Application of inhibitor targeting macrophage transketolase in preparation of medicine for preventing and treating inflammatory diseases

Abstract

The invention discloses application of an inhibitor targeting macrophage transketolase in preparation of drugs for preventing and treating inflammatory diseases, and relates to the technical field of biology. The invention provides application of a transketolase inhibitor in preparation of a medicine for preventing or treating inflammatory diseases. The transketolase inhibitor can inhibit occurrence of infectious shock. The invention discloses an important function and mechanism of the non-oxidized PPP key enzyme TKT for promoting the inflammatory reaction of the macrophages by integrating metabolism and signal pathways, and discloses that the TKT can be used as a potential treatment target for treating the inflammatory diseases mediated by the macrophages.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of an inhibitor targeting macrophage transketolase in preparing medicines for preventing and treating inflammatory diseases. Background Art

[0002] The inflammatory response is usually a complex biological response of the body to infection, injury or other stimuli. This response involves the interaction of multiple cell types, molecular signals and chemicals. Macrophages are an important part of the innate immune system. As key inflammatory effector cells, they 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 proinflammatory cytokines and chemokines to clear invading pathogens, mediate antigen presentation, regulate the extent and duration of inflammatory responses, and participate in tissue repair and remodeling. After activation by lipopolysaccharide (LPS), a cell wall component released by Gram-negative bacteria, TLR4 triggers a downstream signaling cascade involving the myeloid differentiation primary response 88 (MyD88) adaptor protein and TLR domain-containing adaptor-inducing interferon-β (TIF), which subsequently activates an enzyme cascade including the transforming factor-β-activated proteinase 1 (TAK1) pathway, and downstream of that, the mitogen-activated proteinase (MEK) pathway and the nuclear factor kappa light-chain-enhancer of activated B cells (NF-kB) pathway. However, excessive activation of TLRs may lead to uncontrolled inflammatory responses, thereby disrupting immune homeostasis and triggering acute life-threatening sepsis or other chronic diseases, including diabetes, atherosclerosis, ankylosing arthritis, etc. Therefore, identifying the key regulators of macrophage-mediated inflammation is essential for the treatment of such inflammatory diseases.

[0003] When macrophages are activated by pathogens or other potential inflammatory factors, metabolic reprogramming occurs to meet their energy and biosynthetic needs, thereby providing essential metabolic intermediates such as reactive oxygen species (ROS), nitric oxide (NO) for antimicrobial function, and arginine for tissue remodeling. In addition, glycolytic activity increases, while the tricarboxylic acid cycle (TCA cycle), mitochondrial oxidative phosphorylation (OXPHOS), and fatty acid oxidation (FAO) are inhibited, ultimately leading to the acquisition of a pro-inflammatory phenotype by macrophages. In addition, metabolites such as citrate, itaconate, succinate, fumarate, serine, and α-ketoglutarate (α-KG) also play a vital role in the inflammatory response of macrophages. Macrophage metabolism can interfere with epigenetic modifications, signal transduction, redox status, and other regulatory processes, thereby affecting macrophage activation and function. Therefore, targeting macrophage metabolism is considered a potential strategy for treating inflammatory diseases.

[0004] The pentose phosphate pathway (PPP) is an important component of glucose catabolism and is divided into oxidative and nonoxidative branches. In the oxidative branch of the PPP, glucose-6-phosphate (G6P) is oxidized to generate diphosphoglycerol (DPP) and maltose-5-phosphate (Ru5P). The production of DPP in this branch is essential for maintaining the cellular redox balance and supporting anabolic processes. Ru5P and other sugar phosphates are converted into ribose-5-phosphate (R5P) and cellulose-5-phosphate (X5P), which contribute to the generation of pentoses for nucleoside synthesis and provide 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 cellular redox state and thus the inflammatory response of macrophages. However, the role of the nonoxidative PPP in macrophage-mediated inflammation remains largely undefined.

[0005] The nonoxidative pentose phosphate pathway (PPP) has only recently received attention. Transketolase (TKT) is a nonoxidative PPP enzyme that reversibly converts nonoxidative PPP metabolites into glycolytic intermediates, allowing cells to adjust their metabolism to different environmental conditions according to their functional needs. Functionally, TKT catalyzes two reversible reactions: the conversion of D-lignocellulose 5-phosphate (Xu 5 P) and ribose 5-phosphate (R5 P) to glyceraldehyde 3-phosphate (G3 P) and pentose 7-phosphate (S7 P), and the conversion of Xu 5 P and D-erythrose 4-phosphate (E4 P) to G3 P and fructose 6-phosphate (F6 P). Deficiency of TKT in mouse hepatocytes, adipocytes, or T cells affects the phenotype and function of cells and accelerates the progression of diseases, including obesity, hepatic steatosis, and autoimmune diseases. However, the role of TKT in regulating macrophage-mediated inflammatory responses has not been reported. Summary of the invention

[0006] The purpose of the present invention is to provide an inhibitor targeting macrophage transketolase for use in preparing a drug for preventing and treating inflammatory diseases.

[0007] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides use of a transketolase inhibitor in the preparation of a medicament for preventing or treating an inflammatory disease.

[0008] Specifically, the transketolase inhibitor is selected from one or more of hydroxythiamine, siRNA of transketolase, and shRNA of transketolase.

[0009] Specifically, the inflammatory diseases include acute inflammatory diseases and chronic inflammatory diseases.

[0010] Specifically, the inflammatory diseases include but are not limited to sepsis, rheumatoid arthritis, inflammatory bowel disease, neurodegenerative diseases, septic shock, sepsis, systemic lupus erythematosus, pneumonia, acute lung injury, acute pancreatitis, critical illness respiratory distress syndrome, acute appendicitis, acute tonsillitis, asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, atherosclerosis, gout, arthritis, ankylosing spondylitis, Hodgkin's disease, conjunctivitis, iritis, uveitis, dermatitis, atopic dermatitis, eczema, and multiple sclerosis.

[0011] Furthermore, the inflammatory disease is sepsis.

[0012] Specifically, the inflammatory diseases include LPS-induced inflammatory diseases, TLR4-mediated inflammatory diseases, TAK1-NF-κB / MAPK-mediated inflammatory diseases, and gram-negative bacteria Escherichia coli-mediated inflammatory diseases.

[0013] Specifically, the drug inhibits the up-regulation of the expression of inflammatory factors and chemokines.

[0014] Furthermore, the inflammatory factors include IL-6, IL-1β and TNF-α.

[0015] Furthermore, the chemokines include Ccl2, Cxcl2, Cxcl11 and Cxcl15.

[0016] Specifically, the drugs include: (1) transketolase inhibitors; and / or; (2) a pharmaceutically acceptable carrier; and / or; (3) Optionally, one or more other active substances for treating inflammatory diseases.

[0017] Specifically, the drug has at least one of the following effects: (1) Reduce the expression of pro-inflammatory factors in the body; (2) Reduce the expression of chemokines in the body; (3) Increase the expression of anti-inflammatory factors in the body; (4) Alleviate lung damage; (5) Inhibit glycolysis; (6) Enhance oxidative phosphorylation; (7) Increase the levels of polyunsaturated fatty acids in macrophages.

[0018] Furthermore, the drug reduces the expression of pro-inflammatory factors through transketolase enzyme activity.

[0019] Furthermore, the drug reduces the expression of pro-inflammatory factors through the TAK1-NF-κB / MAPK signaling pathway.

[0020] Furthermore, the active substance is selected from one or more of antibiotics, drugs for inhibiting infection, and drugs for inhibiting inflammatory response.

[0021] In yet another aspect, the present invention provides a drug for preventing or treating inflammatory diseases, wherein the drug comprises a therapeutically effective amount of a transketolase inhibitor.

[0022] Specifically, the medicine also includes a pharmaceutically acceptable carrier.

[0023] Furthermore, the pharmaceutically acceptable carrier includes, but is not limited to, excipients, buffers, emulsifiers, stabilizers, diluents, adhesives, preservatives, lubricants, pH regulators, cryoprotectants, flavoring agents, and fillers.

[0024] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, 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.

[0025] Specifically, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0026] Specifically, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, glyceryl oleate, and polyglyceryl-3 polyricinoleate.

[0027] Specifically, the stabilizer is selected from at least one of acacia gum, agar, alginic acid, cellulose ether, and carboxymethyl chitosan.

[0028] Specifically, 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.

[0029] Specifically, the binder is selected from at least one of ethanol, starch slurry, pregelatinized starch, dextrin, syrup, hydroxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, sodium alginate, polyvinyl pyrrolidone, gum arabic, gelatin, and alginic acid.

[0030] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric cyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0031] Specifically, the lubricant is selected from at least one of magnesium stearate, zinc stearate, glyceryl monostearate, polyethylene glycol, stearic acid, talc, sodium chloride, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium stearyl fumarate, and poloxamer.

[0032] Specifically, the pH regulator is selected from at least one of citric acid, fumaric acid, succinic acid, tartaric acid, malic acid and ascorbic acid.

[0033] Specifically, 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 or formamide.

[0034] Specifically, the flavoring agent is selected from at least one of sweet orange flavor, vanilla flavor, strawberry flavor, milk flavor, banana flavor, and cherry flavor.

[0035] Specifically, 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.

[0036] Specifically, the dosage form of the drug is drops, mixtures, tinctures, injections, tablets, powders, oral liquids, capsules, granules, ointments, suspensions, powders, emulsions, pellets, pills, buccal tablets, lyophilized powder injections, gels, suppositories or aerosols.

[0037] The beneficial effects of the present invention are: The transketolase inhibitor provided by the present invention can inhibit the occurrence of septic shock. The transketolase inhibitor hinders the activation of the TAK1-NF-κB / MAPK signaling pathway in macrophages, thereby inhibiting the inflammatory response. The transketolase inhibitor reprograms macrophage metabolism, inhibits glycolysis, promotes oxidative phosphorylation, and significantly increases the level of PUFA, especially ω-3 fatty acids EPA / DHA and ω-6 fatty acids AA. The present invention also designs an AAV-shTKT adenovirus vector targeting lung tissue macrophages TKT, and achieves good therapeutic effects in the treatment and prevention of sepsis, providing potential clinical applications for the management of inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 LPS and other pro-inflammatory stimuli reduce the expression of TKT in macrophages. LPS-induced downregulation of TKT expression depends on the TLR4-NF-κB signaling axis: AC in the figure refers to various inflammatory stimuli. Tkt The mRNA and protein levels of wild-type mice were down-regulated; D refers to wild-type mice injected with PBS or LPS intraperitoneally, and bone marrow cells were extracted from mice 6 hours later and detected by qPCR Tkt and inflammatory factors Il-6 E refers to the expression level of PBMC isolated from human healthy controls or sepsis patients by qPCR analysis. TKT and inflammatory factors Il-1βFI in the figure is the expression level of PM transfected with siRNA or treated with NF-κB inhibitor BMS-345541 and stimulated with LPS for 4 h. Tkt and Il-6 The levels were detected by qPCR.

[0039] Figure 2 To inhibit TKT and reduce macrophage inflammatory response, and to construct and verify TKT myeloid-specific knockout mice: A in the figure shows wild-type PMs pretreated with TKT inhibitor OT for 12 hours, then stimulated with LPS for 4 hours, and qPCR was used to detect inflammatory cytokines Il-6, Il-1β, Tnf-α Expression of inflammatory cytokines; Figure B shows wild-type PMs were transfected with siCtrl or siTKT, and stimulated with LPS 48 h later. Il-6, Il-1β, Tnf-α Expression; C in the figure is Tkt fl / fl Allele targeting strategy; Figure D is the use of mouse toe or tail tip DNA as a template, PCR identification of mouse genotype; Figure E is qPCR and western blot detection of Tkt mRNA and TKT protein expression levels in PMs of WT and KO mice; Figure F refers to FACS detection of F4 / 80 in peritoneal cells of WT and KO mice + CD11b + The proportion of double positive macrophages.

[0040] Figure 3 TKT deficiency in macrophages can inhibit the expression of pro-inflammatory cytokines: A in the figure refers to the qPCR detection of inflammatory cytokines after the inhibitor OT was applied to PBMC cells of sepsis patients. Il-6, Il-1β and Tnf-α Figures B and C show the expression of macrophage inflammation-related genes detected by qPCR (B) and ELISA (C) after TKT-WT-PMs and TKT-KO-PMs were stimulated with LPS for 4 h. IL-6 , IL-1β , TNF-α Figures D and E show the mRNA and protein levels of TKT-WT-PMs and TKT-KO-PMs. E. coli After stimulation, qPCR (D) and ELISA (E) were used to detect the expression of macrophage inflammation-related genes. IL-6 , IL-1β , TNF-α mRNA and protein levels.

[0041] Figure 4 Myeloid-specific TKT deficiency inhibits the progression of sepsis: A and B in the figure refer to intraperitoneal injection of TKT-WT-Mφ and TKT-KO-Mφ mice E. coliOr survival rate after LPS. CE in the figure refers to the levels of inflammatory cytokines IL-6, IL-1β, and TNF-α in serum detected by ELISA, the mRNA levels of each cytokine in lung tissue detected by qPCR, and representative images of H&E staining of lung tissue after intraperitoneal injection of LPS in mice; F in the figure refers to the survival rate of TKT-WT-Mφ and TKT-KO-Mφ mice after CLP surgery. GI in the figure refers to the levels of inflammatory cytokines IL-6, IL-1β, and TNF-α in serum detected by ELISA, and inflammatory cytokines in lung, liver, kidney, and spleen detected by qPCR after CLP surgery in mice. Il-6 Representative images of H&E staining of lung tissues.

[0042] Figure 5 TKT inhibits TAK1-NF-κB / MAPK signaling pathway: A in the figure refers to the effect of TKT on the nuclear translocation of P65 in macrophages; B in the figure refers to the effect of TKT on inflammatory cytokines through P65 Il-6 , Il-1β mRNA levels; CD in the figure refers to the effect of TKT on the activation of NF-κB luciferase reporter gene; EG in the figure refers to the effect of TKT and its inhibitor OT on the activation of TAK1-NF-κB signaling pathway.

[0043] Figure 6 Reprogramming of macrophage metabolic network for TKT knockout: A and B in the figure refer to the effects of macrophage ECAR / OCR caused by TKT deficiency; Figure C is a schematic diagram of non-targeted metabolomics analysis; Figure D refers to the changes in glycolysis (Glycolysis), pentose phosphate pathway (PPP), and tricarboxylic acid cycle (TCA) related metabolites in TKT-WT-PMs and TKT-KO-PMs; Figure E is a non-targeted metabolomics analysis of TKT-WT-PMs and TKT-KO-PMs, screening for differential metabolites with p < 0.05, and enrichment of KEGG metabolic pathways; Figure F refers to the levels of differentially metabolized fatty acids in TKT-WT-PMs and TKT-KO-PMs cells in the α-linolenic acid and linoleic acid metabolic pathways.

[0044] Figure 7 Macrophage TKT affects TAK1 / TAB1 binding by regulating unsaturated fatty acid levels, thereby affecting inflammation: A and B in the figure refer to macrophage TKT affecting inflammatory signals by regulating unsaturated fatty acid levels; C and D in the figure refer to TKT affecting TAK1 / TAB1 binding by changing the level of unsaturated fatty acids.

[0045] Figure 8AAV-shRNA targeting TKT in lung macrophages improves mouse E. coli Induced sepsis: WT mice were injected with AAV adenovirus via tail vein, and three weeks later, the E. coli Figure A shows the flow cytometry sorting strategy for mouse lung macrophages; Figure B shows the silencing level of TKT in the sorted macrophages detected by qPCR and western blot; Figure C shows the qPCR detection of related cytokines in mouse lung tissue Il-6 , Il-1β , Tnf-α as well as Il-10 The expression level of inflammatory cytokines IL-6 and TNF-α in mouse serum detected by ELISA in D; the level of BUN and Scr in mouse serum detected by the kit in E; the level of AST and ALT in mouse serum detected by the kit in F; the dry-wet weight ratio of mouse lung tissue in G; the representative image of H&E staining of mouse lung tissue in H&E; the mRNA level of each cytokine in lung tissue detected by qPCR in I in Figure; and the survival status of mice in J in Figure. DETAILED DESCRIPTION

[0046] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0047] Example 1 Animals, materials, experimental methods and statistical analysis used in the present invention 1 Experimental animals Myeloid-specific knockout TKT mice Tkt fl / fl (C57BL / 6J) mice were purchased from Saiye Bioscience Co., Ltd. (Guangzhou, China) and bred using CRISPR-Cas9-mediated genome editing technology; myeloid knockout tool mice Lyz2-Cre (C57BL / 6J) and Tkt fl / fl Mice were crossed to generate myeloid cell-specific TKT gene knockout mice Tkt fl / fl Lyz2 -Cre + , litter mate Tkt fl / fl Lyz2 -Cre -All mice were housed in a specific pathogen-free (SPF) environment at 22-24°C with unlimited food and water and a 12-h light-dark cycle.

[0048] 2 Cell culture and treatment HEK293T and RAW264.7 cell lines were cultured in complete RPMI-1640 (Gibco) or DMEM (Gibco) at 37°C and 5% CO. 2 The cells were cultured under the conditions of 10% (v / v) FBS (Gibco), 1% penicillin-streptomycin (100 U / ml, Gibco), and 1% ciprofloxacin (Biyuntian). Mouse peritoneal macrophages (PEMs) were obtained by intraperitoneal injection of 1 mL of 3% Brewer's collagenase medium. In the TKT inhibition experiment, cells were treated with thiamine (OT; 50 μM; O4000; Merck) to inhibit TKT activation. To establish the relevant model in vitro, cells were treated with the following conditions: 200 ng / ml LPS-4h, 20 μg / mL Poly(I:C) -12h, 6 μg / mL CpG -12h, 1 ng / mL HT-DNA -12h, serum starvation for 12h, 500 μM AICAR -12h, 50 μM eicosapentaenoic acid (EPA) -12h, 50 μM docosahexaenoic acid (DHA) -12h, 50 μM arachidonic acid (AA) -12h. 3 Human blood samples According to the research protocol approved by the Ethics Review Committee of Tianjin Medical University, the sepsis patients and healthy subjects participating in this study provided informed consent for blood collection in accordance with the requirements of institutional and national guidelines. The research protocol was approved by the Institutional Review Board. The identity, age, and gender of the patients and healthy blood donors were kept confidential in accordance with the regulations of the Ethics Committee.

[0049] Peripheral blood mononuclear cells (PBMCs) from patients with sepsis or healthy blood donors were separated using human peripheral blood lymphocyte separation medium (C0025, Biotech) and used in related experiments.

[0050] 4 Statistical analysis methods Legend All data were statistically and analytically processed using Graph Pad Prism software, and graphs were drawn using Graph Pad Prism software. In order to ensure the accuracy and reliability of the experimental data, all quantitative experimental data were maintained in three groups of biological replicates and three technical replicates, and the differences between samples were shown in the graphs. The values ​​are expressed as mean ± standard error, and the difference analysis was performed using a two-tailed Student's t test. The data between multiple samples were compared using a one-way analysis of variance, and the survival curve was tested using the log-rank test (Mantel-Cox). NS: no statistical difference (p ≥ 0.05), *p ≤0.05, **p ≤ 0.01, ***p ≤ 0.001 were considered statistically significant.

[0051] Example 2 Effect of transketolase TKT on sepsis 1. Experimental methods 1.1 Plasmids and transfection All full-length DNAs of TKT, TAK1, and TAB1 cDNAs were cloned into pLenti-3xFlag or pLenti-HA vectors. Mutations of TKT were generated using a standard PCR cloning strategy with Phusion Hot StartII DNA polymerase (Thermo Fisher Scientific). Expression plasmids for MyD88, TRAF6, Trif, TAK1, TAB1, IKKα, IKKβ, and P65 were constructed using standard molecular biology techniques and cloned into pcdna3.1-3×Flag vectors. Plasmid DNA was transfected into cells using Lipofectamine 2000 reagent (11668019; Thermo Fisher Scientific) according to standard protocols. Short interfering RNA (Sangon Biotechnology Co., Ltd.) targeting specific genes was transfected into cells using Lipofectamine RNAiMAX transfection reagent (13778100; Thermo Fisher Scientific) according to the manufacturer's instructions.

[0052] 1.2 RNA extraction, RT-PCR, and RNA-seq analysis Total RNA was extracted from cells using TRIGene reagent (P118, Genstar) and used for RNA-seq analysis (BGI) or cDNA synthesis (A214, Genstar). Quantitative PCR was performed on a 7500HT Fast qRT-PCR instrument (4351107, Thermo Fisher Scientific) using SYBR Green premix (B21203, Bimake). Data were normalized to β-Actin transcripts and analyzed using the ΔΔCt method.

[0053] 1.3 Protein isolation and immunoblotting analysis Cells were lysed in RIPA lysis buffer supplemented with protease inhibitors (B14001, Bimake). Total protein was quantified using a BCA kit (23225; Thermo Fisher Scientific). Equal amounts of protein from each sample were loaded into 8–12% SDS-polyacrylamide for electrophoresis and then transferred to a PVDF membrane (IPVH00010, MERCK) for immunoblotting, and the target protein levels were analyzed using specific antibodies. Color development was performed using an ECL chemiluminescence kit (Millipore).

[0054] 1.4 Histopathological analysis Mouse organs were fixed with 10% formaldehyde overnight, embedded in paraffin for H&E staining, and histological images were collected using a light microscope.

[0055] 1.5 Immunofluorescence staining For P65 nuclear translocation staining, Raw264.7 cells were cultured on coverslips and then transfected with the indicated plasmids or siRNA. Cells were stimulated with LPS, washed, fixed, permeabilized, and then stained with the indicated antibiotics and DAPI. Images were acquired using a fluorescence microscope.

[0056] 1.6 Co-immunoprecipitation The cells were harvested and lysed with NETN lysis buffer supplemented with protease inhibitors (B14001, Bimake). After centrifugation, an appropriate amount of supernatant was added to 6× SDS buffer and boiled at 100°C for 10 min as input. Anti-FLAG-M2 affinity gel or anti-HA was washed three times, and then 30 μL of the mixture was resuspended in the remaining cell lysis buffer and incubated overnight in a shaker at 4°C. The immunoprecipitate was washed twice with NETN buffer and then washed twice with PBS. 80 μL of 1× SDS buffer was added and boiled at 100°C for 10 min to denature the immunoprecipitated protein, and then subjected to immunoblotting analysis.

[0057] 1.7 Flow cytometry The cells were extracted into a single cell suspension, blocked with a specific surface antibody in a staining solution containing 2% BSA, then washed three times with PBS, incubated with relevant flow cytometry antibodies on ice in the dark for 30 min, washed three times with PBS, and then loaded onto a BD Caliber flow cytometer (BD Biosciences) for flow cytometric analysis and flow sorting using a FACS Aria FusionCell Sorter (BECKMAN, CytoFLEX SRT). Data were analyzed by FlowJo (Tree Star) software.

[0058] 1.8 LPS, Escherichia coli E. coli Infection and CLP models WT and KO mice were injected intraperitoneally E. coli (1×10 6CFU) or LPS (20 mg / kg for survival and 25 mg / kg for acute infection) to induce septic shock. The CLP procedure was performed according to a published protocol (Rittirsch, Danielet al. “Immunodesign of experimental sepsis by cecal ligation and puncture.” Nature protocols vol. 4,1 (2009): 31-6. doi:10.1038 / nprot.2008.214). Male mice aged 8-10 weeks were anesthetized with tribromoethanol (200 mg / kg, intraperitoneal injection). When the mice did not respond to paw pinch, the abdominal hair was shaved and the skin was disinfected with 75% alcohol. A midline abdominal incision was made with a scalpel. The cecum was exposed, ligated from the distal third with a 4-0 silk suture, and the cecal wall was punctured with a 21-gauge needle. The cecum was gently squeezed to reveal a small amount of feces and complete perforation was ensured. The cecum was then returned to the abdominal cavity and the incision was closed. Immediately after surgery, 0.5 mL of warm saline was injected subcutaneously. The mice in the control group underwent anesthesia, laparotomy, and wound suture, but they did not undergo cecal ligation and puncture. The survival rate of the group was monitored and the survival of the mice was observed and recorded in a timely manner, and the mice with relevant pathological injuries were sampled for testing at the corresponding time.

[0059] 1.9 Enzyme-linked immunosorbent assay (ELISA) and kit assay The ELISA kits for mouse IL-6 (Cat#EK0411), IL-1β (Cat#EK0394), and TNF-α (Cat#EK0527) were from Boster, and the kits for BUN (Cat#C013-2-1), SCr (Cat#C011-2-1), AST (Cat#C009-2-1), and ALT (Cat#C010-2-1) were from Nanjing Jiancheng. The levels of related substances in mouse plasma and cell supernatant were detected according to the manufacturer's instructions.

[0060] 1.10 Dual luciferase reporter activity assay The reporter plasmids were transiently transfected into cells using Lipofectamine (Thermo Fisher Scientific, USA) according to the protocol. Transfection efficiency was determined using the pRL-CMV plasmid containing the Renilla luciferase gene (Promega). HEK293T cells were transiently transfected with the reporter plasmids. After 24 h of treatment, the transfected cells were washed twice with PBS and then lysed in lysis buffer (Yeason) with gentle shaking for 20 min at room temperature. The cell lysate was centrifuged at 17,000 x g for 2 min to remove cell debris. The supernatant was transferred to a new tube and the dual-luciferase activity in the cell extract was determined according to the manufacturer's protocol (Promega). Firefly luciferase activity was measured using a fluorimeter programmed with a 2-second delay before each reporter detection and a 10-second measurement. After measuring firefly luciferase activity (Stop&Glo®, Promega), Renilla luciferase assay buffer was added to measure enzyme activity. Each transfection was repeated in duplicate, and all measurements were repeated at least three times.

[0061] 1.11 LC-MS analysis of metabolites Mouse PM (5 x 10 6 After 24 h, cells were washed with ice-cold PBS, and metabolites were extracted with 80% ice-cold methanol. LC-MS was performed by LipidALL Technologies (Changzhou, China), and metabolite abundances were expressed relative to internal standards according to standard protocols.

[0062] 1.12 Mitochondrial and glycolysis stress test Appropriate density of PM from TKT-WT-Mφ or TKT-KO-Mφ mice was inoculated into 24-well seahorseXFe-24 assay plates. After adding appropriate stimuli, cells were washed and cultured in 1640 medium without buffer for 1 hour. Water was added to the probe plate in advance and the analyzer was preheated. Various drugs were added to the drug injection port of the probe plate according to the measured metabolism. Then, the preset program was executed to inject drugs into the cell supernatant in sequence to monitor the real-time dynamic changes of drug-induced cell metabolism.

[0063] 1.13 AAV adenoviral vector infection The AAV Lung X delivery system was used to knock out TKT in mouse liver macrophages. Recombinant adeno-associated virus serotype lung X (AAV Lung X) vectors carrying mouse TKT or NC vectors with CD68 promoter (a promoter targeting macrophages), AAV Lung X-CD68-sh (TKT) or AAV Lung X-CD68-NC were produced by Obio Technology Co. Ltd. (Shanghai, China). Mice were injected via the tail vein with 100 μl of virus containing 4E+11 vg of AAV Lung X vector genome, and relevant disease models were established three weeks later.

[0064] 2. Experimental results 2.1 LPS and other proinflammatory stimuli reduce TKT expression in macrophages, and LPS-induced downregulation of TKT expression depends on the TLR 4-NF-κB signaling axis Macrophage-mediated inflammatory responses are closely related to metabolic processes. TKT-mediated nonoxidative PPP significantly affects the functions of hepatocytes, adipocytes, and regulatory T cells. Therefore, we first investigated whether TKT expression is associated with macrophage-mediated inflammation.

[0065] When mouse peritoneal macrophages (PMs) were exposed to pro-inflammatory stimuli including LPS, Gram-negative bacteria Escherichia coli (E. coli), herring testis DNA (HT-DNA), TLR 3 or 9 agonists Poly (I:C) or CpG, serum starvation (SS), and AMPK agonist 5-imidazole-4-carboxamide ribonucleoside (AICAR), the expression level of TKT was significantly decreased by qPCR and Western blot analysis ( Figure 1 A- Figure 1 In vivo, the mRNA expression of TKT in mouse bone marrow was significantly increased after stimulation with LPS ( Figure 1 D) and in serum peripheral blood mononuclear cells (PBMCs) from patients with systemic inflammatory response syndrome (SIRS) or sepsis ( Figure 1 E) were significantly decreased.

[0066] Upon recognition of LPS or Gram-negative bacteria, TLR4 activates the MyD88-dependent or TRIF-dependent pathway, leading to the activation of NF-κB. We further investigated whether TLR4-NF-κB signaling would reduce TKT expression.

[0067] Studies have shown that silencing MyD88 or TRIF can reactivate the expression of TKT in LPS-stimulated macrophages ( Figure 1 F and Figure 1In addition, treatment with the NF-κB inhibitor BMS-345541 or knockdown of the NF-κB subunit p65 significantly increased the expression of TKT ( Figure 1 H and Figure 1 Overall, we demonstrated that exposure to multiple proinflammatory stimuli significantly inhibited TKT expression both in vitro and in vivo, and that LPS-induced inhibition of TKT expression was dependent on TLR4-NF-κB signaling.

[0068] 2.2 Macrophage TKT deficiency inhibits the expression of proinflammatory cytokines Since TLR4-NF-κB signaling was found to have a regulatory effect on TKT expression, we then sought to determine whether TKT in macrophages in turn affects TLR4-mediated inflammatory responses.

[0069] We found that hydroxythiamine treatment or knockdown of TKT by small interfering RNA (siRNA) significantly reduced mRNA expression of proinflammatory cytokines, including interleukin (IL)-6, IL-1β, and tumor necrosis factor α (TNF-α), in PMs of LPS-treated mice ( Figure 2 A and Figure 2 Consistently, we observed that mRNA levels of pro-inflammatory cytokines were significantly decreased after OT administration to peripheral blood mononuclear cells (PBMCs) from sepsis patients ( Figure 3 These findings suggest that inhibition of TKT can reduce LPS-induced inflammatory responses.

[0070] To further elucidate the role of TKT in macrophages during inflammation, we constructed bone marrow-specific TKT knockout mice ( Tkt fl / fl Lyz2 -Cre + , i.e., TKT-KO-Mφ mice) and littermates ( Tkt fl / fl , i.e., TKT-WT-Mφ mice) ( Figure 2 C and Figure 2 As expected, TKT was efficiently knocked out in PMs isolated from TKT-KO-Mφ mice ( Figure 2 E). TKT-KO-Mφ mice follow Mendelian inheritance and do not show developmental phenotypic defects. Compared with TKT-WT-Mφ mice, their PMs show normal differentiation and number ( Figure 2 F). Next, we treated WT and KO PMs with LPS and found that the mRNA expression levels of Il-6, Il-1β, and Tnf-a in TKT-deficient macrophages were significantly lower than those in WT-PMs ( Figure 3In agreement with these findings, we also confirmed by ELISA that the concentrations of proinflammatory cytokines were reduced in the supernatants of KO-PMs ( Figure 3 C). In infection E. coli After TKT-KO, the inflammatory response was significantly reduced ( Figure 3 D and Figure 3 E), further confirming the key role of TKT in regulating macrophage-mediated inflammation.

[0071] 2.3 Myeloid TKT deficiency prevents septic shock Since overactivation of the TLR4-TAK1-NF-κB / MAPK signaling pathway is a key factor in sepsis, we investigated whether TKT deficiency would affect the development of septic shock in vivo by infecting mice with Escherichia coli.

[0072] We used E. coli, endotoxin, or cecal ligation surgery (CLP) to perform experiments. The results showed that TKT-KO-Mφ mice showed a significant decrease in the number of TKT-WT-Mφ mice after intraperitoneal injection of E. coli ( Figure 4 A and Figure 4 Consistent with this finding, we observed a significant decrease in the concentrations of IL-6, IL-1β, and TNF-α in the serum of TKT-KO-Mφ mice ( Figure 4 In addition, the production of pro-inflammatory cytokines and chemokines was reduced, while the production of the anti-inflammatory cytokine IL-10 was increased in the lungs of TKT-KO-Mφ mice ( Figure 4 D in the figure). Hematoxylin and eosin (H&E) staining results showed that the degree of lung injury in TKT-KO-Mφ mice was significantly milder than that in TKT-WT-Mφ mice ( Figure 4 E in.

[0073] Next, in the case of E. coli or CLP-induced septic shock, the survival rate of TKT-KO-Mφ mice was significantly higher than that of WT littermates ( Figure 4 F). At 20 hours after CLP surgery, the concentrations of proinflammatory factors in the serum of TKT-KO-Mφ mice and their mRNA levels in multiple organs such as lung, liver, kidney and spleen were significantly lower than those of WT littermates ( Figure 4 G and Figure 4 In addition, H&E staining of the lungs showed that the extent of lung injury in TKT-KO-Mφ mice was reduced ( Figure 4 Taken together, these results strongly suggest that myeloid TKT deficiency can suppress the onset of septic shock.

[0074] 2.4 Macrophage TKT promotes the expression of pro-inflammatory cytokines by inhibiting the TAK1-NF-kB / MAPK signaling pathway TLR4-induced NF-κB activation leads to the production of IL-1β, IL-6, and TNF-β. We then focused on whether TKT affects NF-κB activation to enhance LPS-induced inflammatory responses.

[0075] After 20 min of LPS treatment, TKT knockdown reduced, while TKT overexpression increased, the nuclear translocation of p65 ( Figure 5 In addition, p65 knockdown also severely inhibited the upregulation of IL-6 and IL-1β expression induced by TKT overexpression ( Figure 5 Figure 3 (B), indicating that NF-κB activation is critical for the promotion of these proinflammatory cytokines by TKT.

[0076] Since MyD88, TRAF6, TIF, TAK1, TAB1, and IKKa / β are key regulators of NF-κB activation, we next determined which of these molecules acted in coordination with TKT by using an NF-κB luciferase reporter assay. To this end, expression vectors for MyD88, TRAF6, TRIF, TAK1, TAB1, IKKα / β, and p65 were co-transfected with NF-κB-driven luciferase in HEK 293T cells alone, as well as with empty vector or TKT expression vector. Then, NF-κB-driven luciferase activity was compared between cells infected with empty vector and TKT expression vector for each of these signaling molecules, respectively.

[0077] Overexpression of TKT together with MyD88, TRAF6, or TRIF significantly increased NF-κB-driven luciferase activity, and this increase was shown to be dependent on the TKT dose ( Figure 5 C and Figure 5 In contrast, no significant increase was detected when TKT was co-expressed with TAK1, TAB1, IKKa / β, or p65 ( Figure 5 (D) suggests that the node where TKT regulates the signal axis is located upstream of TAK1 / TAB1 and downstream of TIF. Since TAK1 is a key upstream molecule of the MAPK pathway and the IKKa / β-NF-κB pathway, we then examined whether TKT regulates the phosphorylation of TAK1, IKK a / β, and MAPK kinases. Compared with WT macrophages, OT-treated and TKT-deficient primary macrophages showed decreased levels of p-TAK1, p-IKK a / β, p-p65, p-JNK, and p-P38, but not p-ERK, after LPS stimulation ( Figure 5E and Figure 5 However, TKT overexpression promoted the phosphorylation of these pathway proteins ( Figure 5 In conclusion, we found that macrophage TKT promoted the expression of proinflammatory cytokines by inhibiting the TAK1-NF-κB / MAPK signaling pathway.

[0078] 2.5 Loss of TKT reprograms macrophage metabolism and leads to increased levels of polyunsaturated fatty acids Cellular energy is a key factor in regulating immune-mediated inflammation. Therefore, we investigated whether TKT deficiency alters macrophage energy metabolism. Seahorse showed that the extracellular acidification rate (ECAR) of KO PM was lower than that of WT PM at baseline and after LPS stimulation ( Figure 6 Interestingly, TKT-deficient PMs showed an increased oxygen consumption rate (OCR; Figure 6 These results suggest that TKT deficiency causes macrophages to switch from glycolysis to oxidative phosphorylation. To further elucidate the potential mechanism by which TKT promotes inflammation, we also performed metabolomics analysis of PMs of TKT-WT and TKT-KO ( Figure 6 TKT deficiency leads to accumulation of R5P and S7P in the non-oxidative PPP ( Figure 6 D in ). In contrast, the levels of sugar decomposition metabolites F6P and G3P increased. Pyruvate levels decreased, and the levels of TCA cycle intermediates citrate, isocitrate, a-KG, fumarate, and malate did not change significantly. In addition, the metabolomics differential metabolite pathway enrichment results revealed that TKT deficiency significantly altered the fatty acid metabolic pathways in macrophages, especially the α-linolenic acid / linolenic acid (ALA / LA) pathway ( Figure 6 E in the omega-6 fatty acid), while the polyunsaturated fatty acids (PUFAs) of arachidonic acid (AA, 20:4 ω-6), eicosapentaenoic acid (EPA, 20:5 ω-3), and docosahexaenoic acid (EPA, 22:6 ω-3) were identified as the three fatty acids with the largest multiple increases ( Figure 6 F in.

[0079] 2.6 Increased PUFA caused by TKT knockout inhibits the interaction between TAK1 and TAB1, thereby inhibiting the activation of the TAK1-NF-κB / MAPK signaling pathway Earlier studies have shown that polyunsaturated fatty acids such as DHA, EPA, and AA can exert powerful anti-inflammatory effects in macrophages by regulating inflammatory signaling pathways involving GPRs, nuclear receptors, and other mechanisms. To investigate how AA and EPA / DHA affect the effects of TKT on macrophage-mediated inflammatory responses, we pretreated PM with PA, AA, or EPA / DHA before LPS stimulation. Compared with PA treatment, AA or EPA / DHA treatment significantly reduced the increase in proinflammatory cytokine production and TAK1 signaling pathway activation induced by TKT overexpression ( Figure 7 A and Figure 7 B in the figure). Our previous results showed that the node where TKT exerts its pro-inflammatory regulatory effects is located upstream of TAK1 / TAB1 and downstream of TRIF, and DHA has been reported to inhibit the binding of TAK1 / TAB1. Therefore, we speculated that TKT may also affect the binding of TAK1 to TAB1. Our co-immunoprecipitation assay showed that overexpression of TKT promoted the binding of TAK1 to TAB1, and this increase was reversed by pre-supplementation with AA or EPA / SHA ( Figure 7 C and Figure 7 Overall, these findings suggest that TKT promotes the binding of TAK1 and TAB1 downstream of TLR4 by regulating PUFA metabolism, thereby affecting the activation of the TAK1-NF-κB / MAPK signaling pathway and ultimately altering the expression of proinflammatory cytokines.

[0080] 2.7 AAV adenoviral vector targeting macrophage TKT improves mouse E. coli Disease progression of septic shock induced To investigate the therapeutic potential of targeting macrophage TKT for the treatment of sepsis, we used an adeno-associated virus (AAV) delivery system to inject C57BL / 6 mice via the tail vein. AAV8 vectors carrying mouse shTKT (AAV-shTKT) or empty vector (AAV-EGFP-shNC) with the CD68 promoter (a macrophage-targeting promoter) were injected into wild-type mice via the tail vein, and three weeks later, the AAV8 vectors were injected intraperitoneally. E. coli Sepsis model was established, and 12 hours later, lung tissue cells were extracted from mice, and macrophages were sorted by flow cytometry ( Figure 8 The mRNA and protein levels of TKT were further detected, and the results showed that the expression level of TKT in lung macrophages was significantly downregulated ( Figure 8 In B), qPCR detection showed that inflammatory factors in lung macrophages of mice injected with AAV-shTKT Il-6 , Il-1β , Tnf-α The expression of inflammatory cytokines is downregulated, while Il-10 The expression level was upregulated ( Figure 8 In addition, the levels of inflammatory factors IL-6, IL-1β, TNF-α, and liver and kidney injury indicators urea nitrogen, creatinine, AST, and ALT in the serum of mice injected with AAV-shTKT were significantly reduced ( Figure 8 D- Figure 8 F in the figure), the dry-wet weight ratio of lung tissue decreased ( Figure 8 G), H&E staining results showed that the degree of lung injury in mice injected with AAV-shTKT was significantly milder than that in AAV-shNC mice ( Figure 8 The production of pro-inflammatory cytokines and chemokines in the lungs of H) mice was reduced, while the production of anti-inflammatory cytokines Il-10 The production of Figure 8 At the same time, E. coli In the septic shock induced by AAV, the survival rate of mice injected with AAV-shTKT was significantly higher than that of the control group ( Figure 8 Taken together, these results strongly suggest that AAV adenoviral vectors targeting macrophage TKT can effectively inhibit the occurrence of septic shock in mice.

[0081] Altogether, our results suggest that the TKT-mediated non-oxidative PPP integrates the regulation of cellular metabolism and signaling pathways to influence inflammatory responses.

[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 and signal transduction. Importantly, we designed an AAV-shTKT adenovirus vector targeting lung tissue macrophages TKT and achieved good therapeutic effects in the treatment and prevention of sepsis in mice, providing potential clinical applications for the management of acute inflammatory diseases.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a macrophage-targeted transketolase inhibitor in the preparation of a drug for preventing or treating sepsis.

2. The use according to claim 1, characterized in that: The transketolase inhibitor is selected from one or more of hydroxythiamine, siRNA of transketolase, and shRNA of transketolase.

3. The use according to claim 1, characterized in that: The drugs include: (1) transketolase inhibitors; and / or; (2) a pharmaceutically acceptable carrier; and / or; (3) Optionally, one or more other active substances for treating sepsis.

4. A drug for preventing or treating sepsis, characterized in that: The medicine contains a therapeutically effective amount of a macrophage-targeted transketolase inhibitor.

5. The drug according to claim 4, characterized in that The transketolase inhibitor is selected from one or more of hydroxythiamine, siRNA of transketolase, and shRNA of transketolase.

6. The drug according to claim 4, characterized in that The medicine also includes a pharmaceutically acceptable carrier.

7. The drug according to claim 6, characterized in that The pharmaceutically acceptable carrier includes excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH regulators, cryoprotectants, flavoring agents and fillers.

8. The drug according to claim 4, characterized in that The dosage form of the drug is oral preparation or injection.

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