Use of transketolase inhibitor targeting macrophages in preparation of drugs for preventing and treating fatty liver

Abstract

This invention provides the application of a macrophage-targeted transketolase inhibitor in the preparation of drugs for the prevention and treatment of fatty liver, relating to the field of biotechnology. In the application provided by this invention, the transketolase inhibitor is selected from at least one of hydroxythiamine, transketolase siRNA, and transketolase shRNA. Inhibitors targeting macrophage transketolase can eliminate or slow the progression of MAFLD and MASH. Knockdown of macrophage transketolase reduces the level of inflammatory activation, reprograms glucose, lipid, and amino acid metabolism, reduces the α-KG / fumarate and α-KG / succinate ratios, leads to DNA hypermethylation, and reduces the chromatin accessibility of pro-inflammatory genes in macrophages. This invention provides a novel application of macrophage-targeted transketolase inhibitors in the prevention and treatment of fatty liver associated with metabolic dysfunction.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of a macrophage-targeting transketolase inhibitor in the preparation of drugs for the prevention and treatment of fatty liver. Background Technology

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

[0003] Metabolic-dysfunction-associated fatty liver disease (MAFLD) is a clinicopathological syndrome characterized by diffuse macrovesicular steatosis of hepatocellular carcinoma, lipid metabolism disorders, and inflammation induced by lipid peroxidation, caused by factors other than alcohol and other clearly defined hepatotoxic factors. If left uncontrolled, MAFLD can further develop into non-alcoholic steatohepatitis, fatty liver fibrosis, cirrhosis, and even liver cancer. MAFLD is also a major contributing factor to cardiovascular and cerebrovascular diseases, diabetes, heart disease, and chronic kidney disease. The pentose phosphate pathway (PPP) is an important component of glucose catabolism, divided into oxidative and non-oxidative branches. In the oxidative branch of the PPP, glucose-6-phosphate (G6P) is oxidized to glycerol diphosphate (DPP) and ribomaltose-5-phosphate (Ru5P). The production of DPP in this branch is crucial for maintaining cellular redox homeostasis and supporting anabolistic processes. Ru5P and other sugar phosphates are converted into ribose-5-phosphate (R5P) and xylinosyl-5-phosphate (X5P), which facilitate the generation of pentoses for nucleoside synthesis and provide intermediates for glycolysis and other metabolic pathways. Glucose-6-phosphate dehydrogenase (G6PD) and carbohydrate kinase-like protein (CARKL) are key enzymes in the oxidation of PPP and have been shown to affect the redox state of cells, thereby influencing macrophage inflammatory responses. However, the role of non-oxidative PPP in macrophage-mediated inflammation remains unclear.

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

[0005] Therefore, the development of non-oxidative pentose phosphate pathway agents for the application in the preparation of drugs for the prevention and treatment of metabolic diseases is a key focus for researchers in this field. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides the application of a macrophage-targeting transketolase inhibitor in the preparation of a drug for the prevention and treatment of fatty liver.

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

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

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

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

[0011] More preferably, the lipid absorption genes include Fatp1 and Fapp1.

[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 promotes the upregulation of 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) One or more other active substances for treating metabolic diseases.

[0019] Preferably, the drug for preventing and treating fatty liver has at least one of the following effects: (1) Reduce 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) Reduces the expression of lipid synthesis genes in the body; (7) Increase the expression of fat breakdown 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 glutathione transferase (AST) in serum; (12) Reduce the concentration of alanine aminotransferase (ALT) in serum.

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

[0021] Preferably, the drug inhibits the expression of inflammatory genes through the TAK1-NF-κB / MAPK signaling pathway, thereby slowing 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 adjusters, 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, alginate, gelatin, gum arabic, glyceryl monostearate, sodium glycolate starch, 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 polyglycerol-3 polyricinoleate.

[0026] Preferably, the stabilizer is selected from at least one of acacia gum, agar, alginate, cellulose ether and carboxymethyl chitosan.

[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 adhesive is selected from at least one of ethanol, starch paste, pregelatinized starch, dextrin, syrup, hydroxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, sodium alginate, polyvinylpyrrolidone, gum arabic, gelatin, and alginic acid.

[0029] Preferably, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[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 stearate fumarate, and poloxamer.

[0031] Preferably, the pH adjuster 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 flavoring, vanilla flavoring, strawberry flavoring, milk flavoring, banana flavoring, and cherry flavoring.

[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 the following: drops, mixtures, tinctures, injections, tablets, powders, oral liquids, capsules, granules, ointments, suspensions, emulsions, pills, lyophilized powder for injection, gels, suppositories, and aerosols.

[0036] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a non-oxidative pentose phosphate pathway factor, a transketolase inhibitor, capable of eliminating or slowing the progression of MAFLD and MASH. Knockdown of macrophage transketolase reduces inflammation activation levels by inhibiting the TAK1-NF-κB / MAPK signaling pathway, while simultaneously reprogramming glucose, lipid, and amino acid metabolism, decreasing the α-KG / fumarate and α-KG / succinate ratios, leading to DNA hypermethylation and reducing chromatin accessibility of pro-inflammatory genes in macrophages, thereby suppressing inflammation levels. Inflammation is also a contributing factor to the progression of MAFLD / MASH. Inhibition of TKT reduces the expression of inflammatory genes in macrophages, thereby alleviating lipid accumulation in hepatocytes (manifested as decreased expression levels of lipid transport and synthesis genes and increased expression levels of lipid oxidation and utilization genes), inflammatory infiltration (manifested as downregulated expression levels of inflammatory chemokines throughout the liver), and liver fibrosis (decreased expression of liver fibrosis genes). By affecting macrophage TKT expression, inflammation is regulated, thus influencing the progression of fatty liver disease.

[0037] 2. This invention also designed an AAV-shTKT adenovirus vector that targets TKT in liver macrophages, and achieved good therapeutic effects in the treatment and prevention of fatty liver associated with metabolic dysfunction, providing potential clinical applications for the prevention and treatment of metabolic diseases. Attached Figure Description

[0038] Figure 1 This diagram illustrates the inhibition of TKT expression in macrophages under PA stimulation.

[0039] Figure 2 A diagram illustrating the reduction of macrophage inflammatory response by inhibiting TKT. in, A consisted of wild-type PMs pretreated with the TKT inhibitor OT for 12 h, followed by PA stimulation for 12 h. Inflammatory cytokines were detected by qPCR. Il-6 The diagram representing the expression; B consisted of wild-type PMs that were pretreated with the TKT inhibitor OT for 12 h, followed by PA stimulation for 12 h. Inflammatory cytokines were detected by qPCR. Il-1β The diagram representing the expression; C represents the expression of the inflammatory cytokine TNF-α in wild-type PMs after pretreatment with the TKT inhibitor OT for 12 h, followed by stimulation with PA for 12 h, and detection by qPCR. D represents the transfection of wild-type macrophages with siCtrl or siTKT, respectively, followed by PA stimulation 48 h later, and macrophage detection by qPCR. Tkt The diagram shows the mRNA level. E represents the transfection of wild-type macrophages with siCtrl or siTKT, respectively, followed by PA stimulation 48 h later, and qPCR detection of macrophage inflammatory cytokines. Il-6 The diagram shows the mRNA level. F represents the transfection of wild-type PMs with siCtrl or siTKT, respectively, followed by PA stimulation 48 h later, and qPCR detection of macrophage inflammatory cytokines. Il-1β mRNA level map; G was generated by transfecting wild-type PMs with siCtrl or siTKT, respectively, followed by PA stimulation 48 h later, and qPCR was used to detect inflammatory cytokines in macrophages. Tnf-α mRNA level diagram.

[0040] Figure 3 A diagram showing that TKT myeloid knockout can suppress the expression of pro-inflammatory cytokines in macrophages; Among them, A represents TKT-WT-PMs and TKT-KO-PMs stimulated with PA for 12 h, and macrophages were detected by qPCR. Tkt mRNA level map; B consists of TKT-WT-PMs and TKT-KO-PMs stimulated with PA for 12 h, and macrophage inflammation-related genes detected by qPCR. Il-6 mRNA level map; C represents TKT-WT-PMs and TKT-KO-PMs stimulated with PA for 12 h, followed by qPCR detection of macrophage inflammation-related genes. Il-1β mRNA level map; D represents TKT-WT-PMs and TKT-KO-PMs stimulated with PA for 12 h, followed by qPCR detection of macrophage inflammation-related genes. Tnf-α mRNA level diagram.

[0041] Figure 4 A diagram illustrating the progress of myeloid-specific TKT knockout in improving MAFLD induced by a high-fat diet in mice; Where A is the mouse liver weight / body weight ratio graph; B is a graph showing the levels of total cholesterol (TC) and triglycerides (TG) in mouse liver as determined by the kit; C is the H&E staining image of a paraffin section of the liver; D is an Oil Red O staining image of a frozen section of liver; E is a graph showing the expression levels of lipid-related genes in mouse liver detected by qPCR. F is a graph showing the expression levels of inflammation-related genes in mouse liver detected by qPCR; G is the fluorescence graph (left) and quantification (right) of monocytes / macrophages (F4 / 80), IL-6 / IL-1β, and DAPI in mouse liver detected by immunofluorescence. H is a graph showing the levels of AST and ALT in mouse serum detected by the kit.

[0042] Figure 5 A diagram illustrating the progress of myeloid-specific TKT knockout in improving MAFLD induced by a high-fat diet in mice; Where A is a schematic diagram of feeding control diet and high-fat diet; B is a graph showing the continuous monitoring of mouse weight changes; C shows the weight of the mouse liver; D is a line graph of mouse GTT and a bar chart of the area under the curve; E represents the mouse ITT line graph and the area under the curve histogram; F shows the H&E staining of a paraffin section of mouse white adipose tissue.

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

[0044] Figure 7 A figure illustrating the progress of TKT myeloid-specific knockout in improving MASH induced by a high-fat, high-cholesterol diet in mice; A is a schematic diagram of feeding control diet and high-fat diet. Age- and weight-matched male TKT-WT-Mφ and TKT-KO-Mφ mice were fed the corresponding diets. B is a graph showing the continuous monitoring of mouse weight changes; C represents the weight gain of mice; D shows the weight of the mouse liver; E is an H&E staining image of a paraffin section of mouse white adipose tissue.

[0045] Figure 8 Diagram showing the inhibition of the TLR4-NF-κB signaling pathway in macrophages with TKT deficiency.

[0046] Figure 9 TKT deficiency reduces the α-KG / fumarate and α-KG / succinate ratios; In this figure, A represents the changes in glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle-related metabolites in the non-targeted metabolome of TKT-WT-PMs and TKT-KO-PMs. B is a graph showing the ratios of metabolites α-KG / fumaric acid and α-KG / succinic acid in non-targeted metabolomics.

[0047] Figure 10 A diagram illustrating the influence of TKT on the macrophage epigenetic landscape; In this figure, 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 as detected by Western blotting. B is a graph showing the level of 5hmc in TKT-WT-PMs and TKT-KO-PMs cells as determined by ELISA. CE is the ATAC-seq analysis diagram 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 in TKT-WT-PMs and TKT-KO-PMs cells; D is the analysis of genes with downregulated chromatin openness in TKT-KO-PMs. De novo Motif enrichment analysis plot; E is a genome browser view specifying ATAC-seq-related gene loci in TKT-WT-PMs and TKT-KO-PMs cells; F is in TKT-WT-PMs and TKT-KO-PMs cells, p65 in macrophages Il-6 ChIP-qPCR diagram of gene promoter sites; G is present in TKT-WT-PMs and TKT-KO-PMs cells, and cFos is present in macrophages. Il-6 ChIP-qPCR diagram of gene promoter sites.

[0048] Figure 11 AAV-shRNA targeting TKT cells in liver macrophages can improve the MASH map induced by HFHC in mice; In this diagram, A represents a schematic. WT mice were fed HFHF diet and injected with AAV adenovirus via tail vein after eight weeks. GTT and ITT levels were measured after two weeks of feeding. Tissue samples were taken after 16 weeks of feeding. B is a fluorescence image of macrophages (CD68), adenovirus EGFP, and DAPI detected by immunofluorescence in mouse liver; C is a flow cytometry sorting strategy diagram for mouse liver macrophages; D is a graph showing the silencing level of TKT in sorted macrophages as detected by qPCR and Western blot. E represents the detection of relevant cytokines, chemokines, and anti-inflammatory cytokines in macrophages separated by qPCR. Il- 10 The level of expression; F is a graph showing the continuous monitoring of mouse weight changes; G represents the weight of the mouse liver; H represents the mouse liver / body weight ratio; I represents the mouse GTT plot; J represents the mouse ITT plot; K refers to the H&E staining pattern of white adipose tissue and liver tissue; L refers to the graph showing the levels of TC and TG in mouse liver detected by the kit; M refers to the mRNA level map of inflammation-related genes in liver tissue detected by qPCR; N refers to the mRNA level map of lipid metabolism-related genes in liver tissue detected by qPCR; O refers to the mRNA level map of fibrosis-related genes in liver tissue detected by qPCR; P refers to the level graph of AST and ALT in mouse serum detected by the kit.

[0049] Note: Explanation of significant differences in the attached figures: No statistical difference (p > 0.05), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 were considered statistically significant. Detailed Implementation

[0050] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the invention is further illustrated below with specific embodiments. However, these embodiments are merely preferred embodiments and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. It is worth noting that the raw materials used in this invention are all common commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0051] Kit information: 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 methods: All data were statistically analyzed and processed using GraphPad Prism software, and graphs were also created using GraphPad Prism software. To ensure the accuracy and reliability of the experimental data, all quantitative experimental data were replicated three times biologically and three times technically. Differences between samples were displayed in the graphs. Values ​​are expressed as mean ± standard error. Two-tailed Student's t-tests were used for difference analysis, and one-way ANOVA was used for comparisons of data among multiple samples. NS: No statistically significant difference (p > 0.05), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 are considered statistically significant.

[0053] Example 1 Palmitic acid (PA) stimulants reduce TKT expression in macrophages. 1. Experimental Methods Mouse peritoneal macrophages (PMs) were extracted and treated with an inflammatory stimulant (PA 50 μM-12 h). Cells were collected and TKT expression levels were detected by qPCR.

[0054] 2. Experimental Results Macrophage-mediated inflammatory responses are closely related to metabolic processes. TKT-mediated non-oxidative PPP significantly affects the function of hepatocytes, adipocytes, and regulatory T cells. In particular, TKT knockout in hepatocytes significantly improves steatohepatitis and liver fibrosis, but the role of TKT in the progression of steatohepatitis in macrophages remains unknown. Therefore, we first investigated whether TKT expression is associated with macrophage inflammation mediated by the lipotoxic palmitic acid.

[0055] When mouse peritoneal macrophages (PMs) were exposed to the lipotoxicity inducer saturated fatty acid palmitic acid (PA), qPCR analysis showed a significant decrease in TKT expression levels. Figure 1 This indicates that lipotoxicity inhibits TKT expression in macrophages.

[0056] Example 2 Macrophage TKT cells lack expression of pro-inflammatory cytokines. 1. Experimental Methods Mouse peritoneal macrophages (PMs) were extracted, transfected with siTKT, stimulated with PA after 48 hours, and collected after 12 hours. The knockdown efficiency and inflammatory factor expression levels were detected by qPCR.

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

[0058] 2. Experimental Results We then attempted to determine whether TKT in macrophages, in turn, affects the activation of inflammation.

[0059] We found that in PA-treated mouse PM, treatment with hydroxythiamine or knockdown of TKT via small interfering RNA (siRNA) significantly reduced pro-inflammatory cytokines (including interleukins). IL-6, IL-1β and tumor necrosis factor α ( Tnf- α mRNA expression of ()) Figure 2 ,include Figure 2(AG in the sample). These findings suggest that inhibiting TKT can reduce PA-induced inflammatory responses.

[0060] To further elucidate the role of TKT in macrophages during inflammation, we constructed a bone marrow-specific TKT knockout mouse (BMKT knockout mouse). Tkt fl / fl Lyz2 -Cre + (i.e., TKT-KO-Mφ mice) and littermate mice ( Tkt fl / fl (i.e., TKT-WT-Mφ mice). Next, we treated WT and KO PMs with PA and found that TKT-deficient macrophages... Il-6 , Il-1β and Tnf-α The mRNA expression level was significantly lower than that of WT-PM ( Figure 3 ,include Figure 3 The presence of AD in the data further validates the crucial role of TKT in regulating macrophage-mediated inflammation.

[0061] Example 3 Myeloid-specific TKT deficiency alleviates high-fat diet-induced MAFLD Macrophages play a crucial 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 steatohepatitis (MAFLD). MAFLD is initially driven by metabolic syndromes such as obesity, insulin resistance (IR), and type 2 diabetes, progressing from MAFLD characterized by simple steatosis to MAASH caused by an inflammatory response. Macrophages are activated by factors such as LPS (which migrates into the liver due to intestinal barrier dysfunction) from gut bacteria or saturated free fatty acids (PA), significantly promoting severe hepatic lipid accumulation, hepatocyte ballooning, lobar inflammation, and fibrosis through the release of cytokines, thereby exacerbating inflammation. Since the expression of pro-inflammatory cytokines is significantly reduced in TKT-KO macrophages treated with PA or LPS, we hypothesize that TKT in macrophages may influence the progression of MAFLD.

[0062] 1. Experimental Methods Nonalcoholic fatty liver disease (MAFLD) and nonalcoholic steatohepatitis (MASH) models: Mice were housed under specific pathogen-free (SPF) conditions at 22-24°C with unrestricted food and water for a 12-hour light-dark cycle. Six-week-old male WT and KO mice were randomly divided into groups of 8-10 mice each. The control group was fed a standard diet (CD), the MAFLD group was fed a high-fat diet (HFD) for 24 weeks, and the MASH group was fed a high-fat and high-cholesterol diet (HFHC) for 24 weeks. Body weight was continuously monitored throughout the period, and relevant metabolic indicators were measured later. After the rearing period, samples were collected for subsequent experimental analysis.

[0063] Glucose tolerance test (GTT): Mice were subjected to glucose tolerance testing in the later stage of special diet feeding. Before the test, they were fasted for 16 h and changed to new cages. After 16 h, blood was collected from the tail tip of the mice, and the 0 h blood glucose of the mice was measured using a Roche blood glucose meter. At the same time, a glucose solution of 1 U / kg body weight was injected intraperitoneally. The blood glucose level of the mice was measured at different time points after the injection (15 min, 30 min, 60 min, 90 min and 120 min), and the readings were recorded and statistically analyzed.

[0064] Insulin Tolerance Test (ITT): Mice underwent the insulin tolerance test in the later stages of a specially fed diet, some time after the GTT test. Mice were fasted for 4 hours and placed in new cages before the test. Glucose solution was prepared simultaneously with the insulin solution to prevent dangerous hypoglycemia. The 0-hour blood glucose level of the mice was measured using a Roche glucometer. Simultaneously, 0.75 U / kg body weight of insulin solution was injected intraperitoneally. Blood glucose levels were measured at different time points after injection (15 min, 30 min, 60 min, 90 min, and 120 min), and the readings were recorded and statistically analyzed.

[0065] Hematoxylin-eosin (H&E) staining: After cervical dislocation and necropsy of mice, appropriate tissue samples 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; anhydrous ethanol I - 30 min; anhydrous ethanol II - 40 min; xylene I - 20 min; xylene II - 35 min). The tissue was then sequentially immersed in paraffin I for 30 min and paraffin II overnight at 65°C. After paraffin embedding, the tissue was sectioned into 5 μm sections using a tissue sectioner, spread in a 40°C water bath, and baked at 65°C for 1 h. Dewaxing paraffin sections: Xylene I - 10 min; Xylene II - 10 min; 100% anhydrous ethanol I - 5 min; 100% anhydrous ethanol II - 5 min; 95% ethanol - 10 min; 80% ethanol - 10 min; rinse with running water for 5 min. Hematoxylin staining for 2 min, rinse with running water for 5 min. Dedifferentiation with hydrochloric acid and alcohol: 0.5% hydrochloric acid and alcohol for 3 s; tap water for 3 s; rinse with running water for 5 min. Eosin counterstaining for 4-5 min. Dehydration and clearing: 80% ethanol - 1 s; 95% ethanol - 1 s; anhydrous ethanol I - 10 min; anhydrous ethanol II - 10 min; xylene I - 10 min; xylene II - 10 min, air dry, and mount with neutral resin, taking care to remove air bubbles. Observe and photograph under a microscope.

[0066] Oil Red O staining: Fresh liver tissue of appropriate size was taken, blotted dry with filter paper, and placed in OCT embedding medium. After flash freezing in liquid nitrogen, it was stored at -80℃. Frozen sections (8 μm) were then placed in distilled water for 2 min to warm, followed by the addition of isopropanol for 2 min. Staining solution A:B = 3:2 was prepared, allowed to stand for 10 min, and then filtered. The solution was prepared fresh and used immediately, stored away from light. Staining was performed for 10-20 min in a sealed container with the staining solution added. The sections were washed with 60% isopropanol and then distilled water for 2 min. Hematoxylin and eosin staining was added for 2 min, followed by washing with distilled water and then rinsing with tap water for 5 min to achieve a blue reversal. After air-drying, the sections were mounted with glycerol-gelatin mounting medium.

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

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

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

[0070] 2. Experimental Results Since MASH is a late stage of MAFLD, we also investigated the role of TKT in a mouse model of MASH induced by a high-fat, high-cholesterol (HFHC) diet. Compared with the WT littermate control group, TKT-KO-Mφ mice fed an HFHC diet had lower body weight, liver weight, and liver-to-body weight ratio. Figure 6 A and Figure 7 In addition, the liver TG / TC levels in TKT-KO-Mφ mice were lower than those in TKT-WT-Mφ mice (AD). Figure 6 (B in the text). Consistent with results in the HFD model, myeloid TKT knockout significantly attenuated the pathological features of MASH—including hepatic steatosis ( Figure 6 C and Figure 6 D in the text), the size of lipid droplets in adipocytes ( Figure 7 E), liver mRNA levels of genes related to fatty acid absorption and synthesis or inflammation ( Figure 6 E and Figure 6 In the F), liver infiltration of F4 / 80 positive inflammatory macrophages (F4 / 80 positive inflammatory macrophages) Figure 6 Masson staining and serum AST / ALT concentration indicate liver fibrosis and damage. Figure 6 H and Figure 6 (I in the text). Overall, these findings suggest that TKT deficiency in macrophages can also improve MASH progression.

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

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

[0073] Example 6 TKT deficiency reduces the α-KG / fumarate and α-KG / succinate ratios. 1. Experimental Methods Non-targeted metabolomics detection: Extraction of mouse PMs (5×10 6 Cells were seeded in 10 cm cell culture plates and cultured in complete DMEM medium. After 24 h, 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 incubation at -80℃ for 20 min, the cells were scraped off on dry ice and collected into pre-chilled 1.5 mL EP tubes. 500 μL of methanol-water solution was added again to rinse the remaining cells and collect them. LC-MS mass spectrometry analysis of the samples was performed by LipidALL Technologies (Changzhou, China). The relative abundance of metabolites in the samples was compared according to the internal standard metabolite standard protocol. After quality control and normalization, the t-test was used to determine the significance of changes. Metabolites with P < 0.05 were predefined as significantly different metabolites.

[0074] 2. Experimental Results To further elucidate the potential mechanism by which TKT promotes inflammation, we also performed metabolomics analysis on the PM of TKT-WT and TKT-KO. TKT deficiency led to the accumulation of R5P and S7P in non-oxidative PPP (…). Figure 9 In contrast, the levels of glucose catabolites F6P and G3P increased. Pyruvate levels decreased, while the levels of TCA cycle intermediates citrate, isocitrate, α-KG, fumarate, and malate did not change significantly; however, succinate accumulation ( Figure 9 (A in the text). Notably, TKT knockout in macrophages led to a significant decrease in the α-KG / fumarate and α-KG / succinate ratios (A in the text). Figure 9 (B in the middle).

[0075] Example 7 DNA hypermethylation highlights the disruption of chromatin accessibility and transcriptome changes in TKT-deficient macrophages. 1. Experimental Methods Chromatin transposase accessibility sequencing (ATAC-seq): After cell sample collection, a library was built using the Novizan Hyperactive ATAC-Seq Library Prep Kit for Illumina. The eluted DNA was then subjected to library quality control and ATAC-seq, which was performed by Novogene.

[0076] Chromatin immunoprecipitation assay (ChIP-qPCR): After cell samples were collected, they were tested using CST's 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 10-11 transport hydroxylase (Tet) family of DNA dioxygenases), while 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 immunoblotting analysis. Figure 10 (A) TET enzymes catalyze the conversion of 5-methylcytidine (5mC) to 5-hydroxymethylcytidine (5hmC), thereby reducing DNA methylation and increasing hydroxymethylation levels. Maintaining a high intracellular α-KG ratio is crucial for maintaining the activities of TET1, TET2, and TET3 enzymes. ELISA analysis showed reduced 5hmC levels in TKT-deficient macrophages, indicating increased DNA hypermethylation (…). Figure 10 (B in the middle).

[0078] To investigate the epigenetic changes in macrophages after TKT knockout, we studied genome-wide chromatin accessibility using ATAC-seq analysis in WT and KO-PM cells. Figure 10 (C) Compared to TKT-WT-PM, we observed reduced chromatin accessibility for a large number of genes in TKT-KO-PM. Motif analysis of differentially open chromosomal regions revealed enrichment of many inflammation-related transcription factors, including AP-1 and NF-κB (C). Figure 10 (D in the text). Furthermore, in some inflammatory genes... Il-6 Reduced chromatin accessibility was observed at the sites ( Figure 10 (E in the text). Subsequently, using chromatin immunoprecipitation combined with qPCR (ChIP-qPCR), we found that KO-PMs expressed transcription factors NF-κB-p65 and AP-1-cFOS in... Il-6 Enrichment decreases at the promoter ( Figure 10F and Figure 10 These results indicate that DNA hypermethylation in TKT-deficient macrophages leads to disruption of chromatin accessibility and alterations in the transcriptome.

[0079] Example 8 AAV adenovirus vector targeting liver macrophage TKT ameliorated disease progression in diet-induced MASH in mice with high liver function hypersensitivity (HFHC). 1. Experimental Methods TKT was knocked down in mouse liver macrophages using the AAV8 delivery system. Recombinant adeno-associated virus serotype 8 (AAV8) vectors carrying mouse shTKT or empty vectors with the CD68 promoter (a promoter targeting macrophages) were used; AAV8-CD68-NC or AAV8-CD68-shTKT were manufactured by Obio Technology Co. Ltd. (Shanghai, China). Mice were injected with the virus via tail vein.

[0080] 2. Experimental Results To explore the therapeutic potential of targeting macrophage TKT in treating MAFLD, we administered adeno-associated virus (AAV) delivery systems via tail vein injection to C57BL / 6 mice. AAV8 vectors carrying mouse shTKT (AAV-shTKT) or an empty vector (AAV-EGFP-shNC) with the CD68 promoter (a promoter targeting macrophages) were injected via tail vein into wild-type mice that had been fed an HFHC diet for eight weeks. HFHC feeding continued, and GTT and ITT levels were measured during this period. Figure 11 (A) In this study, mice were fed this vaccine until 16 weeks of age, at which point tissue samples were extracted for analysis. Immunofluorescence results in the liver showed that the adenovirus successfully targeted macrophages (A). Figure 11 (B in the text) Mouse liver macrophages were sorted by flow cytometry, and the TKT knockdown efficiency was detected by qPCR and Western blot. Figure 11 C and Figure 11 (D in the text), and qPCR detection revealed inflammatory factors in the liver macrophages of mice injected with AAV-shTKT. Il-6 , Il-1β , Tnf-α and chemokines Ccl2 , Cxcl9 Downregulation of expression levels, while suppressing inflammation Il-10 Upregulation of expression level ( Figure 11 E in the text). Continuous monitoring of body weight revealed that mice in the TKT group with knocked-down liver macrophages had lighter body weight than the WT group, and their liver weight, liver / body weight ratio, GTT, and ITT levels were all lower than those in the control group. Figure 11 F- Figure 11In the J), ​​H&E staining results showed that the accumulation of lipid droplets in the fat and liver tissue of mice injected with AAV-shTKT was significantly less than that in AAV-shNC mice (J). Figure 11 K), serum TC and TG levels are lower ( Figure 11 In addition, qPCR results from liver tissue showed that the knockout group had lower expression levels of lipid metabolism-related genes, inflammatory cytokines, chemokines, and genes related to liver fibrosis that promote lipid droplet infiltration, while anti-inflammatory cytokines were lower. Il-10 The level of expression is relatively high. Figure 11 M- Figure 11 In addition, the kit showed that the serum AST and ALT levels in mice injected with AAV-shTKT were significantly reduced. Figure 11 (P in the text). In summary, these results indicate that the AAV adenovirus vector targeting macrophage TKT can effectively alleviate the development of MASH in mice induced by a high-fat diet.

[0081] In summary, the above findings indicate that TKT-mediated non-oxidative PPP integrates the regulation of cellular metabolism, epigenetic modifications, and signaling pathways to influence pro-inflammatory responses.

[0082] The results of this invention demonstrate that the transketolase-mediated nonoxidative pentose phosphate pathway plays a crucial role in driving macrophage-mediated inflammatory responses by integrating metabolic, epigenetic, and signal transduction regulation. Importantly, we designed an AAV-shTKT adenovirus vector targeting hepatic 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, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of a macrophage-targeting transketolase inhibitor in the preparation of a drug for the prevention and treatment of fatty liver, characterized in that, The transketolase inhibitor is hydroxythiamine, and the fatty liver is metabolic-associated fatty liver disease.

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

3. The application according to claim 2, characterized in that, The pharmaceutically acceptable carrier is selected from at least one of stabilizers, binders, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

4. The application according to claim 1, characterized in that, The effects of the drug include at least one of the following: (1) Reduce 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) Reduces the expression of lipid synthesis genes in the body; (7) Increase the expression of fat breakdown 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.

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

6. The application 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 activity of transketolase.

7. The application 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 the progression of fatty liver.

8. The application according to claim 1, characterized in that, The drug inhibits the upregulation of the expression of lipid absorption genes, lipid transport genes, and lipid synthesis genes; the drug promotes the upregulation of the expression of lipolysis genes, lipid β-oxidation genes, and lipid utilization genes.

Images