mRNA level, (C) protein levels of TKT, p-C / EBPα, and C / EBPα, and (D) relative quantification of TKT protein in primary hepatocytes of wild-type mice after treatment with 10 nM insulin (n = 4).

[0086] (E) Binding ability of p-C / EBPα and C / EBPα to the TKT promoter in primary hepatocytes of wild-type mice after treatment with 10 nM insulin (n = 3).

[0087] (F-H) Primary hepatocytes from liver-specific insulin receptor knockout mice after treatment with 10 nM insulin, (F) TKT mRNA levels, (G) relative quantification of TKT, p-C / EBPα, C / EBPα protein levels, and (H) TKT protein (n = 3).

[0088] (I-M) Mice with liver-specific insulin receptor knockout were fed a normal diet or a high-fat diet for 90 days. Protein and (J) mRNA levels of liver (I) TKT, H&E staining of liver tissue (K), (L) Oil Red O, and (M) Sirius Red staining (n = 3-4).

[0089] Figure 9 Significant accumulation of R5P and inosine was observed in TKT-deficient hepatocytes.

[0090] (A and B) Principal component analysis (PCA) of differential metabolites and (B) KEGG metabolic pathway enrichment analysis in primary hepatocytes from wild-type and liver-specific TKT knockout mice (n = 5).

[0091] (C) Heatmap of differential metabolites in the purine metabolism pathway (n = 5).

[0092] (D) 13 Structural diagram of 13 C-labeled inosine (

[0093] (E) Metabolic flux analysis of primary hepatocytes from wild-type and liver-specific TKT knockout mice cultured in medium containing 5.6 mM 13 C5-inosine for 6 hours (n = 3).

[0094] (F) Isotope labeling levels of R5P, G6P / F6P, and S7P in primary hepatocytes from wild-type and liver-specific TKT knockout mice (n = 3).

[0095] (G) Analysis of Glucose and G6P / F6P labeling after culturing primary hepatocytes from wild-type and liver-specific TKT knockout mice in medium containing 15 mM 1,2 13 C2-glucose for 30 minutes, analysis of Sed7P, R5P, and R1P labeling after 6 hours, and analysis of inosine labeling after 48 hours (n = 3).

[0096] (H and I) Labeling of R5P, G6P / F6P, and S7P in primary hepatocytes from wild-type and liver-specific TKT knockout mice (n = 3).

[0097] Figure 10, TKT deficiency enhances mitochondrial function through the inosine-PKA-CREB pathway.

[0098] (A) Expression levels of p-PKA substrate, p-CREB, CREB, UCP1, and TKT in primary hepatocytes of wild-type and liver-specific TKT-deficient mice (n = 3).

[0099] (B-F) Analysis of mitochondrial morphology and quantity in liver tissues 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).

[0100] (G-J) Analysis of mitochondrial function in primary hepatocytes of wild-type and liver-specific TKT-deficient 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).

[0101] (K and L) Hypoxanthine riboside and (L) OCR levels in primary hepatocytes of wild-type mice after treatment with 10 nM insulin (n = 6).

[0102] (M) Hypoxanthine riboside levels in primary hepatocytes of wild-type, liver-specific TKT-deficient, and liver-specific insulin receptor-deficient mice after treatment with 10 nM insulin (n = 4 - 6).

[0103] Figure 11 , TKT deficiency can enhance mitochondrial function through the inosine-PKA-CREB pathway.

[0104] (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 hypoxanthine riboside for 6 hours (n = 3).

[0105] (B-D) (B) Mitochondrial DNA (mtDNA) content, (C) protein, and (D) mRNA expression levels of mitochondrial genes in primary hepatocytes of wild-type and liver-specific TKT-deficient mice (n = 4 in B and D, n = 3 in C).

[0106] (E) Immunofluorescence staining of TOM20 protein in liver tissues of wild-type and liver-specific TKT-deficient mice (n = 3).

[0107] (F) Western blot analysis of p-PKA substrate and TKT protein in primary hepatocytes of wild-type mice after treatment with 56 μM inosine and 10 μM H89 for 6 hours.

[0108] (G and H) Analysis of (G) OCR, (H) ATP, and remaining respiratory capacity in primary hepatocytes of wild-type and liver-specific TKT-deficient mice after treatment with 56 μM inosine for 6 hours (n = 3).

[0109] (I) Changes in OCR in primary hepatocytes of wild-type mice after treatment with 56 μM inosine and 10 μM H89 for 6 hours (n = 3).

[0110] All data: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. Data are presented as mean ± SEM. Two-tailed t-tests were used for B and D.

[0111] Figure 12 Inosine upregulates PC synthesis by activating the PKA-CREB-ChoKβ axis in hepatocytes.

[0112] (A) Heatmap of the correlation matrix of characteristic genes. Each row or column corresponds to a characteristic gene, including the module number and TKT protein. Red and blue indicate positive and negative correlations, respectively. Modules 1, 3, and 7 show Pearson correlation coefficients > 0.72, p < 0.01.

[0113] (B-D) Network diagrams of the top 10 lipids with the highest connectivity in (B) module 1, (C) module 3, and (D) module 7. Changes in module eigengenes (ME) in the livers of NAFLD patients 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.

[0114] (E) Levels of 6 lipids in modules 1, 3, and 7 in the livers of NAFLD patients and healthy individuals (n = 24).

[0115] (F) Schematic diagram of the phosphatidylcholine synthesis pathway.

[0116] (G and H) Levels of (G) phosphatidylcholine (PC) and (H) CDP-choline pathway metabolites in primary hepatocytes of wild-type and liver-specific TKT-deficient mice (n = 6 in G, n = 4 in H).

[0117] (I and J) Levels of PC in primary hepatocytes (I) and OCR (J) after wild-type and liver-specific TKT-deficient mice were fed a low-choline diet (20% choline content of the NCD diet) for 3 weeks (n = 4 in I, n = 3 in J).

[0118] (K) Relative levels of CDP-choline pathway metabolites in primary hepatocytes of wild-type mice after treatment with 56 μM inosine for 6 hours (n = 3).

[0119] (L and M) mRNA and (M) protein levels of ChoK in primary hepatocytes of wild-type and liver-specific TKT-deficient mice (L = 4, M = 2).

[0120] (N) Relative levels of CDP-choline pathway metabolites in primary hepatocytes of wild-type mice after treatment with 56 μM inosine and 10 μM H89 for 6 hours (N = 3).

[0121] Figure 13 The target gene of the present invention and the signal pathways related to its upstream and downstream regulation.

[0122] Figure 14 TKT deficiency enhances hepatocyte mitochondrial function by increasing phosphatidylcholine synthesis.

[0123] (A) Protein levels of PGC1α, NRF2, and DRP1 in TKT-deficient hepatocytes.

[0124] (B) Lipidomics results of mitochondria in TKT-deficient hepatocytes.

[0125] (C-F) Levels of choline, phosphatidylcholine, CDP-choline, and PC in liver tissues of mice in the low-choline diet and normal diet groups.

[0126] (G-J) Mitochondrial membrane potential, ATP, mitochondrial ROS, and OCR levels in hepatocytes of mice in the low-choline diet group and normal diet group.

[0127] (K-P) Levels of (K) choline, (L) PC, and (M) CDP-choline in primary hepatocytes of wild-type and liver-specific TKT-deficient mice after feeding a low-choline diet, and levels of (N) ATP, (O) mitochondrial membrane potential, and (P) mitochondrial ROS in hepatocytes. Detailed implementation manners

[0128] The present invention first reveals a novel target gene closely related to the remission or treatment of metabolic-related fatty liver disease and the signal pathways related to its upstream and downstream regulation. The present invention discloses a new mechanism for regulating metabolic-related fatty liver disease participated by this signal pathway. The present invention also discloses preferably remission / treatment drugs.

[0129] Transketolase (TKT) or the insulin / InsR-C / EBPα-TKT signaling pathway containing TKT and its regulation

[0130] As used in the present invention, the terms "(signaling) pathway" and "(signaling) route" can be used interchangeably.

[0131] As used in the present invention, the "(signaling) pathway" refers to a signal system formed by the mutual restriction or interaction between a series of genes or proteins or their metabolites (synthetic products or processed products), and also includes the interaction between pathway proteins and other intracellular components or organelles, and sometimes also includes the co-participation of their upstream and downstream genes or proteins, which generally leads to the occurrence of some cellular events. The insulin / InsR-C / EBPα-TKT signaling pathway mainly includes the following elements: TKT gene / protein, C / EBPα gene / protein, InsR gene / protein; preferably, it also includes their upstream and downstream regulatory genes / proteins or chemical molecules.

[0132] The nucleotide sequence of the TKT gene is shown, for example, as Gene ID: 7086 (human) and Gene ID: 21881 (mouse); its protein amino acid sequence is shown, for example, as NCBI Reference Sequence: NP_001055.1 (human) or NCBI Reference Sequence: NP_033414.1 (mouse).

[0133] The nucleotide sequence of the C / EBP gene is shown, for example, as Gene ID: 1050 (human) and Gene ID: 12606 (mouse); its protein amino acid sequence is shown, for example, as NCBI Reference Sequence: NP_004355.2 (human), NCBI Reference Sequence: NP_031704.2 (mouse).

[0134] The nucleotide sequence of the InsR gene is shown, for example, as Gene ID: 3643 (human) and Gene ID: 16337 (mouse); its protein amino acid sequence is shown, for example, as NCBI Reference Sequence: NP_000199.2 (human), NCBI Reference Sequence: NP_034698.2 (mouse).

[0135] In the present invention, unless otherwise stated, the protein / gene information in some upstream and downstream signaling pathways related to the insulin / InsR-C / EBPα-TKT signaling pathway is known in the art.

[0136] Within hepatocytes, downstream of the insulin / InsR-C / EBPα-TKT signaling pathway, there exists a series of further regulatory pathways. By downregulating the said TKT or the insulin / InsR-C / EBPα-TKT signaling pathway containing TKT, it is further possible to: reduce the inosine level, enhance mitochondrial function, thereby improving hepatic lipid metabolism and alleviating or treating metabolic associated fatty liver disease. More specifically, after downregulating the said TKT or the insulin / InsR-C / EBPα-TKT signaling pathway containing TKT, it can prevent R5P derived from inosine from entering glycolysis, promote the synthesis of inosine from R5P derived from glucose, increase the intracellular inosine level; thereby promoting the inosine-PKA-CREB pathway and activating the PKA-CREB-ChoKβ axis, increasing the synthesis of phosphatidylcholine (PC), enhancing mitochondrial function, improving hepatic lipid metabolism, and alleviating or treating metabolic associated fatty liver disease; preferably, the increase in the synthesis of phosphatidylcholine (PC) is mediated by the CDP-choline pathway.

[0137] As used in the present invention, unless otherwise stated, the target genes / proteins under discussion and the signaling pathways they participate in are those in the liver (cells).

[0138] When used as a target for artificial regulation or when establishing a screening system artificially, the above proteins or coding genes can be naturally occurring, for example, they can be purified and isolated from mammals; or they can be recombinantly prepared, for example, recombinant proteins can be produced according to conventional gene recombination techniques. In addition, any variant forms that do not affect the biological activity of these proteins are available, such as derivatives or variants whose functions have not changed.

[0139] The above proteins (polypeptides) also include their 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, still more preferably 1 - 8, 1 - 5) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. Any protein with high homology to the said protein (such as having a homology of 70% or higher with the polypeptide sequence; preferably 80% or higher; more preferably 90% or higher, 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 a protein truncation, as long as the mutant protein or truncation substantially retains the function of the full-length protein.

[0140] The sequences of the above genes also include sequences degenerate thereto. The polynucleotides (genes) encoding proteins can be natural genes or their degenerate sequences.

[0141] As used in the present invention, the terms "downregulation (agent)" and "inhibition (agent)" can be used interchangeably, and also include: blocking (agent), antagonizing (agent), etc.

[0142] The inventors of the present invention have found that in the said signal pathway, downregulating TKT, downregulating the C / EBPα-TKT interaction, and downregulating the insulin / InsR-C / EBPα interaction can alleviate or treat metabolic associated fatty liver disease. Therefore, drugs suitable for targeted regulation can be screened or designed through this mode of action.

[0143] It should be understood that after knowing the function of the insulin / InsR-C / EBPα-TKT signal pathway (preferably, also including its upstream and downstream proteins or genes), various methods well-known to those skilled in the art can be used to regulate the insulin / InsR-C / EBPα-TKT signal pathway. For example, various methods well-known to those skilled in the art can be used to regulate the expression of pathway proteins or cause their loss of expression.

[0144] The present invention provides a method for downregulating the insulin / InsR-C / EBPα-TKT signal pathway, including targeting mutations, gene editing, or gene recombination of the TKT, C / EBPα, or InsR genes in the insulin / InsR-C / EBPα-TKT signal pathway, so as to achieve downregulation.

[0145] As a more specific implementable mode, a method for downregulating the expression of the TKT, C / EBPα, or InsR genes is provided, including: transferring interfering molecules that interfere with the expression of the TKT, C / EBPα, or InsR genes into cells, or treating cells through a suitable pathway to introduce them into cells, for example, designing a transmembrane functional domain to enable it to have the ability to penetrate membranes. RNA interference technology is a technology for silencing gene expression. The principle of RNA interference technology is that longer double-stranded RNA is cut and processed by the specific nuclease Dicer into small interfering RNAs consisting of 21-23 nt of sense and antisense strands. The small interfering RNAs then form a silencing complex (RNA-induced silencing complex, RISC) and unwind into single strands. The antisense strand guides the silencing complex to specifically bind to the target mRNA through base pairing, causing the mRNA to decompose.

[0146] As another embodiment of the present invention, the CRISPR / Cas system is used for gene editing to knockdown, knockout or downregulate target genes. Appropriate sgRNA target sites can bring higher gene editing efficiency. Therefore, before starting gene editing, appropriate target sites need to be designed and found. After designing specific target sites, in vitro cell activity screening is also required to obtain effective target sites for subsequent experiments. Using this method, the TKT, C / EBPα or InsR gene can be transformed into a truncated or mutant form with loss of function.

[0147] Regulatory reagents and pharmaceutical compositions

[0148] As a preferred embodiment of the present invention, downregulators for downregulating the expression or activity of TKT, downregulating the C / EBPα-TKT interaction, or downregulating the insulin / InsR-C / EBPα interaction are provided. The downregulators refer to any substances that can reduce the activity, reduce the stability, downregulate the expression, reduce the activation level (such as reducing the phosphorylation level), reduce the effective action time, inhibit their transcription and translation of TKT, C / EBPα or InsR. These substances can all be used in the present invention as potentially useful substances for alleviating or treating metabolic associated fatty liver disease. They can be compounds, small chemical molecules, biomolecules. The biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

[0149] As used in the present invention, the "metabolic associated fatty liver disease" includes fatty liver.

[0150] As used in the present invention, the "metabolic associated fatty liver disease" includes two different indications: non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0151] In the present invention, the downregulators can be: nucleic acid inhibitors, protein inhibitors, antibodies, ligands, compounds, nucleases, nucleic acid binding molecules, etc., provided that they can downregulate the expression of TKT, C / EBPα or InsR, inhibit their activity or function, or downregulate the C / EBPα-TKT interaction or downregulate the insulin / InsR-C / EBPα interaction. The nucleic acid inhibitors include: shRNA, antisense nucleic acids, small interfering RNAs, microRNAs that target the coding gene of TKT, C / EBPα or InsR or its transcript, or constructs that can express or form the shRNA, antisense nucleic acids, small interfering RNAs, microRNAs.

[0152] 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.

[0153] As a preferred embodiment of the present invention, the down-regulator is an interfering molecule that specifically interferes with the expression of TKT. Short hairpin RNA (shRNA) is an RNA sequence that forms a sharp turn structure and can silence genes via 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 an siRNA molecule targeting TKT, C / EBPα or InsR genes.

[0154] Around the target of TKT, the inventors have conducted in-depth experimental demonstrations, including demonstrations at the cellular level and the animal level. On this basis, the inventors have optimized and obtained a series of siRNA reagents with excellent effects. The siRNA is an siRNA with a nucleotide sequence such as SEQ ID NO:1 to SEQ ID NO:50; more preferably, the siRNA is an siRNA with a nucleotide sequence such as SEQ ID NO:37 (No. 1115), SEQ ID NO:25 (No. 773), SEQ ID NO:50 (siTKT16) or SEQ ID NO:49 (siTKT10). The siRNA can be used in multiple species, has a very ideal targeted regulatory effect, no non-specific regulation, and has high clinical application value.

[0155] 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.

[0156] 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.

[0157] As another optional mode 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, by any of the above methods, TKT, C / EBPα or InsR is converted into its mutant so that it no longer functions.

[0158] The present invention also provides a pharmaceutical composition for alleviating or treating metabolic associated fatty liver disease, comprising an effective amount of the down-regulator described in the present invention.

[0159] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals as used herein.

[0160] As used herein, a "pharmaceutically acceptable" component is a substance that is suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance having a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents.

[0161] The present invention also provides a kit for alleviating or treating metabolic associated fatty liver disease, the kit comprising: an effective amount of the down-regulator described in the present invention. More preferably, the kit further comprises: an instruction manual to guide clinicians to administer the drug in a correct and reasonable manner.

[0162] For convenient administration, the down-regulator is made into a unit dosage form and placed in a kit. The "unit dosage form" refers to a dosage form prepared for convenient medication, which is the dosage form required for single-dose medication, including but not limited to various liquid preparations (such as injections), solid preparations (such as tablets), capsules, and sustained-release preparations. In addition, the down-regulator can also be independently placed in different containers and mixed and applied when needed.

[0163] Although in the specific embodiments of the present invention, a dosing regimen for animals such as mice is given. It should be understood that it is easy for those skilled in the art to convert the dosing dose from animals such as mice to the dosing dose applicable to humans. For example, it can be calculated according to the Meeh-Rubner formula: Meeh-Rubner formula: A = k×(W 2 / 3 ) / 10,000. In the formula, A is the body surface area, calculated in m 2 ; W is the body weight, calculated in g; K is a constant, which varies with the 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 according to different drugs and clinical situations, the conversion of the dosing dose can vary according to the evaluation of an experienced pharmacist.

[0164] Drug screening based on the InsR-C / EBPα-TKT signaling pathway

[0165] Based on the new findings of the present inventors, the research on the (insulin / )InsR-C / EBPα-TKT signaling pathway has various uses, including: screening substances that regulate this signaling pathway, with the expectation of using them to alleviate or treat metabolic associated fatty liver disease. Among them, the regulation includes: downregulating TKT, downregulating the C / EBPα-TKT interaction, downregulating the insulin / InsR-C / EBPα interaction, etc.

[0166] The present invention provides a method for screening downregulators that regulate the InsR-C / EBPα-TKT signaling pathway. By adding a candidate substance to be screened into a system containing the InsR-C / EBPα-TKT signaling pathway and observing the changes or interactions of each protein or gene in the InsR-C / EBPα-TKT signaling pathway for screening. If the candidate substance has the effects of downregulating TKT, downregulating the C / EBPα-TKT interaction, downregulating the insulin / InsR-C / EBPα interaction, etc., then the candidate substance is a substance useful for alleviating or treating metabolic associated fatty liver disease.

[0167] As used herein, "inhibition", "downregulation", etc. all refer to "inhibition", "downregulation" with statistical significance. That is: significantly "inhibit", "downregulate". Compared with the protein activity, protein expression, protein binding or methylation level of the control group, significantly "inhibit" or "downregulate" by more than 10%, 20%, 30%, 40%, 50%; more preferably, more than 60%, 70%, 80%.

[0168] The system containing the InsR-C / EBPα-TKT signaling pathway is selected from: cell system (or cell culture system), subcellular system (or subcellular culture system), solution system, animal system or tissue system (or tissue culture system). Preferably, the system containing the InsR-C / EBPα-TKT signaling pathway is hepatocytes (or cell culture).

[0169] As a preferred embodiment of the present invention, the method further includes: performing further cell experiments and / or animal tests on the obtained potential substances to further select and determine substances useful for alleviating or treating metabolic associated fatty liver disease from the candidate substances.

[0170] When performing screening, various techniques well-known in the art can be used to determine the changes and interactions of proteins or their coding genes.

[0171] A variety of conventional techniques can be employed to identify the transcription or expression of genes in a system. These techniques include, but are not limited to: oligonucleotide hybridization techniques (such as probes), polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, etc. A variety of techniques well-known to those skilled in the art can be used to detect the interaction between proteins and the strength of the interaction, such as co-immunoprecipitation technique, GST precipitation technique, phage display technique or yeast two-hybrid system. The nuclear localization of proteins is also a technique well-known in the art.

[0172] The substances preliminarily screened by the above methods can form a screening library, so that people can finally screen out the substances that are truly useful for alleviating or treating metabolic associated fatty liver disease from it.

[0173] The present invention also provides potential substances obtained by using the said screening method and can be used for alleviating or treating metabolic associated fatty liver disease.

[0174] The present invention also provides a method for preparing a drug for alleviating or treating metabolic associated fatty liver disease (especially alleviating or treating metabolic associated fatty liver disease), the method comprising: synthesizing and / or purifying the substances obtained by the above screening and useful for alleviating or treating metabolic associated fatty liver disease as a drug for alleviating or treating metabolic associated fatty liver disease.

[0175] The substances obtained and useful for alleviating or treating metabolic associated fatty liver disease can be used to prepare a pharmaceutical composition, as described in the following text of the present invention.

[0176] The methods for screening substances acting on a target with a protein or a gene or a specific region thereof as the target are well-known to those skilled in the art, and these methods can all be used in the present invention. The said candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, organic small molecules, inorganic small molecules and nucleic acid sequences, etc. Those skilled in the art know clearly how to select the applicable screening methods according to the types of substances to be screened.

[0177] Applications for diagnosis or prognostic assessment

[0178] In the present invention, it is found that the upregulation of TKT in the liver is a common feature of human and mouse NAFLD. The upregulation of TKT promotes the progression of NAFLD in mice, the upregulation of TKT promotes non-alcoholic steatohepatitis (NASH), and TKT is involved in a new signaling pathway: the insulin / InsR-C / EBPα-TKT signaling pathway. Therefore, TKT and the signaling pathway it participates in can be used as molecular markers for guiding the diagnosis or prognosis (including drug use guidance) of metabolic-related fatty liver diseases: (i) for disease typing and differential diagnosis; (ii) for evaluating the therapeutic drugs, drug efficacy, prognosis of relevant populations, and selecting appropriate treatment methods. For example, populations with abnormal (increased) TKT gene expression can be isolated, so as to carry out more targeted treatment.

[0179] The disease prognosis of the subject providing the sample to be evaluated can be predicted by judging the expression or activity of TKT or the pathway genes / proteins of the signaling pathway it participates in, 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 treatment plan for inhibiting TKT can be considered. The threshold is easy to determine for those skilled in the art. For example, by comparing and analyzing the general TKT expression of patients with metabolic-related fatty liver diseases or the expression of normal healthy people, the threshold for abnormal TKT expression can be obtained.

[0180] Therefore, the present invention provides the use of TKT or the pathway genes / proteins of the signaling pathway it participates in for preparing reagents or kits for evaluating the prognosis of metabolic-related fatty liver diseases. Various techniques known in the art can be used to detect the presence 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 and expression of genes or proteins in an analyte. Preferably, when detecting at the gene level, specific amplification primers or probes for specific recognition can be used to determine the presence of the target gene; when detecting at the protein level, antibodies or ligands that specifically bind to the protein can be used to determine the expression of the target protein.

[0181] 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, enzymes, control solution, color development solution, washing solution, etc. In addition, the kit may also include an instruction manual and / or nucleic acid sequence analysis software, etc.

[0182] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following embodiments, they are generally carried out according to the conventional conditions such as those described in "Molecular Cloning: A Laboratory Manual", Third Edition, edited by J. Sambrook et al., Science Press, or according to the conditions recommended by the manufacturer.

[0183] Example 1. Expression characteristics of TKT in liver tissue

[0184] Liver samples were obtained from liver transplant donors of patients with non-alcoholic fatty liver disease (NAFLD) or healthy individuals, and the sample source information is as Figure 2 A, and they were divided into the NAFLD group (HS) and the healthy control group (Control) according to clinical pathological diagnosis.

[0185] Metabolomics analysis showed that compared with the control group, the pentose phosphate pathway (PPP) in NAFLD livers was significantly altered ( Figure 1 A).

[0186] According to further proteomics analysis of PPP and glycolytic enzymes, the non-oxidative PPP metabolic enzyme TKT showed the most significant difference between NAFLD livers and the control group ( Figure 1 B).

[0187] Regarding the expression of the non-oxidative PPP metabolic enzyme TKT, Western blot and quantitative PCR analyses were performed. The results showed that TKT was significantly upregulated in NAFLD livers ( Figure 1 C-E).

[0188] Immunohistochemistry (IHC) analysis was performed on a randomly selected group of human liver samples (n = 6), and the results showed that the staining of TKT in the NAFLD group was deeper than that in the healthy control group ( Figure 1 F).

[0189] Furthermore, NAFLD was induced in wild-type mice by feeding them a high-fat diet (HFD). The mice in the HFD group were fed a high-fat diet every day and allowed to eat freely; the control NCD group mice were fed a normal diet every day and allowed to eat freely. This simulated the occurrence and development process of human NAFLD patients. After 3 months, lipid accumulation and TKT upregulation were observed in the livers of the mice ( Figure 1 G-J).

[0190] Therefore, these findings indicate that the upregulation of TKT in the liver is a common feature of NAFLD in humans and mice.

[0191] Example 2. Upregulation of TKT promotes the progression of NAFLD in mice

[0192] 1. Upregulation of TKT in the liver promotes non - alcoholic fatty liver (NAFL)

[0193] To investigate whether TKT upregulation drives the progression of NAFLD, an adenovirus (adeno - associated virus, AAV) - mediated gene overexpression system was constructed to overexpress TKT in the mouse liver.

[0194] Tail - vein injection of AAV - Flag - TKT into mice significantly increased the TKT level in the liver ( Figure 1 K and Figure 2 B).

[0195] After feeding mice a high - fat diet (HFD) for 10 weeks, mice injected with AAV - Flag - TKT showed more severe hepatic steatosis ( Figure 1 L and Figure 2 C), as well as higher liver and serum triglyceride levels ( Figure 1 M).

[0196] Overexpression of TKT in the liver also increased the total blood cholesterol, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in mice ( Figure 2 D).

[0197] These results indicate that upregulation of TKT in the liver promotes high - fat - diet - induced non - alcoholic fatty liver (NAFL).

[0198] 2. Upregulation of TKT in the liver promotes non - alcoholic steatohepatitis (NASH)

[0199] Methionine - and choline - deficient (MCD) diet is commonly used to induce NASH in mice, which is a progressive form of NAFLD. Mice were fed an MCD diet daily (ad libitum), and various parameters of animals injected with AAV - Flag - TKT and control (AAV - ctrl) were observed.

[0200] The results showed that after 4 weeks of MCD diet, mice injected with AAV - Flag - TKT exhibited more severe hepatocyte ballooning degeneration, lipid accumulation, inflammation, and fibrosis than the control group ( Figure 2 E), accompanied by elevated liver triglyceride (TG) and total cholesterol (TC) levels, as well as serum aspartate aminotransferase (AST) level ( Figure 2 F), indicating that animals injected with AAV - Flag - TKT had a more significant progression of NASH than the control.

[0201] Therefore, upregulation of TKT in the liver significantly promotes the progression of NAFL and NASH.

[0202] Example 3. Hepatocyte - specific TKT knockout improves NAFLD in mice

[0203] 1. Downregulation of TKT in the liver improves non - alcoholic fatty liver (NAFL)

[0204] To investigate whether TKT is crucial for the development of NAFLD, a mouse strain with hepatocyte - specific TKT deletion (TKT flox / flox Alb cre , abbreviated as TKT fl / fl Alb cre ) was generated.

[0205] Generation of TKT flox / flox Alb cre 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 the TKT flox / flox mouse strain ( Figure 3 S). This strain was then crossed with AlbCre mice to obtain TKT flox / flox Alb cre mice.

[0206] Generation of TKT + / + Alb cre mice: Wild - type C57BL / 6 mice were crossed with AlbCre mice to obtain TKT + / + Alb cre mice.

[0207] Under normal chow diet (NCD) conditions, TKT flox / flox Alb cre mice exhibited normal liver morphology and function. TKT flox / flox Alb cre and TKT + / + Alb cre mice were fed a high - fat diet (HFD) for 90 days. Compared with obese TKT + / + Alb cre mice, TKT flox / flox Alb cre mice had a slower body weight gain rate ( Figure 4 A). After 90 days of HFD feeding, compared with control mice, TKT flox / flox Alb cre mice had lower body weight and liver weight ( Figure 4 B - D).

[0208] After HFD feeding, TKT flox / flox Alb creMice showed less hepatic steatosis than the control group, as confirmed by magnetic resonance imaging (MRI), liver appearance photographs, and H&E and Oil Red O staining results ( Figure 3 A-C).

[0209] Moreover, the hepatic triglyceride level (TG), serum triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in TKT flox / flox Alb cre mice were lower than those in the control group ( Figure 3 D-H), while the total hepatic cholesterol remained unchanged ( Figure 3 I).

[0210] Therefore, hepatocyte-specific TKT deletion in mice could significantly improve NAFL induced by HFD.

[0211] 2. Downregulation of TKT in the liver alleviates non-alcoholic steatohepatitis (NASH)

[0212] Investigate whether hepatocyte-specific TKT knockout can alleviate NASH induced by MCD diet. Mice were fed an MCD diet, and the disease progression of TKT flox / flox Alb cre ) knockout mice was observed compared with that of control mice (TKT + / + Alb cre ).

[0213] After 4 weeks of MCD diet, TKT flox / flox Alb cre ) knockout mice in the liver showed significantly reduced hepatic lipid accumulation ( Figure 3 J, K), while the mRNA levels of lipid synthase genes remained unchanged ( Figure 3 L).

[0214] TKT flox / flox Alb cre mice had far fewer inflammatory cell infiltrations in the liver than the control group ( Figure 3 M, N), accompanied by downregulation of inflammation-related genes ( Figure 3 O).

[0215] MCD diet caused severe fibrosis in the liver of the control group, which was confirmed by Sirius red staining and Masson staining, while knockout of TKT in the liver alleviated this condition ( Figure 3 P and Figure 4 E).

[0216] In addition, TKT deletion also reduced hepatocyte necrosis related to the progression of NASH ( Figure 4 F).

[0217] Therefore, TKT deficiency in hepatocytes can improve NAFL and NASH in mice.

[0218] 3. Role of TKT in the liver in the development of NAFLD

[0219] To further demonstrate that TKT is a driver of NAFLD, an adenovirus overexpression system was used to re-express TKT in the livers of flox / flox Alb cre mice. HFD-induced NAFL and MCD-induced NASH models were established in mice with liver-specific deletion of TKT (TKT flox / flox Alb cre ), and then adenoviruses AAV-Flag-TKT and AAV-Ctrl were injected via the tail vein, respectively.

[0220] During HFD feeding, re-expression of TKT in the liver promoted hepatic lipid accumulation ( Figure 4 G, H), elevated serum and hepatic triglyceride, total cholesterol levels, as well as serum ALT and AST levels ( Figure 4 I-N).

[0221] Consistently, re-expression of TKT in the liver restored MCD-induced hepatic lipid accumulation and fibrosis, upregulated hepatic triglyceride and cholesterol levels, as well as serum ALT and AST levels ( Figure 4 P-U).

[0222] In summary, upregulation of TKT in the liver is both necessary and sufficient for the development of NAFLD in mice.

[0223] Example 4. Analysis of the regulatory role of TKT in the liver and the involved regulatory pathways

[0224] 1. Insulin-InsR signaling drives TKT upregulation and lipid accumulation during NAFLD progression

[0225] The inventors investigated the mechanism of TKT upregulation in NAFLD livers, which is usually associated with hyperinsulinemia and insulin resistance. Mice were fed with HFD, and elevated serum insulin levels were observed ( Figure 8 A). In primary hepatocytes treated with insulin, the elevated levels of TKT mRNA and protein peaked at 40 minutes and then gradually declined ( Figure 8 B-D), accompanied by fluctuations in the levels of phosphorylated transcription factor CCAAT / enhancer-binding protein α (C / EBPα) ( Figure 8 C). Analysis suggested that C / EBPα is a candidate transcription factor for the TKT gene promoter.

[0226] It was confirmed by chromatin immunoprecipitation (ChIP) that insulin enhanced the binding ability of phosphorylated C / EBPα to the region of 643 - 795 bp upstream of the TKT promoter through InsR( Figure 8 E).

[0227] To further investigate the role of insulin receptor (InsR) in insulin-induced TKT expression, a mouse strain (InsR flox / flox Alb cre ) was generated in which InsR in hepatocytes was specifically deleted.

[0228] InsR flox / flox Alb cre Mice were purchased from The Jackson laboratory.

[0229] InsR flox / flox Alb cre Primary hepatocytes did not show fluctuations in insulin-stimulated TKT and phosphorylated C / EBPα( Figure 8 F - H), suggesting that insulin promotes TKT expression through InsR. Subsequently, InsR flox / flox Alb cre mice were fed with HFD, and it was found that HFD neither increased the levels of hepatic TKT mRNA and protein( Figure 8 I, J), nor caused obvious lipid accumulation and fibrosis in the livers of InsR - / - mice( Figure 2 K - M).

[0230] Therefore, during the progression of NAFLD, insulin-InsR signaling drives TKT upregulation and lipid accumulation.

[0231] 2. TKT-deficient hepatocytes significantly increase the accumulation of R5P and inosine

[0232] Primary hepatocytes were isolated from TKT flox / flox Alb cre and TKT + / + Alb cre mice for metabolomics analysis. The PCA results showed significant differences between the two groups( Figure 9 A).

[0233] Through metabolic pathway enrichment, it was found that TKT deficiency significantly altered the purine metabolic pathway and also the PPP( Figure 9 B). 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 mitochondrial function in adipose tissue as an extracellular signal through the cAMP-PKA-UCP1 pathway.

[0234] Next, how TKT deficiency regulates hepatic inosine metabolism was investigated. Primary hepatocytes from 13 C5]-inosine-fed TKT flox / flox Alb cre and TKT + / + Alb cre mice, in which only ribose contained 13 C, were used for metabolic flux analysis.

[0235] The results confirmed that 13 C5]-labeled inosine could be taken up by primary hepatocytes without interference from TKT deficiency ([[]]END]] Figure 9 D-inosine). TKT deficiency blocked carbon transfer between R5P, S7P, and F6P, resulting in an increased relative abundance of inosine-derived R5P ([[]]END]] Figure 9 F), while the relative abundances of F6P and S7P were decreased ([[]]END]] Figure 9 F). The elevated R5P level may have inhibited the degradation rate of PNP enzyme, leading to a decreased isotopic labeling efficiency of R1P and R5P ([[]]END]] Figure 9 E). These results indicate that TKT deficiency in hepatocytes impairs the entry of pentoses derived from inosine into central carbon metabolism by blocking the non-oxidative PPP.

[0236] In hepatocytes, glucose is metabolized through the PPP to generate R5P for the resynthesis of IMP, which is dephosphorylated to form inosine. Next, how TKT deficiency affects the synthesis of glucose-derived inosine was investigated. [1,2- 13 C2]-glucose-treated TKT flox / flox Alb cre and TKT + / + Alb cre primary hepatocytes were used for glucose metabolic flux analysis. Considering the two reactions catalyzed by TKT, the deletion of TKT not only blocked carbon transfer between F6P and X5P, resulting in an increased relative abundance and isotopic labeling efficiency of F6P ([[]]END]] Figure 9 G and 9H), but decreased the relative abundance and isotopic labeling efficiency of S7P ([[]]END]] Figure 9 I). In addition, the major isotopic labeling form of R5P changed from M+2 to M+1 ([[]]END]] Figure 9 I), indicating that compensatory oxidative PPP may produce a higher abundance of R5P ([[]]END]] Figure 9H). Overall, the accumulation of R5P in TKT-deficient hepatocytes provides more pentose for inosine synthesis, leading to an increase in inosine levels ( Figure 9 C).

[0237] In summary, TKT deficiency in hepatocytes not only prevents R5P from the inosine source from entering glycolysis but also promotes the synthesis of inosine from R5P derived from glucose, ultimately resulting in an increase in intracellular inosine levels.

[0238] 3. TKT deletion enhances mitochondrial function by promoting the inosine-PKA-CREB pathway

[0239] It has been reported that inosine promotes energy consumption in adipose tissue through the PKA-CREB-UCP1 pathway.

[0240] Next, the inventors investigated the role of inosine in hepatocytes. By treating wild-type primary hepatocytes with inosine, it was found that inosine activated the PKA-CREB signaling pathway but did not increase the expression of UCP1 protein ( Figure 11 A). Interestingly, an increase in the activity of the PKA-CREB axis in flox / flox Alb cre hepatocytes was observed, which was consistent with the accumulation of inosine caused by TKT deletion ( Figure 10 A). The inventors further investigated whether TKT deletion affects the mitochondrial morphology and function of hepatocytes.

[0241] Hepatocytes with TKT deletion had a higher mitochondrial DNA content ( Figure 11 B), 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 ( Figure 11 C-E). Transmission electron microscopy results of liver tissues showed that TKT deficiency increased mitochondrial density, diameter, and surface area ( Figure 10 B-F). Hepatocytes with TKT deletion exhibited higher mitochondrial membrane potential, ATP production capacity, and mitochondrial ROS levels ( Figure 10 g-1). The results of the Seahorse experiment showed that the oxygen consumption rate (OCR) of hepatocytes with TKT deletion was enhanced ( Figure 10 J).

[0242] The inventors found that inosine treatment promoted mitochondrial activity in hepatocytes, and the effect was more obvious in hepatocytes with TKT deletion. After adding inosine, the OCR level, ATP production capacity, and spare respiratory capacity of TKT knockout hepatocytes were significantly upregulated ( Figure 11G, H). Therefore, TKT deficiency promotes hepatocyte mitochondrial activity and sensitivity to inosine nucleoside stimulation, improving hepatic lipid metabolism.

[0243] To determine whether inosine nucleoside 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 The levels of phosphorylated PKA substrates in primary hepatocytes ( Figure 11 F). Importantly, inosine nucleoside could not upregulate mitochondrial function in primary hepatocytes treated with H89 ( Figure 11 i).

[0244] It has been reported that hyperinsulinemia interferes with mitochondrial function. The inventors found that insulin decreased intracellular inosine nucleoside levels ( Figure 10 K), inhibiting mitochondrial activity in WT primary hepatocytes ( Figure 10 L). To investigate whether InsR and TKT play important roles in insulin-reduced inosine nucleoside, primary hepatocytes were isolated from InsR flox / flox Alb cre and TKT flox / flox Alb cre mice and control mice, and it was found that insulin could not affect inosine nucleoside levels in InsR- or TKT-deficient hepatocytes ( Figure 10 M).

[0245] 4. TKT deficiency promotes hepatocyte mitochondrial function by increasing phosphatidylcholine (PC) synthesis

[0246] To investigate the regulatory role of mitochondrial morphological and functional changes caused by TKT deficiency in hepatocytes, the inventors compared the levels of PGC1α, NRF2, and DRP1 in TKT flox / flox Alb cre and TKT + / + Alb cre primary hepatocytes, which regulate mitochondrial fission and fusion. However, TKT deletion did not change the levels of PGC1α, NRF2, and DRP1 in hepatocytes ( Figure 14 A).

[0247] It has been reported that alterations in cellular lipid composition affect mitochondrial morphology and function. Therefore, lipidomic analysis was performed on human liver samples (n = 48) to analyze candidate lipids that were altered by TKT deletion and might affect mitochondrial function. Approximately 700 lipids in 10 classes were identified. All lipids were divided into 15 modules using weighted gene co-expression network analysis (WGCNA)Figure 12 A), and perform a correlation analysis between these modules and the TKT protein level. Three lipid modules were negatively correlated with the TKT protein level, with a Pearson correlation coefficient > 0.8 and a p-value < 0.01( Figure 12 A).

[0248] By analyzing the central lipids of these 3 modules, 6 lipids such as phosphatidylcholine (PC), sphingomyelin (SM), N-acylethanolamine (NAE), ceramide (Cer), triglyceride (TG) and diacylglycerol (DG) were identified( Figure 12 B - D). PC is the most abundant phospholipid in the mitochondrial membrane and is also the most significantly downregulated lipid in patients with NAFLD( Figure 12 e).

[0249] In addition, the deletion of TKT in mouse hepatocytes led to an upregulation of PC( Figure 12 G). Intracellular PC synthesis occurs through the phosphatidylethanolamine (PE) methylation pathway or the CDP-choline pathway( Figure 12 F), and PC is crucial for mitochondrial function. The results of the inventors' lipidomics study showed that the mitochondrial PC level rather than the PE level was upregulated in TKT-deficient hepatocytes( Figure 14 B), indicating that the PE methylation pathway dependent 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 all upregulated in primary hepatocytes( Figure 12 H), indicating that TKT deficiency increased the mitochondrial PC level by promoting the CDP-choline pathway.

[0250] To investigate whether the intracellular PC level is crucial for the mitochondrial function of mouse hepatocytes, the inventors reduced the dietary choline intake by 80% to limit PC biosynthesis. After 3 weeks of a low-choline diet, compared with the normal diet group, the levels of liver choline, phosphatidylcholine, CDP-choline and PC all decreased( Figure 14 C - F). Importantly, low choline intake impaired the mitochondrial membrane potential, ATP production capacity, mitochondrial ROS and OCR levels of hepatocytes( Figure 14 G - J). In addition, choline-restricted diet eliminated the flox / flox Alb cre and + / + Alb cre levels of CDP-choline pathway metabolites between TKT Figure 12 I and Figure 14 K - M) and mitochondrial activity( Figure 12 J andFigure 14 Difference between (N - P).

[0251] The above results indicate that liver TKT deficiency enhances mitochondrial activity and improves hepatic lipid metabolism by increasing intracellular PC synthesis.

[0252] 5. Inosine stimulates PC synthesis by activating the PKA - CREB - ChoKβ axis in hepatocytes

[0253] Next, it was investigated whether and how inosine regulates PC synthesis in hepatocytes. Inosine upregulated the levels of CDP - choline pathway metabolites in WT primary hepatocytes ( Figure 12 K). Choline kinase is a key enzyme in the CDP - choline pathway, catalyzing the first step of the CDP - choline pathway. Compared with the control group, the mRNA and protein levels of ChoKβ in TKT flox / flox Alb cre primary hepatocytes were increased ( Figure 12 L, M).

[0254] Using PROMO to predict the potential transcription factors of ChoKβ, it was found that CREB might regulate the transcriptional activity of ChoKβ. Importantly, the PKA inhibitor H89 abolished the inosine - induced CDP - choline pathway activity ( Figure 12 N).

[0255] These data indicate that in the hepatocytes of TKT - deficient mice, the accumulation of inosine promotes intracellular PC synthesis and mitochondrial function and improves hepatic lipid metabolism by activating the PKA - CREB - ChoKβ pathway.

[0256] 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 the level of inosine in hepatocytes. TKT deficiency promotes the hepatic inosine level, and the accumulation of inosine promotes intracellular PC synthesis and mitochondrial function by activating the PKA - CREB - ChoKβ pathway ( Figure 13 ).

[0257] Example 5. Development and function of GalNAc - siRNA targeting liver TKT

[0258] 1. Development of GalNAc - siRNA and its regulation of hepatocyte TKT

[0259] Targeted knockout and other methods require altering the genome and treating at the pre-embryonic stage, which lacks practicality for clinical treatment. To explore the possibility of targeting liver TKT for the treatment of NAFLD, the inventors conducted screening and research analysis and obtained two siRNA reagents.

[0260] The sequence of siTKT10:

[0261] sense: CCGUGGACAUUGCUAACAUTT (SEQ ID NO:49);

[0262] antisense: AUGUUAGCAAUGUCCACGGTT (SEQ ID NO:51).

[0263] The sequence of siTKT16:

[0264] sense: GCUGCAGAGAGUCUAAAGATT (SEQ ID NO:50);

[0265] antisense: UCUUUAGACUCUCUGCAGCTT (SEQ ID NO:52).

[0266] The sequence of siNC: irrelevant sequence.

[0267] Covalently bind the 3'-end of the siRNA sense strand to N-acetylgalactosamine (GalNAc), and the preparation method adopts the solid-phase synthesis method with a CPG carrier.

[0268] The GalNAc group can be specifically recognized by the receptor ASGPR on the liver cell membrane, and the siRNA double strand is co-endocytosed into the cell. Therefore, the covalently linked product can achieve hepatocyte-targeted delivery of siRNA. Two GalNAc conjugates of siTKT, GalNAc siTKT10 and GalNAc siTKT16, were obtained, and GalNAc siNC was prepared as a control.

[0269] Two GalNAc conjugates of siTKT can effectively knockdown TKT in primary mouse hepatocytes without lipid nanoparticle coating ( Figure 6 A, B).

[0270] The inventors conducted in vivo experiments in mice with GalNAc-siTKT16. Subcutaneous injection of GalNAc-siTKT 16 can reduce the level of TKT protein in the liver but does not affect other tissues ( Figure 6 C-H).

[0271] The structure of GalNAc-siTKT16 is as follows:

[0272] AS (antisense): UmCfUmUmUmAfGmAmCmUmCmUmCmUfGmCfAmGmCmAmGmCmCm;

[0273] SS (sense): CmUmGmCmUmGmCfAmGfAfGfAmGmUmCmUmAmAmAmGmAm - L96.

[0274] Therefore, GalNAc - siRNA targeting liver TKT has a significant down - regulation effect on TKT and an ideal specific regulatory effect, thus having the ability to effectively improve NAFL and NASH.

[0275] 2. Effects of GalNAc - siTKT on NAFL

[0276] At the animal level, the therapeutic effect of GalNAc - siTKT on NAFL was studied. Mice were fed with HFD for 90 days, and then a single injection of GalNAc - siTKT was given subcutaneously to the mice on days 3, 7, 14, and 21 ( Figure 5 A), and analyzed on day 30.

[0277] The results showed that GalNAc - siTKT significantly reduced the level of TKT in the liver ( Figure 5 B), reduced hepatic lipid accumulation ( Figure 5 C), decreased the levels of serum and hepatic triglycerides and total cholesterol ( Figure 5 D - G), as well as the levels of serum ALT and AST ( Figure 5 H, I).

[0278] 3. Therapeutic effect of GalNAc - siTKT on NASH

[0279] The preventive potential of GalNAc - siTKT against MCD - induced NASH in mice was further studied. At the beginning of MCD, a single subcutaneous injection of GalNAc - siTKT (10 mg / kg) was given, and then once on days 3, 7, 14, and 21 ( Figure 5 J).

[0280] On day 30, it was found that GalNAc - siTKT decreased the level of TKT in the liver ( Figure 5 K), alleviated hepatic steatosis, and relieved liver fibrosis ( Figure 5 L). GalNAc - siTKT intervention also decreased the levels of serum and hepatic triglycerides and total cholesterol ( Figure 5 M - P), as well as the levels of serum ALT and AST ( Figure 5 Q, R).

[0281] In addition, GalNAc-s iTKT treatment reduced the levels of genes related to inflammation and fibrosis, but did not affect fatty acid synthesis genes ( Figure 5 S-U).

[0282] In summary, GalNAc-s iTKT specifically targets TKT in hepatocytes, significantly improves NAFL and NASH, and provides a treatment strategy for treating human NAFLD.

[0283] Example 6, Screening and Effect Analysis of Human-Mouse-Monkey Homologous Sequences

[0284] To obtain the siTKT sequence with the best knockdown effect, the inventors performed a full-sequence screening and experimental analysis on the human-mouse-monkey homologous regions of the TKT gene; at the same time, other gene sequences in the whole genome were also considered to exclude non-specific occurrences. From a large number of candidate siRNAs, approximately 48 groups of siRNAs were initially screened (Table 1).

[0285] Table 1

[0286]

[0287]

[0288] By comparing the mRNA and protein knockdown effects in primary mouse hepatocytes and human LO2 cell lines, sequences 773 and 1115 were selected to have the best knockdown effect ( Figure 7 A).

[0289] GalNAc modification was performed on sequences 773 and 1115 to obtain GalNAc-si773 and GalNAc-si1115, which were used for in vivo experiments in mice. GalNAc-si773 and GalNAc-si1115 were dissolved in PBS and subcutaneously injected into the backs of mice at a dose of 10 mg / kg.

[0290] After 7 days of normal diet in mice, the mice were euthanized to obtain liver tissues for detection of TKT expression levels, and it was found that the in vivo knockdown effect of sequence 1115 was better than that of 773 ( Figure 7 B and C).

[0291] Subsequently, pharmacokinetic experiments of 1115 in mice were carried out. The experimental results showed that after subcutaneous injection of GalNAc-siTKT at doses of 5 mg / kg and 10 mg / kg respectively, the peak blood drug concentration in mice was around 60 minutes, and the in vivo half-life of the drug was about 4 hours ( Figure 7 D).

[0292] Therefore, GalNAc-siTKT 773 and GalNAc-si1115 of the human-mouse-monkey homologous sequences show excellent knockdown effects in vitro and in vivo.

[0293] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

Claims

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

2. The use according to claim 1, wherein The downregulator includes those selected from: a TKT downregulator, a downregulator of the C / EBPα-TKT interaction, and a downregulator of the InsR-C / EBPα interaction; Preferably, the TKT downregulator includes: a reagent for silencing, knocking down or knocking out the TKT gene, and a reagent for inhibiting the activity of the TKT protein; more preferably, it includes: an interfering molecule specifically interfering with the expression of the TKT gene, a CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent targeting the TKT gene, and the reagent causes a loss-of-function mutation of TKT; Preferably, the downregulator of the C / EBPα-TKT interaction includes: a reagent for weakening the binding of C / EBPα to the TKT promoter, a reagent for silencing, knocking down or knocking out the C / EBPα gene, and a reagent for inhibiting the activity of the C / EBPα protein; more preferably, it includes: a reagent for reducing the phosphorylation level of the C / EBPα protein, an interfering molecule specifically interfering with the expression of the C / EBPα gene, a CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent targeting the C / EBPα gene, and the reagent causes a loss-of-function mutation of C / EBPα; Preferably, the downregulator of the InsR-C / EBPα interaction includes: a reagent for silencing, knocking down or knocking out the InsR gene, a reagent for inhibiting the activity of the InsR protein, and a reagent for weakening the binding of insulin to InsR; more preferably, it includes: an interfering molecule specifically interfering with the expression of the InsR gene, a CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent targeting the InsR gene, and the reagent causes a loss-of-function mutation of InsR.

3. The use according to claim 2, characterized in that, The downregulator is an interfering reagent for silencing the TKT gene; preferably, the interfering reagent is siRNA; preferably, the siRNA is an siRNA with a nucleotide sequence as shown in SEQ ID NO:1 to SEQ ID NO:50; more preferably, the siRNA is an siRNA with a nucleotide sequence as shown in SEQ ID NO:37, SEQ ID NO:25, SEQ ID NO:50 or SEQ ID NO:49; or The downregulator is a homologous recombination reagent for knocking down or knocking out the TKT gene, which terminates gene encoding; or The downregulator is a homologous recombination reagent for knocking down or knocking out the InsR gene, which terminates gene encoding. Preferably, the siRNA forms an siRNA preparation for hepatocyte-targeted delivery; preferably, the siRNA is covalently bound to N-acetylgalactosamine (GalNAc) to form a conjugate.

4. The use according to claim 1, characterized in that, The metabolic associated fatty liver disease includes: non-alcoholic fatty liver disease, non-alcoholic steatohepatitis; or The down-regulator of the TKT or the InsR-C / EBPα-TKT signaling pathway increases the inosine level, enhances mitochondrial function, and thus improves hepatic lipid metabolism and alleviates or treats metabolic associated fatty liver disease; preferably, the down-regulator: blocks the entry of R5P derived from inosine into glycolysis, promotes the synthesis of inosine from R5P derived from glucose, increases the intracellular inosine level, enhances mitochondrial function, improves hepatic lipid metabolism, and alleviates or treats metabolic associated fatty liver disease; preferably, the increase in phosphatidylcholine (PC) synthesis is mediated by the CDP-choline pathway.

5. An siRNA or an siRNA preparation for alleviating or treating metabolic associated fatty liver disease, wherein the siRNA comprises siRNAs with nucleotide sequences as shown in SEQ ID NOs: 1 to 50; preferably, the siRNA is an siRNA with a nucleotide sequence as shown in SEQ ID NO: 37, SEQ ID NO: 25, SEQ ID NO: 50 or SEQ ID NO: 49; the siRNA preparation is a stable siRNA preparation for hepatocyte-targeted delivery; preferably, the siRNA is fluorinated and methoxylated and then covalently bound to N-acetylgalactosamine to form a conjugate.

6. A pharmaceutical composition or a kit for alleviating or treating metabolic associated fatty liver disease, which comprises the siRNA or the siRNA preparation for alleviating or treating metabolic associated fatty liver disease according to claim 5, and a pharmaceutically acceptable carrier or excipient.

7. Use of the InsR-C / EBPα-TKT signaling pathway for screening substances for alleviating or treating metabolic associated fatty liver disease.

8. A method for screening substances for alleviating or treating metabolic associated fatty liver disease, comprising: (1) contacting a candidate substance with a system containing the InsR-C / EBPα-TKT signaling pathway; (2) screening out substances that down-regulate the InsR-C / EBPα-TKT signaling pathway, and the substances are useful for alleviating or treating metabolic associated fatty liver disease; wherein, the down-regulation includes: down-regulating TKT, down-regulating the C / EBPα-TKT interaction, and down-regulating the insulin / InsR-C / EBPα interaction.

9. The method according to claim 8, wherein Step (1) includes: adding a candidate substance to a system containing the InsR-C / EBPα-TKT signaling pathway; Step (2) includes: detecting the changes of each protein or its coding 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 down-regulates TKT, down-regulates the C / EBPα-TKT interaction or down-regulates the insulin / InsR-C / EBPα interaction, then the candidate substance is a useful substance for alleviating or treating metabolic associated fatty liver disease.

10. Use of liver TKT protein or its coding gene in the preparation of a diagnostic reagent for diagnosing or predicting the prognosis of metabolic associated fatty liver disease; preferably, the diagnosis or prognosis includes: Based on the expression of liver TKT protein, determine the occurrence or progression of its metabolic associated fatty liver disease, or determine whether it is suitable for a treatment regimen using a "downregulator of the TKT or InsR-C / EBPα-TKT signaling pathway containing TKT"; if the TKT protein is highly expressed, then this treatment regimen is applicable.

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