Application of transketolase inhibitor of targeted macrophages in preparation of medicine for preventing and treating fatty liver

Abstract

The invention provides application of a transketolase inhibitor targeting macrophages in preparation of a medicine for preventing and treating fatty liver, and relates to the technical field of biology. In the application provided by the invention, the transketolase inhibitor is selected from at least one of hydroxythiamine, siRNA (small interfering Ribonucleic Acid) of transketolase and shRNA (short hairpin Ribonucleic Acid) of transketolase. Inhibitors that target macrophage transketolase can eliminate or slow down the progression of MAFLD and MASH. After macrophage transketolase is knocked down, the activation level of inflammation can be reduced, metabolism of glucose, lipid and amino acid is reprogrammed, the proportion of alpha-KG / fumarate and alpha-KG / succinate is reduced, DNA hypermethylation is caused, and chromatin accessibility of pro-inflammatory genes in macrophages is reduced. The invention provides a novel application of a targeted macrophage transketolase inhibitor in prevention and treatment of metabolic dysfunction related fatty liver.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to the application of a transketolase inhibitor targeting macrophages in the preparation of a drug for preventing and treating fatty liver. Background Art

[0002] Macrophages are an important part of the innate immune system and are widely distributed in various organs and tissues of humans. When Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), transcription factors in macrophages are activated, thereby initiating the production of a variety of pro-inflammatory cytokines and chemokines to eliminate invading pathogens, mediate antigen presentation, regulate the degree and duration of the inflammatory response, and participate in tissue repair and remodeling. However, the over-activation of TLRs may lead to out-of-control inflammatory responses, thereby disrupting immune homeostasis and triggering acute life-threatening sepsis or other chronic diseases, including metabolic dysfunction-associated fatty liver disease (MAFLD), diabetes, atherosclerosis, ankylosing spondylitis, etc. Therefore, identifying key regulatory factors that modulate macrophage-mediated inflammation is crucial for the treatment of such inflammatory or metabolic diseases.

[0003] Metabolic-dysfunction-associated fatty liver disease (MAFLD) is caused by factors other than alcohol and other well-defined liver-damaging factors, and is mainly characterized by a clinical and pathological syndrome mainly manifested by diffuse hepatocyte macrovesicular steatosis, lipid metabolism disorders, and inflammation induced by lipid peroxidation. If not controlled, MAFLD can further develop into non-alcoholic steatohepatitis, hepatic fibrosis, cirrhosis, and even liver cancer and other diseases. At the same time, MAFLD is also one of the main causes of related diseases such as cardiovascular and cerebrovascular diseases, diabetes, heart diseases, and chronic kidney diseases. The pentose phosphate pathway (PPP) is an important part of glucose catabolism and is divided into an oxidative branch and a non-oxidative branch. In the oxidative branch of the PPP, glucose-6-phosphate (G6P) is oxidized to generate diphosphoglycerate (DPP) and ribulose-5-phosphate (Ru5P). The production of DPP in this branch is crucial for maintaining the redox balance of cells and supporting anabolic processes. Ru5P and other sugar phosphates are converted into 5-phosphoribose (R5P) and 5-phosphoxylulose (X5P), which helps generate pentoses for nucleotide synthesis and provides intermediates that can be used for glycolysis and other metabolic pathways. Glucose-6-phosphate dehydrogenase (G6PD) and carbohydrate kinase-like protein (CARKL) are key enzymes in the oxidative PPP and have been shown to affect the redox state of cells, thereby affecting the inflammatory response of macrophages. However, the role of the non-oxidative PPP in macrophage-mediated inflammation remains unclear.

[0004] The non-oxidative pentose phosphate pathway (PPP) has only recently received attention. Transketolase (TKT) is a non-oxidative PPP enzyme that can reversibly convert non-oxidative PPP metabolites into glycolytic intermediates, enabling cells to adjust their metabolism according to their functional needs to adapt to different environmental conditions. Functionally, TKT catalyzes two reversible reactions: the conversion of D-xylulose 5-phosphate (Xu5P) and ribose 5-phosphate (R5P) into glyceraldehyde 3-phosphate (G3P) and sedoheptulose 7-phosphate (S7P); and the conversion of Xu5P and D-erythrose 4-phosphate (E4P) into G3P and fructose 6-phosphate (F6P). The lack of TKT in mouse hepatocytes, adipocytes, or T cells affects cell phenotype and function and accelerates the progression of diseases, including obesity, hepatic steatosis, and autoimmune diseases. However, the role of the macrophage TKT-mediated non-oxidative pentose phosphate pathway in regulating metabolism-related diseases has not been reported.

[0005] Based on this, the development of the application of non-oxidative pentose phosphate pathway acting factors in the preparation of drugs for preventing and treating metabolic diseases is the focus of researchers in this field. Summary of the Invention

[0006] In view of the above problems, the present invention provides an application of a transketolase inhibitor targeting macrophages in the preparation of a drug for preventing and treating fatty liver.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides an application of a transketolase inhibitor targeting macrophages in the preparation of a drug for preventing and treating fatty liver, and the transketolase inhibitor is selected from at least one of hydroxythiamine, siRNA of transketolase, and shRNA of transketolase.

[0008] Preferably, the fatty liver is selected from at least one of simple fatty liver, metabolic associated fatty liver disease (MAFLD), and metabolic associated fatty hepatitis (MASH).

[0009] More preferably, the fatty liver is selected from at least one of metabolic associated fatty liver disease (MAFLD) and metabolic associated fatty hepatitis (MASH).

[0010] Preferably, the drug inhibits the up-regulation of the expression of lipid absorption genes, lipid transport genes, and lipid synthesis genes.

[0011] More preferably, the lipid absorption genes include Fatp1 and Fabp1.

[0012] More preferably, the lipid transport gene includes CD36.

[0013] More preferably, the lipid synthesis genes include Fasn, Pparγ, Scd1, Srebf1, and Acca.

[0014] Preferably, the drug upregulates the expression of lipolysis genes, lipid β-oxidation genes, and lipid utilization genes.

[0015] More preferably, the lipolysis genes include Atgl, Mgl, and Hsl.

[0016] More preferably, the lipid β-oxidation genes include Pparα and Cpt1α.

[0017] More preferably, the lipid utilization gene includes Ucp2.

[0018] Preferably, the drug for preventing and treating fatty liver includes a transketolase inhibitor and at least one of the following components: (1) a pharmaceutically acceptable carrier; (2) optionally one or more other active substances for treating metabolic diseases.

[0019] Preferably, the effects of the drug for preventing and treating fatty liver include at least one of the following: (1) reducing body weight; (2) reducing liver weight; (3) reducing the ratio of liver weight to body weight; (4) reducing the expression of lipid absorption genes in the body; (5) reducing the expression of lipid transport genes in the body; (6) reducing the expression of lipid synthesis genes in the body; (7) increasing the expression of lipolysis genes in the body; (8) increasing the expression of lipid β-oxidation genes in the body; (9) increasing the expression of lipid utilization genes in the body; (10) alleviating liver injury; (11) reducing the concentration of aspartate aminotransferase (AST) in the serum; (12) reducing the concentration of alanine aminotransferase (ALT) in the serum.

[0020] Preferably, the drug inhibits the expression of lipid absorption genes, lipid transport genes, and lipid synthesis genes by inhibiting the activity of transketolase.

[0021] Preferably, the drug inhibits the expression of inflammatory genes through the TAK1-NF-κB / MAPK signaling pathway, thereby slowing down the progression of fatty liver.

[0022] Preferably, the pharmaceutically acceptable carrier is selected from at least one of excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH regulators, cryoprotectants, flavoring agents, and fillers.

[0023] Preferably, the excipient is selected from at least one of microcrystalline cellulose, lactose, cyclodextrin, carboxymethyl cellulose, mannitol, magnesium stearate, starch, calcium phosphate, ethyl cellulose, methyl cellulose, alginic acid, gelatin, gum arabic, glyceryl monostearate, sodium starch glycolate, guar gum, glycerol, and propylene glycol.

[0024] Preferably, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0025] Preferably, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, glyceryl monooleate, and polyglyceryl-3 polyricinoleate.

[0026] Preferably, the stabilizer is selected from at least one of acacia gum, agar, alginic acid, cellulose ether, and carboxymethyl chitin ester.

[0027] Preferably, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0028] Preferably, the binder is selected from at least one of ethanol, starch paste, pregelatinized starch, dextrin, syrup, hydroxypropyl methyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, sodium alginate, polyvinylpyrrolidone, gum arabic, gelatin, and alginic acid.

[0029] Preferably, the preservative is selected from at least one of methyl paraben, propyl paraben, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethyl paraben.

[0030] Preferably, the lubricant is selected from at least one of magnesium stearate, zinc stearate, glyceryl monostearate, polyethylene glycol, stearic acid, talc, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium stearyl fumarate, and poloxamer.

[0031] Preferably, the pH regulator is selected from at least one of citric acid, fumaric acid, succinic acid, tartaric acid, malic acid, and ascorbic acid.

[0032] Preferably, the cryoprotectant is selected from at least one of sucrose, glucose, mannitol, fructose, trehalose, dextrose, lactose, glycerol, methanol, ethanol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), acetamide, and formamide.

[0033] Preferably, the flavoring agent is selected from at least one of sweet orange flavor, vanilla flavor, strawberry flavor, milk flavor, banana flavor, and cherry flavor.

[0034] Preferably, the filler is selected from at least one of mannitol, xylitol, sorbitol, maltose, microcrystalline cellulose, glucose, lactose, sucrose, dextrin, starch, sodium alginate, and sodium bicarbonate.

[0035] Preferably, the dosage form of the drug is selected from at least one of drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, emulsion, pill, freeze-dried powder injection, gel, suppository, and aerosol.

[0036] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a non-oxidative pentose phosphate pathway acting factor, a transketolase inhibitor, which can eliminate or slow down the progression of MAFLD and MASH. After knocking down macrophage transketolase, the activation level of inflammation is reduced by inhibiting the TAK1-NF-κB / MAPK signaling pathway. At the same time, the metabolism of glucose, lipids, and amino acids is reprogrammed, and the ratios of α-KG / fumarate and α-KG / succinate are reduced, resulting in DNA hypermethylation and reduced chromatin accessibility of pro-inflammatory genes in macrophages, thereby inhibiting the inflammation level. Inflammation is also one of the factors contributing to the progression of MAFLD / MASH. Inhibiting TKT will reduce the expression of macrophage inflammatory genes, thus alleviating lipid accumulation in hepatocytes (manifested as reduced expression levels of lipid transport and synthesis genes throughout the liver and increased expression levels of lipid oxidation and utilization genes), inflammatory infiltration (manifested as downregulated expression levels of inflammatory factor chemokines throughout the liver), and liver fibrosis (reduced expression of liver fibrosis genes). By regulating inflammation by affecting the expression of macrophage TKT, the progression of fatty liver in the liver is further affected.

[0037] 2. The present invention also designs an AAV-shTKT adenovirus vector targeting liver macrophage TKT and has achieved good therapeutic effects in the treatment and prevention of metabolic dysfunction-related fatty liver, providing potential clinical applications for the prevention and treatment of metabolic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a diagram showing the inhibition of TKT expression in macrophages under PA stimulation.

[0039] Figure 2 Figure for inhibiting macrophage inflammatory response by reducing TKT Among them, A shows the expression of inflammatory cytokines detected by qPCR after wild-type PMs were pretreated with the TKT inhibitor OT for 12 h and then stimulated with PA for 12 h. Il-6 Figure B shows the expression of inflammatory cytokines detected by qPCR after wild-type PMs were pretreated with the TKT inhibitor OT for 12 h and then stimulated with PA for 12 h. Il-1β Figure C shows the expression of the inflammatory cytokine TNF-α detected by qPCR after wild-type PMs were pretreated with the TKT inhibitor OT for 12 h and then stimulated with PA for 12 h. D shows the mRNA levels of macrophages detected by qPCR after wild-type PMs were transfected with siCtrl or siTKT respectively and stimulated with PA 48 h later. Tkt Figure E shows the mRNA levels of macrophage inflammatory cytokines detected by qPCR after wild-type PMs were transfected with siCtrl or siTKT respectively and stimulated with PA 48 h later. Il-6 Figure F shows the mRNA levels of macrophage inflammatory cytokines detected by qPCR after wild-type PMs were transfected with siCtrl or siTKT respectively and stimulated with PA 48 h later. Il-1β Figure G shows the mRNA levels of macrophage inflammatory cytokines detected by qPCR after wild-type PMs were transfected with siCtrl or siTKT respectively and stimulated with PA 48 h later. Tnf-α Figure

[0040] Figure 3 Figure for the expression of macrophage pro-inflammatory cytokines being inhibited by TKT myeloid knockout Among them, A shows the mRNA levels of macrophages detected by qPCR after TKT-WT-PMs and TKT-KO-PMs were stimulated with PA for 12 h. Tkt Figure B shows the mRNA levels of macrophage inflammation-related genes detected by qPCR after TKT-WT-PMs and TKT-KO-PMs were stimulated with PA for 12 h. Il-6 Figure C shows the mRNA levels of macrophage inflammation-related genes detected by qPCR after TKT-WT-PMs and TKT-KO-PMs were stimulated with PA for 12 h. Il-1β Figure D shows the mRNA levels of macrophage inflammation-related genes detected by qPCR after stimulating TKT-WT-PMs and TKT-KO-PMs with PA for 12 h. Tnf-α The figure of mRNA levels.

[0041] Figure 4 The figure showing that myeloid-specific knockout of TKT improves the progression of MAFLD induced by high-fat diet in mice; Among them, A is the figure of the ratio of mouse liver weight to body weight; B is the figure of the contents of total cholesterol (TC) and triglyceride (TG) detected by kit in mouse liver; C is the H&E staining figure of liver paraffin sections; D is the Oil Red O staining figure of liver frozen sections; E is the figure of the expression levels of lipid-related genes detected by qPCR in mouse liver; F is the figure of the expression levels of inflammation-related genes detected by qPCR in mouse liver; G is the fluorescence figure (left side) and quantification (right side) of immunofluorescence detection of mononuclear macrophages (F4 / 80), IL-6 / IL-1β, and DAPI in mouse liver; H is the figure of the levels of AST and ALT detected by kit in mouse serum.

[0042] Figure 5 The figure showing that myeloid-specific knockout of TKT improves the progression of MAFLD induced by high-fat diet in mice; Among them, A is the schematic diagram of feeding control diet and high-fat diet; B is the figure of continuously monitoring the body weight change of mice; C is the figure of mouse liver weight; D is the line graph of mouse GTT and the bar graph of the area under the curve; E is the line graph of mouse ITT and the bar graph of the area under the curve; F is the H&E staining figure of mouse white adipose tissue paraffin sections.

[0043] Figure 6 The figure showing that myeloid-specific knockout of TKT improves the progression of MASH induced by high-fat and high-cholesterol diet in mice; Among them, A is the figure of the ratio of mouse liver weight to body weight; B is the figure of the contents of TC and TG detected by kit in mouse liver; C is the H&E staining figure of liver paraffin sections; D is the Oil Red O staining figure of liver frozen sections; E is the figure of the expression levels of lipid-related genes detected by qPCR in mouse liver; F is a graph showing the expression levels of inflammation-related genes in the livers of mice detected by qPCR; G is a fluorescence image (left) and quantification (right) of monocytes / macrophages (F4 / 80), IL-6 / IL-1β, and DAPI in the livers of mice detected by immunofluorescence; H is a graph showing the levels of AST and ALT in the sera of mice detected by a kit.

[0044] Figure 7 It is a graph showing the improvement of the progression of MASH induced by a high-fat and high-cholesterol diet in mice by myeloid-specific knockout of TKT; A is a schematic diagram of the feeding of control diet and high-fat diet, and age- and weight-matched male mice of TKT-WT-Mφ and TKT-KO-Mφ are fed the corresponding diets; B is a graph showing the continuous monitoring of the body weight changes of mice; C is a graph showing the weight gain of mice; D is a graph showing the liver weights of mice; E is an H&E staining image of paraffin sections of white adipose tissue in mice.

[0045] Figure 8 It is a graph showing the inhibition of the TLR4-NF-κB signaling pathway by TKT deficiency in macrophages;

[0046] Figure 9 It is a graph showing that TKT deficiency reduces the α-KG / fumarate and α-KG / succinate ratios; Among them, A is a graph showing the changes in metabolites related to glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle in the untargeted metabolome of TKT-WT-PMs and TKT-KO-PMs; B is a graph showing the ratios of the metabolites α-KG / fumarate and α-KG / succinate in untargeted metabolomics.

[0047] Figure 10 It is a graph showing the influence of TKT on the macrophage epigenetic landscape; Among them, A is a graph showing the changes in the levels of histone methylation modifications H3K27me3, H3K4me3, H3K9me3, and H3K79me3 in TKT-WT-PMs and TKT-KO-PMs cells detected by immunoblotting; B is a graph showing the levels of 5hmc in TKT-WT-PMs and TKT-KO-PMs cells measured by ELISA; C-E are ATAC-seq analysis diagrams of TKT-WT-PMs and TKT-KO-PMs cells (cell sets of 4 mice per group); C is the ATAC-seq signal map of open chromatin regions of TKT-WT-PMs and TKT-KO-PMs cells; D is the De novo motif enrichment analysis diagram of genes with downregulated chromatin openness in TKT-KO-PMs; E is the genomic browser view of specified ATAC-seq-related gene loci in TKT-WT-PMs and TKT-KO-PMs cells; F is the ChIP-qPCR diagram of p65 at the gene promoter locus in macrophages of TKT-WT-PMs and TKT-KO-PMs cells Il-6 ; G is the ChIP-qPCR diagram of cFos at the gene promoter locus in macrophages of TKT-WT-PMs and TKT-KO-PMs cells Il-6 ;

[0048] Figure 11 AAV-shRNA targeting TKT in liver macrophages can improve MASH induced by HFHC in mice; Among them, A is a schematic diagram. WT mice are fed HFHF diet, and after 8 weeks, AAV adenovirus is injected via the tail vein. After 2 weeks, GTT and ITT levels are detected, and tissue samples are taken after 16 weeks of feeding; B is the fluorescence diagram of immunofluorescence detection of macrophages (CD68), adenovirus EGFP, and DAPI in the mouse liver; C is the flow sorting strategy diagram of mouse liver macrophages; D is the diagram of the silencing level of TKT in sorted macrophages detected by qPCR and western blot; E is the diagram of the expression levels of related cytokines, chemokines, and anti-inflammatory cytokines detected by qPCR in sorted macrophages Il- 10 ; F is the diagram of continuously monitoring the body weight change of mice; G is the diagram of the mouse liver weight; H is the diagram of the mouse liver / body weight ratio; I is the diagram of the mouse GTT; J is the diagram of the mouse ITT; K refers to the H&E staining diagram of white adipose tissue and liver tissue; L refers to the diagram of the contents of TC and TG detected in the mouse liver by the kit; M refers to the diagram of the mRNA levels of inflammation-related genes detected by qPCR in liver tissue; N refers to the figure of the mRNA levels of genes related to lipid metabolism detected by qPCR in liver tissues; O refers to the figure of the mRNA levels of genes related to fibrosis detected by qPCR in liver tissues; P refers to the figure of the levels of AST and ALT detected in the sera of mice using a kit.

[0049] Note: Explanation of significant differences involved in the attached figures: No statistical difference (p > 0.05), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 are considered to be statistically significant. Detailed implementation manners

[0050] In order to make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further clarified below with reference to specific embodiments. However, the following embodiments are only the preferred embodiments of the present invention and not all of them. Based on the embodiments in the implementation manners, other embodiments obtained by those skilled in the art without creative efforts all fall within the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all ordinary commercially available products, and no specific limitation is made on their sources. The technical and scientific terms used in the embodiments have the meanings commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0051] Information of the kit: Oil red O staining kit, Solarbio - Cat #G1261; ALT detection kit, Nanjing Jiancheng - Cat #C009 - 2 - 1; ALT detection kit, Nanjing Jiancheng - Cat #C010 - 2 - 1; TC content detection kit, Solarbio - Cat #BC1985; TG detection kit, Solarbio - Cat #BC0625.

[0052] Statistical method: All data statistics and analysis are processed using Graph Pad Prism software, and GraphPad Prism software is used for chart drawing; in order to ensure the accuracy and reliability of experimental data, all quantitative experimental data are maintained with three biological replicates and three technical replicates, and the difference between samples is shown in the chart; the values are expressed as mean ± standard error, and two-tailed Student's t-test is used for differential analysis, and one-way ANOVA is used for data comparison among multiple samples; NS: No statistical difference (p > 0.05), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 are considered to be statistically significant.

[0053] Example 1 Palmitic acid (PA) stimulant reduces the expression of TKT in macrophages 1. Experimental method Mouse peritoneal macrophages (PMs) were extracted, then treated with inflammatory stimulants (PA 50 μM - 12 h), the cells were collected, and the expression level of TKT was detected by qPCR.

[0054] 2. Experimental results Macrophage-mediated inflammatory responses are closely related to metabolic processes. The non-oxidative PPP mediated by TKT significantly affects the functions of hepatocytes, adipocytes, and regulatory T cells. In particular, knocking out TKT in hepatocytes can significantly improve steatohepatitis and liver fibrosis. However, the role of TKT in macrophages in the progression of steatohepatitis remains unknown. Therefore, we first investigated whether the expression of TKT is related to macrophage inflammation mediated by the lipotoxic agent palmitic acid.

[0055] When mouse peritoneal macrophages (PMs) were exposed to the lipotoxic inducer saturated fatty acid palmitic acid (PA), the expression level of TKT was significantly decreased by qPCR analysis ( Figure 1 ), indicating that lipotoxicity inhibits the expression of TKT in macrophages.

[0056] Example 2 Lack of macrophage TKT inhibits the expression of pro-inflammatory cytokines 1. Experimental method Mouse peritoneal macrophages (PMs) were extracted, transfected with siTKT, the cells were stimulated with PA 48 hours later, the cells were collected 12 hours later, and the knockdown efficiency and the expression levels of inflammatory factors were detected by qPCR.

[0057] Mouse peritoneal macrophages (PMs) were extracted, pretreated with oxythiamine (OT, a selective enzyme inhibitor of TKT) for 12 hours, then stimulated with PA, the cells were collected 12 hours later, and the expression levels of inflammatory factors were detected by qPCR.

[0058] 2. Experimental results Subsequently, we attempted to determine whether TKT in macrophages in turn affects the activation of inflammation.

[0059] We found that in PA-treated mouse PMs, treatment with oxythiamine or knockdown of TKT by small interfering RNA (siRNA) could significantly reduce the mRNA expression of pro-inflammatory cytokines (including interleukin IL-6, IL-1β and tumor necrosis factor α ( Tnf- α )) ( Figure 2 , including Figure 2in A-G). These findings indicate that inhibiting TKT can reduce PA-induced inflammatory responses.

[0060] To further clarify the role of TKT in inflammation in macrophages, we generated bone marrow-specific TKT knockout mice ( Tkt fl / fl Lyz2 -Cre + , namely TKT-KO-Mφ mice) and littermate mice ( Tkt fl / fl , namely TKT-WT-Mφ mice). Next, we treated WT and KO PMs with PA and found that the mRNA expression levels of Il-6 , Il-1β and Tnf-α in TKT-deficient macrophages were significantly lower than those in WT-PM ( Figure 3 , including Figure 3 in A-D), further verifying the key role of TKT in regulating macrophage-mediated inflammation.

[0061] Example 3 Myeloid-specific TKT deficiency alleviates high-fat diet-induced MAFLD Macrophages play a key role not only in the development of acute inflammatory sepsis but also in the progression of chronic inflammation-related diseases (such as metabolic dysfunction-associated fatty liver disease, MAFLD). MAFLD is initially driven by metabolic syndromes such as obesity, insulin resistance (IR), and type 2 diabetes, and then progresses from metabolic dysfunction-associated fatty liver (MAFLD) characterized by simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH) caused by inflammatory responses. Macrophages are activated by factors such as LPS derived from gut bacteria (which migrates into the liver due to intestinal barrier dysfunction) or saturated free fatty acids (such as PA), and significantly promote severe hepatic lipid accumulation, hepatocyte ballooning, lobular inflammation, and fibrosis by releasing cytokines, thus exacerbating inflammation. Since the expression of pro-inflammatory cytokines is significantly reduced in TKT-KO macrophages treated with PA or LPS, we hypothesized that TKT in macrophages might affect the progression of MAFLD.

[0062] 1. Experimental methods Models of non-alcoholic fatty liver disease (MAFLD) and non-alcoholic steatohepatitis (MASH): Mice were housed under specific pathogen-free (SPF) conditions throughout the experiment. They were maintained on a 12-hour light-dark cycle at 22-24°C with unrestricted access to food and water. Male wild-type (WT) and knockout (KO) mice at 6 weeks of age were randomly divided into groups of 8-10 mice each. Mice in the normal group were fed a control diet (CD), mice in the MAFLD group were continuously fed a high-fat diet (HFD) for 24 weeks, and mice in the MASH group were continuously fed a high-fat and high-cholesterol diet (HFHC) for 24 weeks. During the experiment, the body weight of the mice was continuously monitored, and relevant metabolic indices were measured later. After the feeding period ended, samples were taken for subsequent experimental tests.

[0063] Glucose tolerance test (GTT): Glucose tolerance was measured in mice during the later stage of feeding with special diets. Before the test, the mice were fasted for 16 h and transferred to a new cage. After 16 h, blood was collected from the tip of the mouse's tail, and the blood glucose level at 0 h was measured using a Roche blood glucose meter. Meanwhile, a glucose solution was intraperitoneally injected at a dose of 1 U / kg body weight. The blood glucose levels of the mice were measured at different time points (15 min, 30 min, 60 min, 90 min, and 120 min) after injection, and the readings were recorded and statistically analyzed.

[0064] Insulin tolerance test (ITT): The insulin tolerance test was performed in mice during the later stage of feeding with special diets, at a time interval after the GTT experiment. Before the test, the mice were fasted for 4 h and transferred to a new cage. While preparing the insulin solution, a glucose solution was also prepared to prevent the risk of hypoglycemia in the mice. The blood glucose level at 0 h was measured using a Roche blood glucose meter. Meanwhile, an insulin solution was intraperitoneally injected at a dose of 0.75 U / kg body weight. The blood glucose levels of the mice were measured at different time points (15 min, 30 min, 60 min, 90 min, and 120 min) after injection, and the readings were recorded and statistically analyzed.

[0065] Hematoxylin and eosin staining (H&E staining): After the mice were sacrificed by cervical dislocation, appropriate-sized tissues were fixed in 4% paraformaldehyde solution for at least 48 h. Gradient dehydration and clearing were performed (70% ethanol I - 1 h; 70% ethanol II - 2 h; 80% ethanol - 30 min; 90% ethanol - 30 min; 95% ethanol I - 30 min; 95% ethanol II - 30 min; absolute ethanol I - 30 min; absolute ethanol II - 40 min; xylene I - 20 min; xylene II - 35 min). Then the tissues were successively immersed in paraffin I for 30 min and paraffin II at 65 °C overnight. After paraffin embedding, the tissues were cut into 5-μm sections with a microtome, the sections were floated on a 40 °C water bath, and baked at 65 °C for 1 h. Deparaffinization of paraffin sections: xylene I - 10 min; xylene II - 10 min; 100% absolute ethanol I - 5 min; 100% absolute ethanol II - 5 min; 95% ethanol - 10 min; 80% ethanol - 10 min; rinsed with running water for 5 min. Stained with hematoxylin for 2 min and rinsed with running water for 5 min. Differentiated with hydrochloric acid alcohol: 0.5% hydrochloric acid alcohol for 3 s; tap water for 3 s; rinsed with running water for 5 min. Counterstained with eosin for 4 - 5 min. Dehydrated and cleared: 80% ethanol - 1 s; 95% ethanol - 1 s; absolute ethanol I - 10 min; absolute ethanol II - 10 min; xylene I - 10 min; xylene II - 10 min. After air-drying, the sections were sealed with neutral balsam, and care was taken to expel air bubbles. Observed and photographed under a microscope.

[0066] Oil Red O staining: Appropriate-sized fresh liver tissues were taken, blotted dry with filter paper, placed in OCT embedding medium, snap-frozen in liquid nitrogen, and stored at -80 °C. After frozen sections (8 μm) were made, the sections were placed in distilled water for 2 min for rewarming, and then isopropanol was added for 2 min. Prepare staining solution A:B = 3:2, let stand for 10 min and then filter with a filter, prepare and use immediately, and store in the dark. Drop the staining solution and stain in a sealed manner for 10 - 20 min. Wash with 60% isopropanol, add distilled water for 2 min. Stain with hematoxylin staining solution for 2 min, wash with distilled water and then rinse with tap water for 5 min to blue. After air-drying, the sections were sealed with glycerin gelatin mounting medium.

[0067] 2. Experimental results We first investigated the function of TKT in a mouse model of MAFLD based on 24-week high-fat diet (HFD) feeding. Compared with TKT-WT-Mφ mice, the body weight, liver weight, and the ratio of liver weight to body weight of TKT-KO-Mφ mice fed HFD for 24 weeks were all lower ( Figure 4 A in Figure 5 A- in Figure 5in C). In addition, compared with TKT-WT-Mφ control mice fed with HFD, TKT-KO-Mφ mice fed with HFD showed less insulin resistance, as supported by the results of glucose tolerance test (GTT) and insulin tolerance test (ITT) ( Figure 5 in D and Figure 5 in E).

[0068] In addition, the degree of lipid accumulation in the liver and peritoneal white adipose tissue (WAT) of TKT-KO-Mφ mice fed with a high-fat diet (HFD) was lower than that of TKT-WT-Mφ mice, as evidenced by the triglyceride (TG) / total cholesterol (TC) concentration ( Figure 4 in B), H&E staining ( Figure 4 in C and Figure 5 in F), and Oil Red O staining ( Figure 4 in D). Consistently, qPCR analysis of the liver showed that TKT-KO-Mφ mice fed with HFD had decreased gene expression levels related to lipid uptake (such as Fatp1 , Fabp1 ), lipid transport ( CD36 ), and lipid synthesis (such as Fasn , Pparγ , Scd1 , Srebf1 and Acca ), while gene expression involved in lipolysis (such as Atgl , Mgl and Hsl ), lipid β-oxidation (such as Pparα and Cpt1α ), and lipid utilization (such as Ucp2 ) was increased ( Figure 4 in E). The liver of TKT-KO-Mφ mice fed with HFD showed less severe inflammation, as demonstrated by the reduced expression of cytokines Il-6 and Il-1β and chemokines Ccl2, Cxcl2, Cxcl11 and Cxcl15 ( Figure 4 in F). Immunofluorescence staining results showed that compared with the WT control group, the liver sections of TKT-KO-Mφ mice fed with HFD had significantly fewer IL-1β or IL-6 positive macrophages ( Figure 4 in G). Finally, the liver injury of TKT-KO-Mφ mice fed with HFD was alleviated, and the concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were lower ( Figure 4 in H). In summary, myeloid-specific TKT deletion eliminated the progression of HFD-induced MAFLD.

[0069] Example 4 Myeloid-specific TKT deletion improves MASH induced by high-fat / high-cholesterol diet 1. The experimental method was the same as that in Example 3.

[0070] 2. Experimental results Since MASH is the advanced stage of MAFLD, we also investigated the role of TKT in a mouse MASH model induced by high-fat and high-cholesterol (HFHC) diet feeding. Compared with the WT littermate control group, the body weight, liver weight, and liver-to-body weight ratio of TKT-KO-Mφ mice fed with HFHC diet were lower ( Figure 6 A in Figure 7 A-D in Figure 6 In addition, the liver TG / TC levels of TKT-KO-Mφ mice were lower than those of TKT-WT-Mφ mice ( Figure 6 B in Figure 6 Consistent with the results in the HFD model, myeloid TKT knockout significantly attenuated the pathological features of MASH - including hepatic steatosis ( Figure 7 C in Figure 6 E in Figure 6 D in Figure 6 E in Figure 6 H in Figure 6 I in

[0071] Example 5 Macrophage TKT deficiency inhibits the TLR4-NF-κB signaling pathway 1. Experimental method Peritoneal macrophages (PMs) from WT and KO mice were extracted, and the cells were collected after stimulating the cells with PA for 15 min and 30 min. Western blot was used to detect the knockdown efficiency and the activation level of the signaling pathway.

[0072] 2. Experimental results As the chronic inflammatory disease MAFLD progresses, macrophages recognize PA as a danger signal that triggers TLR4-NF-κB signaling. Subsequently, we investigated whether TKT regulates PA-mediated inflammatory responses. We found that TKT deletion significantly inhibited the activation of the TLR4-NF-κB signaling pathway in PA-induced macrophages ( Figure 8). In summary, these results strongly suggest that TKT deficiency in macrophages inhibits the TAK1-NF-κB / MAPK-mediated inflammatory response upon PA stimulation.

[0073] Example 6 TKT deficiency reduces the α-KG / fumarate and α-KG / succinate ratios 1. Experimental method Untargeted metabolomics detection: Mouse PMs (5×10 6 ) were seeded in a 10 cm cell plate and cultured in complete DMEM medium. After 24 h, the cells were washed with ice-cold PBS, and 500 μL of cold methanol:H2O (4:1, v / v) was added to completely cover the cells. After standing at -80 °C for 20 min, the cells were scraped off on dry ice and collected into a pre-chilled 1.5 mL EP tube. Another 500 μL of methanol aqueous solution was added to rinse the remaining cells and collect them. LC-MS mass spectrometry analysis of the samples was performed by LipidALL Technologies (Changzhou, China). According to the standard protocol of internal standard metabolites, the relative abundances of metabolites in the samples were compared. After data quality control and normalization, the t-test method was used to determine the significant changes. Metabolites with a pre-defined P < 0.05 were defined as significantly different metabolites.

[0074] 2. Experimental results To further clarify the potential mechanism by which TKT promotes inflammation, we also performed metabolomics analysis on PMs of TKT-WT and TKT-KO. TKT deficiency led to the accumulation of R5P and S7P in the non-oxidative PPP ( Figure 9 A in). In contrast, the levels of glycolytic metabolites F6P and G3P increased. The pyruvate level decreased, and the contents of TCA cycle intermediates citrate, isocitrate, α-KG, fumarate, and malate did not change significantly; however, succinate accumulated ( Figure 9 A in). Notably, TKT knockout in macrophages led to a significant decrease in the α-KG / fumarate and α-KG / succinate ratios ( Figure 9 B in).

[0075] Example 7 DNA hypermethylation highlights the disruption of chromatin accessibility and transcriptome changes in TKT-deficient macrophages 1. Experimental method Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq): After the collection of cell samples, a self-built library was prepared using the Novoprotein Hyperactive ATAC-Seq Library Prep Kit for Illumina. The eluted DNA was subjected to library quality control and ATAC-seq, and the specific implementation was carried out by NovoGene Corporation.

[0076] Chromatin immunoprecipitation assay (ChIP-qPCR): After the collection of cell samples, detection was carried out using the CST SimpleChIp Plus Sonication Chromatin Ip Kit #56383.

[0077] 2. Experimental results α-KG is an essential cofactor for α-KG-dependent dioxygenases, such as histone demethylases and the ten-eleven translocation hydroxylase (Tet) family of DNA dioxygenases, and succinate and fumarate inhibit these enzymes in an α-KG-dependent manner. Although α-KG is known to regulate histone methylation in macrophages, we did not observe significant changes in histone methylation in TKT-KO-PM compared to WT-PM by immunoblot analysis ( Figure 10 in A). TET enzymes catalyze the conversion of 5-methylcytidine (5mC) to 5-hydroxymethylcytidine (5hmC), thereby reducing DNA methylation and increasing the hydroxymethylation level. Maintaining a high proportion of intracellular α-KG is crucial for maintaining the activity of TET1, TET2, and TET3 enzymes. ELISA analysis showed a decrease in the 5hmC level in TKT-deficient macrophages, indicating DNA hypermethylation ( Figure 10 in B).

[0078] To explore the epigenetic changes in macrophages after TKT knockout, we investigated genome-wide chromatin accessibility by performing ATAC-seq analysis in WT and KO-PM ( Figure 10 in C). Compared with TKT-WT-PM, we observed a decrease in chromatin accessibility of a large number of genes in TKT-KO-PM. Motif analysis of differentially open chromosomal regions revealed the enrichment of many inflammation-related transcription factors, including AP-1 and NF-κB ( Figure 10 in D). In addition, a decrease in chromatin accessibility was observed at the loci of some inflammatory genes Il-6 ( Figure 10 in E). Subsequently, we used chromatin immunoprecipitation combined with qPCR (ChIP-qPCR) and found that KO-PMs showed a decrease in the enrichment of the transcription factors NF-κB-p65 and AP-1-cFOS at the Il-6 promoter ( Figure 10F in Figure 10 and G in). These results indicate that DNA hypermethylation in TKT-deficient macrophages leads to disrupted chromatin accessibility and changes in the transcriptome.

[0079] Example 8 An AAV adenovirus vector targeting hepatic macrophage TKT improves the disease progression of MASH induced by HFHC diet in mice 1. Experimental method Use the AAV8 delivery system to knockdown TKT in mouse liver macrophages. The recombinant adeno-associated virus serotype 8 (AAV8) vector carries mouse shTKT or an empty vector with a CD68 promoter (a promoter specific for macrophages), AAV8-CD68-NC or AAV8-CD68-shTKT was produced by Obio Technology Co., Ltd. (Shanghai, China). Mice were injected with the virus via the tail vein.

[0080] 2. Experimental results To explore the therapeutic potential of targeting macrophage TKT in the treatment of MAFLD, we injected C57BL / 6 mice via the tail vein using an adeno-associated virus (AAV) delivery system. An AAV8 vector carrying mouse shTKT (AAV-shTKT) or an empty vector with a CD68 promoter (a promoter specific for macrophages) (AAV-EGFP-shNC) was injected via the tail vein into wild-type mice that had been fed an HFHC diet for eight weeks, and HFHC feeding was continued. During this period, the GTT and ITT levels of the mice were measured ( Figure 11 A in), and when fed for 16 weeks, mouse tissues were extracted for detection. Liver immunofluorescence results showed that the adenovirus successfully targeted macrophages ( Figure 11 B in), mouse liver macrophages were sorted by flow cytometry, and the knockdown efficiency of TKT was detected by qPCR and western blot ( Figure 11 C and in Figure 11 D in), and at the same time, qPCR detection found that inflammatory factors in the liver macrophages of mice injected with AAV-shTKT Il-6 , Il-1β , Tnf-α and chemokines Ccl2 , Cxcl9 showed decreased expression levels, while the expression level of Il-10 that inhibits inflammation was upregulated ( Figure 11 E in). Continuous monitoring of body weight found that the body weight of mice in the group with knockdown of liver macrophage TKT was lighter than that of the WT group, and the liver weight, liver / body weight ratio, GTT, and ITT levels were all lower than those of the control group ( Figure 11 F - in Figure 11In J), the results of H&E staining showed that the degree of lipid droplet accumulation in the adipose and liver tissues of mice injected with AAV-shTKT was significantly lower than that of AAV-shNC mice ( Figure 11 In K), the levels of TC and TG in the serum were lower ( Figure 11 In L), at the same time, the qPCR results of liver tissues showed that the expression levels of lipid metabolism-related genes, inflammatory cytokines, chemokines, and liver fibrosis-related genes promoting lipid droplet infiltration in the knockout group were lower, while the expression levels of anti-inflammatory cytokines Il-10 were higher ( Figure 11 In M- Figure 11 In O), in addition, the levels of AST and ALT in the serum of mice injected with AAV-shTKT detected by the kit were significantly decreased ( Figure 11 In P). In summary, these results indicate that the AAV adenovirus vector targeting macrophage TKT can effectively alleviate the occurrence and development of MASH in mice induced by a high-fat diet.

[0081] All in all, the above research results indicate that TKT-mediated non-oxidative PPP integrates the regulation of cell metabolism, epigenetic modification, and signaling pathways to affect the pro-inflammatory response.

[0082] The research results of the present invention indicate that the transketolase-mediated non-oxidative pentose phosphate pathway plays a key role in driving macrophage-mediated inflammatory responses by integrating the regulation of metabolism, epigenetics, and signal transduction. Importantly, we designed an AAV-shTKT adenovirus vector targeting liver macrophage TKT and achieved good therapeutic effects in the treatment and prevention of non-alcoholic fatty liver disease in mice, providing potential clinical applications for the management of chronic inflammatory diseases.

[0083] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a macrophage-targeted transketolase inhibitor in the preparation of a drug for preventing and treating fatty liver, characterized in that: The transketolase inhibitor is selected from at least one of hydroxythiamine, siRNA of transketolase and shRNA of transketolase.

2. The use according to claim 1, characterized in that: The fatty liver is selected from at least one of simple fatty liver, metabolism-related fatty liver disease and metabolism-related fatty hepatitis.

3. The use according to claim 2, characterized in that: The fatty liver is selected from at least one of metabolism-related fatty liver disease and metabolism-related steatohepatitis.

4. The use according to claim 1, characterized in that: The drug comprises a transketolase inhibitor and at least one of the following ingredients: (1) a pharmaceutically acceptable carrier; (2) Optional one or more other active substances for treating metabolic diseases.

5. The use according to claim 4, characterized in that: The pharmaceutically acceptable carrier is selected from at least one of an excipient, a buffer, an emulsifier, a stabilizer, a diluent, a binder, a preservative, a lubricant, a pH adjuster, a cryoprotectant, a flavoring agent, and a filler.

6. The use according to claim 1, characterized in that: The effects of the drug include at least one of the following: (1) Reduce body weight; (2) Reduce liver weight; (3) Reduce the ratio of liver weight to body weight; (4) Reduce the expression of lipid absorption genes in the body; (5) Reduce the expression of lipid transport genes in the body; (6) Reduce the expression of lipid synthesis genes in the body; (7) Increase the expression of fat decomposition genes in the body; (8) Increase the expression of lipid β-oxidation genes in the body; (9) Increase the expression of lipid utilization genes in the body; (10) Reduce liver damage; (11) Reduce the concentration of aspartate aminotransferase in serum; (12) Reduce the concentration of alanine aminotransferase in serum.

7. The use according to claim 1, characterized in that: The dosage form of the drug is selected from any one of drops, mixtures, tinctures, injections, tablets, powders, oral liquids, capsules, granules, ointments, suspensions, emulsions, pills, freeze-dried powder injections, gels, suppositories, and aerosols.

8. The use according to claim 1, characterized in that: The drug inhibits the expression of lipid absorption genes, lipid transport genes and lipid synthesis genes by inhibiting the transketolase enzyme activity.

9. The use according to claim 1, characterized in that: The drug inhibits the expression of inflammatory genes through the TAK1-NF-κB / MAPK signaling pathway, thereby slowing down the progression of fatty liver.

10. The use according to claim 1, characterized in that: The drug inhibits the up-regulation of the expression of lipid absorption genes, lipid transport genes and lipid synthesis genes; the drug promotes the up-regulation of the expression of lipolysis genes, lipid β-oxidation genes and lipid utilization genes.

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