Use of an inhibitor targeting macrophage transketolase in the preparation of a medicament for preventing and treating inflammatory diseases

Abstract

The present invention discloses the application of an inhibitor targeting macrophage transketolase in the preparation of a drug for preventing and treating inflammatory diseases, and relates to the field of biotechnology. The present invention provides the application of a transketolase inhibitor in the preparation of a drug for preventing or treating inflammatory diseases, and the transketolase inhibitor can inhibit the occurrence of septic shock. The present invention reveals the important role and mechanism of the non-oxidative PPP key enzyme TKT in promoting macrophage inflammatory response by integrating metabolism and signal pathways, and reveals that TKT can be used as a potential therapeutic target for treating macrophage-mediated inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to the application of an inhibitor targeting macrophage transketolase in the preparation of a drug 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 chemical substances. Macrophages are an important part of the innate immune system and, as key inflammatory effector cells, 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 within macrophages are activated, thereby initiating the production of a variety of 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. After being activated by lipopolysaccharide (LPS), a cell wall component released by Gram-negative bacteria, TLR4 triggers downstream signal cascades involving the myeloid differentiation primary response 88 (MyD88) adaptor protein and the Toll / interleukin-1 receptor domain-containing adaptor inducing interferon-β (TRIF), subsequently activating an enzyme cascade including the transforming growth factor-β-activated kinase 1 (TAK1) pathway, downstream of which are the mitogen-activated protein kinase (MEK) pathway and the nuclear factor-kappa B (NF-κB) pathway. However, the over-activation of TLRs may lead to an out-of-control inflammatory response, thereby disrupting immune homeostasis and triggering acute life-threatening sepsis or other chronic diseases, including diabetes, atherosclerosis, ankylosing spondylitis, etc. Therefore, identifying key regulatory factors that modulate macrophage-mediated inflammation is crucial 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 demands, thereby providing essential metabolic intermediates such as reactive oxygen species (ROS), nitric oxide (NO) for antibacterial 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. Moreover, metabolites such as citrate, itaconate, succinate, fumarate, serine, and alpha-ketoglutarate (α-KG) also play crucial roles in the inflammatory response of macrophages. Macrophage metabolism can interfere with epigenetic modifications, signal transduction, redox status, and other regulatory processes, thus affecting macrophage activation and function. Therefore, targeting macrophage metabolism is regarded as a potential strategy for treating inflammatory diseases.

[0004] The pentose phosphate pathway (PPP) is an important part of glucose catabolism, divided into an oxidative branch and a non-oxidative branch. In the oxidative branch of the PPP, glucose-6-phosphate (G6P) is oxidized to generate diphosphoglycerate (DPP) and ribulose-5-phosphate (Ru5P). The production of DPP in this branch is crucial for maintaining the redox balance of cells and supporting anabolic processes. Ru5P and other sugar phosphates are converted to ribose-5-phosphate (R5P) and xylulose-5-phosphate (X5P), which helps generate pentoses for nucleotide synthesis and provides intermediates that can be used for glycolysis and other metabolic pathways. Glucose-6-phosphate dehydrogenase (G6PD) and carbohydrate kinase-like protein (CARKL) are key enzymes in the oxidative PPP and have been shown to affect the redox status of cells, thus influencing the inflammatory response of macrophages. However, the role of the non-oxidative PPP in macrophage-mediated inflammation remains largely unclear.

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

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

[0007] To achieve the above object of the invention, the technical solution of the present invention is as follows:

[0008] On the one hand, the present invention provides the use of a transketolase inhibitor in the preparation of a drug for preventing or treating inflammatory diseases.

[0009] Specifically, the transketolase inhibitor is selected from one or more of thiamine hydrosulfide, siRNA of transketolase, and shRNA of transketolase.

[0010] Specifically, the inflammatory diseases include acute inflammatory diseases and chronic inflammatory diseases.

[0011] Specifically, the inflammatory diseases include but are not limited to sepsis, rheumatoid arthritis, inflammatory bowel disease, neurodegenerative diseases, septic shock, septicemia, systemic lupus erythematosus, pneumonia, acute lung injury, acute pancreatitis, severe disease 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, multiple sclerosis.

[0012] Further, the inflammatory disease is sepsis.

[0013] Specifically, the inflammatory diseases include LPS-induced inflammatory diseases, TLR4-mediated inflammatory diseases, TAK1-NF-κB / MAPK-mediated inflammatory diseases, and Gram-negative bacterium Escherichia coli-mediated inflammatory diseases.

[0014] Specifically, the drug inhibits the upregulation of the expression of inflammatory factors and chemokines.

[0015] Furthermore, the inflammatory factors include IL-6, IL-1β, and TNF-α.

[0016] Furthermore, the chemokines include Ccl2, Cxcl2, Cxcl11, and Cxcl15.

[0017] Specifically, the drug includes:

[0018] (1) Transketolase inhibitors; and / or;

[0019] (2) Pharmaceutically acceptable carriers; and / or;

[0020] (3) Optionally, one or more other active substances for treating inflammatory diseases.

[0021] Specifically, the functions of the drug are at least one of the following:

[0022] (1) Reducing the expression of pro-inflammatory factors in the body;

[0023] (2) Reducing the expression of chemokines in the body;

[0024] (3) Increasing the expression of anti-inflammatory factors in the body;

[0025] (4) Alleviating lung injury;

[0026] (5) Inhibiting glycolysis;

[0027] (6) Enhancing oxidative phosphorylation;

[0028] (7) Increasing the level of polyunsaturated fatty acids in macrophages.

[0029] Furthermore, the drug reduces the expression of pro-inflammatory factors through the reduction of transketolase enzyme activity.

[0030] Furthermore, the drug reduces the expression of pro-inflammatory factors through the TAK1-NF-κB / MAPK signaling pathway.

[0031] Furthermore, the active substance is selected from one or more of antibiotics, drugs for inhibiting infection, and drugs for inhibiting inflammatory reactions.

[0032] In another aspect, the present invention provides a drug for preventing or treating inflammatory diseases, and the drug contains a therapeutically effective amount of a transketolase inhibitor.

[0033] Specifically, the drug further includes a pharmaceutically acceptable carrier.

[0034] Furthermore, the pharmaceutically acceptable carrier includes, but is not limited to, excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH regulators, cryoprotectants, flavoring agents, and fillers.

[0035] 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.

[0036] Specifically, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0037] 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.

[0038] Specifically, the stabilizer is selected from at least one of acacia gum, agar, alginic acid, cellulose ether, and carboxymethyl chitin ester.

[0039] 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.

[0040] Specifically, the binder is selected from at least one of ethanol, starch paste, pregelatinized starch, dextrin, syrup, hydroxypropyl methyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, sodium alginate, polyvinylpyrrolidone, gum arabic, gelatin, and alginic acid.

[0041] Specifically, the preservative is selected from at least one of methyl paraben, propyl paraben, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, parachlorometacresol, benzalkonium bromide, benzalkonium chloride, and ethyl paraben.

[0042] Specifically, the lubricant is selected from at least one of magnesium stearate, zinc stearate, glycerol monostearate, polyethylene glycol, stearic acid, talcum powder, sodium chloride, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium stearyl fumarate, and poloxamer.

[0043] Specifically, the pH regulator is selected from at least one of citric acid, fumaric acid, succinic acid, tartaric acid, malic acid, and ascorbic acid.

[0044] Specifically, the cryoprotectant is selected from at least one of sucrose, glucose, mannitol, fructose, trehalose, dextran, lactose, glycerol, methanol, ethanol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), acetamide, or formamide.

[0045] 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.

[0046] 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.

[0047] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, dripping pill, pill, buccal tablet, freeze-dried powder injection, gel, suppository, or aerosol.

[0048] The beneficial effects of the present invention are as follows:

[0049] The transketolase inhibitor provided by the present invention can inhibit the occurrence of septic shock. The transketolase inhibitor blocks 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 levels of PUFAs, especially the ω-3 fatty acids EPA / DHA and the ω-6 fatty acid AA. The present invention also designs an AAV-shTKT adenovirus vector targeting macrophage TKT in lung tissue and has achieved good therapeutic effects in the treatment and prevention of sepsis, providing potential clinical applications for the management of inflammatory diseases. Description of the Drawings

[0050] Figure 1 To show that LPS and other pro-inflammatory stimulants reduce the expression of TKT in macrophages, the downregulation of LPS-induced TKT expression depends on the TLR4-NF-κB signaling axis: A-C in the figure refer to various inflammatory stimuli TktThe mRNA and protein levels were downregulated; D refers to wild-type mice intraperitoneally injected with PBS or LPS. After 6 h, bone marrow cells were extracted from the mice, and qPCR was used to detect Tkt and inflammatory factors Il-6 expression levels; E refers to qPCR analysis of TKT and inflammatory factors Il-1β expression levels in PBMCs isolated from healthy human controls or sepsis patients. Figures F-I in the graph show qPCR detection of Tkt and Il-6 levels in PMs transfected with siRNA or treated with the NF-κB inhibitor BMS-345541 and stimulated with LPS for 4 h.

[0051] Figure 2 For the inhibition of TKT to reduce macrophage inflammatory response and the construction and verification of TKT myeloid-specific knockout mice: In Figure A, wild-type PMs were pretreated with the TKT inhibitor OT for 12 h and then stimulated with LPS for 4 h, and qPCR was used to detect the expression of inflammatory cytokines Il-6, Il-1β, Tnf-α ; In Figure B, wild-type PMs were transfected with siCtrl or siTKT respectively. After 48 h, they were stimulated with LPS, and qPCR was used to detect the expression of inflammatory cytokines Il-6, Il-1β, Tnf-α ; In Figure C is Tkt fl / fl allele targeting strategy; In Figure D, mouse toe or tail tip DNA was used as a template, and PCR was used to identify the mouse genotype; In Figure E, qPCR and western blot were used to detect the expression levels of Tkt mRNA and TKT protein in PMs of WT and KO mice; In Figure F, it refers to FACS detection of F4 / 80 in peritoneal cells of WT and KO mice + CD11b + proportion of double-positive macrophages.

[0052] Figure 3 For the lack of macrophage TKT to inhibit the expression of pro-inflammatory cytokines: In Figure A, after the inhibitor OT was applied to PBMC cells of sepsis patients, qPCR was used to detect the levels of inflammatory cytokines Il - 6, Il-1β and Tnf-α ; In Figures B and C, TKT-WT-PMs and TKT-KO-PMs were stimulated with LPS for 4 h, and qPCR (B) and ELISA (C) were used to detect the IL-6 , IL-1β , TNF-α mRNA levels and protein levels of macrophage inflammation-related genes. In Figures D and E, TKT-WT-PMs and TKT-KO-PMs were stimulated with E.coli and then qPCR (D) and ELISA (E) were used to detect macrophage inflammation-related genesIL-6 , IL-1β , TNF-α mRNA and protein levels.

[0053] Figure 4 Myeloid-specific TKT deletion inhibits the progression of sepsis: A and B in the figure refer to the survival rates of TKT-WT-Mφ and TKT-KO-Mφ mice after intraperitoneal injection of E. coli or LPS. C-E in the figure respectively refer to the levels of inflammatory cytokines IL-6, IL-1β, and TNF-α in serum detected by ELISA, the mRNA levels of various cytokines in lung tissue detected by qPCR, and representative images of H&E staining of lung tissue in mice after intraperitoneal injection of LPS; F in the figure refers to the survival rates of TKT-WT-Mφ and TKT-KO-Mφ mice after CLP surgery. G-I in the figure respectively refer to the levels of inflammatory cytokines IL-6, IL-1β, and TNF-α in serum detected by ELISA, the mRNA levels of inflammatory cytokines Il-6 in lung, liver, kidney, and spleen detected by qPCR, and representative images of H&E staining of lung tissue.

[0054] Figure 5 TKT inhibits the 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; C-D in the figure refer to the effect of TKT on the activation of NF-κB luciferase reporter gene; E-G in the figure refer to the effect of TKT and its inhibitor OT on the activation of the TAK1-NF-κB signaling pathway.

[0055] Figure 6 TKT knockout reprograms the macrophage metabolic network: A and B in the figure refer to the effects of TKT deficiency on macrophage ECAR / OCR;

[0056] C in the figure is a schematic diagram of non-targeted metabolomics analysis; D in the figure refers to the changes in metabolites related to glycolysis, pentose phosphate pathway (PPP), and tricarboxylic acid cycle (TCA) in TKT-WT-PMs and TKT-KO-PMs; E in the figure is the non-targeted metabolomics analysis of TKT-WT-PMs and TKT-KO-PMs, screening for differential metabolites with p < 0.05 and performing KEGG metabolic pathway enrichment; F in the figure 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.

[0057] Figure 7Macrophage TKT affects inflammation by regulating the level of unsaturated fatty acids, thereby influencing the binding of TAK1 / TAB1: In the figure, A and B refer to macrophage TKT affecting inflammatory signals by regulating the level of unsaturated fatty acids; C and D in the figure refer to TKT affecting the binding of TAK1 / TAB1 by changing the level of unsaturated fatty acids.

[0058] Figure 8 AAV-shrna targeting lung macrophage TKT can improve mice E.coli induced sepsis: WT mice were injected with AAV adenovirus via the tail vein, and three weeks later, intraperitoneally injected with E.coli , in the figure, A is the flow cytometry sorting strategy for mouse lung macrophages; B is the detection of the silencing level of TKT in the sorted macrophages by qPCR and western blot; C in the figure is the detection of the mRNA levels of related cytokines Il-6 , Il-1β , Tnf-α and Il-10 in the mouse lung tissue by qPCR; D in the figure is the detection of the levels of inflammatory cytokines IL-6 and TNF-α in the mouse serum by ELISA; E in the figure is the detection of the levels of BUN and Scr in the mouse serum by kit; F in the figure refers to the detection of the levels of AST and ALT in the mouse serum by kit; G in the figure refers to the wet / dry weight ratio of the mouse lung tissue; H in the figure refers to the representative image of H&E staining of the mouse lung tissue; I in the figure is the detection of the mRNA levels of various cytokines in the lung tissue by qPCR; J in the figure refers to the survival of the mice. Detailed implementation manners

[0059] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further clarified below in conjunction with specific embodiments. However, the following embodiments are only the preferred embodiments of the present invention and not all. Based on the embodiments in the implementation manners, other embodiments obtained by those skilled in the art without creative efforts all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the used operation methods are all conventional operation methods, the used equipment is all conventional equipment, and the equipment materials used in each embodiment are the same.

[0060] Example 1 Animals, materials, experimental methods and statistical analysis used in the present invention

[0061] 1 Experimental animals

[0062] Myeloid-specific knockout TKT mice Tkt fl / fl(C57BL / 6J) mice were purchased from Cyagen Biosciences Inc. (Guangzhou, China) and generated using CRISPR-Cas9-mediated genome editing technology; Myeloid knockout tool mice Lyz2-Cre (C57BL / 6J) were crossed with Tkt fl / fl mice to generate myeloid cell-specific TKT gene knockout mice Tkt fl / fl Lyz2 -Cre + , and littermate Tkt fl / fl Lyz2 -Cre - mice were used as control mice. All mice were housed in a specific pathogen-free (SPF) environment and maintained on a 12 h light / dark cycle at 22 - 24 °C with unrestricted access to food and water.

[0063] 2 Cell culture and treatment

[0064] HEK293T and RAW264.7 cell lines were cultured in complete RPMI-1640 (Gibco) or DMEM (Gibco) medium at 37 °C and 5% CO2, supplemented with 10% (v / v) FBS (Gibco), 1% penicillin-streptomycin (100 U / ml, Gibco), and 1% ciprofloxacin (Beyotime). Mouse peritoneal macrophages (PEMs) were obtained by intraperitoneal injection of 1 mL of 3% Brewer's collagenase medium. In the TKT inhibition assay, cells were treated with thiamine pyrophosphate (OT; 50 μM; O4000; Merck) to inhibit TKT activation. To establish relevant models in vitro, cells were treated under the following conditions: 200 ng / ml LPS - 4 h, 20 μg / mL Poly(I:C) - 12 h, 6 μg / mL CpG - 12 h, 1 ng / mL HT-DNA - 12 h, serum starvation for 12 h, 500 μM AICAR - 12 h, 50 μM eicosapentaenoic acid (EPA) - 12 h, 50 μM docosahexaenoic acid (DHA) - 12 h, 50 μM arachidonic acid (AA) - 12 h.

[0065] 3 Human blood samples

[0066] According to the research protocol approved by the Tianjin Medical University Ethics Review Committee, sepsis patients and healthy individuals participating in this study provided informed consent for blood collection in accordance with institutional and national guidelines. The research protocol was approved by the institutional review board. The identities, ages, and genders of patients and healthy blood donors were kept confidential in accordance with the regulations of the ethics committee.

[0067] Peripheral blood mononuclear cells (PBMCs) from septic patients or healthy donors were isolated using human peripheral blood lymphocyte separation medium (C0025, BioTianmei) and used for related experiments.

[0068] 4 Statistical analysis methods

[0069] Legend All data were statistically analyzed and processed using GraphPad Prism software, and graphs were plotted using GraphPad Prism software. To ensure the accuracy and reliability of the experimental data, all quantitative experimental data were maintained with three biological replicates and three technical replicates, and the differences between samples were shown in the graphs. Values were expressed as mean ± standard error, and two-tailed Student's t-test was used for differential analysis. One-way analysis of variance was used for data comparison among multiple samples, 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.

[0070] Example 2 Effect of transketolase TKT on sepsis

[0071] 1. Experimental methods

[0072] 1.1 Plasmids and transfection

[0073] All full-length DNAs of TKT, TAK1, and TAB1 cDNAs were cloned into the pLenti-3xFlag or pLenti-HA vectors. Mutations of TKT were generated using standard PCR cloning strategies with Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific). Expression plasmids of MyD88, TRAF6, Trif, TAK1, TAB1, IKKα, IKKβ, and P65 were constructed by standard molecular biology techniques and cloned into the pcdna3.1-3×Flag vector. Plasmid DNA was transfected into cells using Lipofectamine 2000 reagent (11668019; Thermo Fisher Scientific) according to the standard protocol. Short interfering RNAs targeting specific genes (Sangon) were transfected into cells using Lipofectamine RNAiMAX transfection reagent (13778100; Thermo Fisher Scientific) according to the manufacturer's instructions.

[0074] 1.2 RNA Extraction, RT-PCR, and RNA-seq Analysis

[0075] Total RNA was extracted from cells using the TRIGene reagent (P118, Genstar) for RNA-seq analysis (BGI Co., Ltd.) or for cDNA synthesis (A214, Genstar Biotech Co., Ltd., Beijing). Quantitative PCR was performed using SYBR Green Premix (B21203, Bimake) on a 7500HT Fast qRT-PCR instrument (4351107, Thermo Fisher Scientific). Data were normalized to β-Actin transcription and analyzed using the ΔΔCt method.

[0076] 1.3 Protein Isolation and Immunoblot Analysis

[0077] 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 onto 8 - 12% SDS - polyacrylamide for electrophoresis and then transferred to a PVDF membrane (IPVH00010, MERCK) for immunoblotting to analyze the target protein level. ECL chemiluminescence kit (Millipore) was used for color development.

[0078] 1.4 Histopathological analysis

[0079] Mouse organs were fixed in 10% formaldehyde overnight. Paraffin embedding was used for H&E staining, and histological images were acquired using a light microscope.

[0080] 1.5 Immunofluorescence staining

[0081] For P65 nuclear translocation staining, Raw264.7 cells were cultured on coverslips and then transfected with the indicated plasmids or siRNAs. Cells were stimulated with LPS, washed, fixed, permeabilized, and then stained with the indicated antibodies and DAPI. Images were acquired using a fluorescence microscope.

[0082] 1.6 Co - immunoprecipitation

[0083] Cells were harvested and lysed in 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℃ for 10 min as the Input sample. 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℃. The immunoprecipitates were washed twice with NETN buffer and then twice with PBS. 80 μL of 1× SDS buffer was added, and the immunoprecipitated proteins were denatured by boiling at 100℃ for 10 minutes, followed by immunoblot analysis.

[0084] 1.7 Flow cytometry

[0085] The cells were extracted into single-cell suspensions, blocked with specific surface antibodies in staining solution containing 2% BSA, then washed three times with PBS, incubated with relevant flow antibodies in the dark on ice for 30 min, washed three times with PBS and then analyzed by flow cytometry using a BD Caliber flow cytometer (BD Biosciences), and sorted by flow cytometry using a FACS Aria Fusion Cell Sorter (BECKMAN, CytoFLEX SRT). The data were analyzed by FlowJo (Tree Star) software.

[0086] 1.8 LPS, Escherichia coli E.coli Infection and CLP models

[0087] WT and KO mice were intraperitoneally injected E.coli (1×10 6 CFU) or LPS (20 mg / kg for survival, 25 mg / kg for acute infection) to induce septic shock. The CLP procedure was performed according to the published protocol (Rittirsch, Daniel et 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 8-10 weeks old were anesthetized with tribromoethanol (200 mg / kg, intraperitoneally). When the mice showed no response to toe pinching, the abdominal hair was shaved off and the skin was disinfected with 75% alcohol. A midline abdominal incision was made with a scalpel. The cecum was exposed, ligated with 4-0 silk suture from the distal third, and perforated with a 21-gauge needle through the cecal wall. The cecum was gently squeezed to expose a small amount of feces to ensure complete perforation. Then the cecum was returned to the abdominal cavity and the incision was closed. Immediately after surgery, 0.5 mL of warm saline was injected subcutaneously. Control group mice received anesthesia, laparotomy, and wound suturing, but they did not receive cecal ligation and puncture surgery. The survival of mice in the group for detecting survival rate was observed and recorded in a timely manner, and specimens of mice for detecting relevant pathological injuries were collected for detection at the corresponding time.

[0088] 1.9 Enzyme-linked immunosorbent assay (ELISA) and kit assay

[0089] ELISA kits for mouse IL-6 (Cat# EK0411), IL-1β (Cat# EK0394), and TNF-α (Cat# EK0527) were from Boster, and 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 relevant substances in mouse plasma and cell supernatants were detected according to the manufacturer's instructions.

[0090] 1.10 Dual-luciferase reporter activity assay

[0091] According to the operating procedure, the reporter plasmid was transiently transfected into cells using Lipofectamine (Thermo Fisher Scientific, USA). The transfection efficiency was determined using the pRL-CMV plasmid (Promega) containing the Renilla luciferase gene. HEK293T cells were transiently transfected with the reporter plasmid. After 24 hours of treatment, the transfected cells were washed twice with PBS and then lysed in lysis buffer (Yeason), gently shaken at room temperature for 20 min. The cell lysate was centrifuged at 17,000 x g for 2 minutes 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). The firefly luciferase activity was measured using a luminometer, and the program was set to delay for 2 seconds before each reporter detection and then measure for 10 seconds. After measuring the firefly luciferase activity (Stop&Glo®, Promega), the Renilla luciferase measurement buffer was added to measure the enzyme activity. Each transfection had duplicate wells, and all measurements were repeated at least three times.

[0092] 1.11 LC-MS analysis of metabolites

[0093] Mouse PM (5 x 10 6 cells) were cultured in complete DMEM. After 24 hours, the cells were rinsed with ice-cold PBS and then 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 the standard protocol.

[0094] 1.12 Mitochondrial and glycolytic stress test assays

[0095] Inoculate the PMs of TKT-WT-Mφ or TKT-KO-Mφ mice with appropriate density into a 24-well Seahorse XFe-24 assay plate. After adding appropriate stimulants, wash the cells and culture them in 1640 medium without buffer for 1 hour. Add water to the probe plate in advance and preheat the analyzer. Add various drugs to the drug addition ports of the probe plate according to the measured metabolism. Then, execute the preset program to inject the drugs into the cell supernatant in sequence, and monitor the real-time dynamic changes of drug-induced cell metabolism.

[0096] 1.13 AAV adenovirus vector infection

[0097] The AAV Lung X delivery system was used to knockout TKT in mouse liver macrophages. The recombinant adeno-associated virus serotype lung X (AAV Lung X) vector carried the mouse TKT or the NC vector with a CD68 promoter (a promoter specific for macrophages). AAV Lung X-CD68-sh(TKT) or AAV Lung X-CD68-NC was 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 the AAV Lung X vector genome, and a relevant disease model was established three weeks later.

[0098] 2. Experimental results

[0099] 2.1 LPS and other pro-inflammatory stimulants reduce the expression of TKT in macrophages, and LPS-induced down-regulation of TKT expression depends on the TLR 4-NF-κB signaling axis

[0100] Macrophage-mediated inflammatory responses are closely related to metabolic processes. TKT-mediated non-oxidative PPP significantly affects the functions of hepatocytes, adipocytes, and regulatory T cells. Therefore, we first investigated whether the expression of TKT was related to macrophage-mediated inflammation.

[0101] When mouse peritoneal macrophages (PMs) were exposed to pro-inflammatory stimulants including LPS, Gram-negative bacterium Escherichia coli (E. coli), herring testis DNA (HT-DNA), TLR 3 or 9 agonist Poly(I:C) or CpG, serum starvation (SS), AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), the expression level of TKT was significantly decreased by qPCR and Western blot analysis ( Figure 1 in A- Figure 1 in C). In vivo, the mRNA expression of TKT in mouse bone marrow was decreased after stimulation with LPS ( Figure 1in D) and in serum peripheral blood mononuclear cells (PBMCs) of patients with systemic inflammatory response syndrome (SIRS) or sepsis ( Figure 1 were significantly reduced in E).

[0102] After 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 the expression of TKT.

[0103] Studies have shown that silencing MyD88 or TRIF can reactivate the expression of TKT in LPS-stimulated macrophages ( Figure 1 in F and Figure 1 in G). In 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 in H and Figure 1 in I). Overall, we demonstrated that exposure to multiple pro-inflammatory stimuli significantly inhibited the expression of TKT both in vitro and in vivo, and the LPS-induced inhibition of TKT expression was dependent on TLR4-NF-κB signaling.

[0104] 2.2 Inhibition of macrophage TKT deficiency suppresses the expression of pro-inflammatory cytokines

[0105] Since it was found that TLR4-NF-κB signaling regulates TKT expression, we then attempted to determine whether TKT in macrophages in turn affects TLR4-mediated inflammatory responses.

[0106] We found that in LPS-treated mouse PMs, treatment with hydroxythiamine or knockdown of TKT by small interfering RNA (siRNA) significantly reduced the mRNA expression of pro-inflammatory cytokines, including interleukin (IL)-6, IL-1β, and tumor necrosis factor α (TNF-α) ( Figure 2 in A and Figure 2 in B). Consistently, we observed a significant decrease in the mRNA levels of pro-inflammatory cytokines after OT administration to peripheral blood mononuclear cells (PBMCs) of septic patients ( Figure 3 in A). These findings indicate that inhibition of TKT can reduce LPS-induced inflammatory responses.

[0107] To further clarify the role of TKT in inflammation in macrophages, we generated bone marrow-specific TKT knockout mice ( Tkt fl / fl Lyz2 -Cre + , namely TKT-KO-Mφ mice) and littermate mice (Tkt fl / fl , namely TKT-WT-Mφ mice) ( Figure 2 C in Figure 2 and D in Figure 2 As expected, TKT was effectively knocked out in PMs isolated from TKT-KO-Mφ mice ( Figure 2 E in Figure 3 The birth of TKT-KO-Mφ mice followed Mendel's genetic law and showed no developmental phenotypic defects. Compared with TKT-WT-Mφ mice, their PMs showed normal differentiation and number ( Figure 3 F in E. coli 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-PM ( Figure 3 B in Figure 3 C in

[0108] 2.3 Myeloid TKT deficiency prevents septic shock

[0109] Since the overactivation of the TLR4-TAK1-NF-κB / MAPK signaling pathway is a key factor in sepsis, we investigated whether TKT deficiency would affect the occurrence of septic shock in vivo by infecting mice with Escherichia coli.

[0110] We used Escherichia coli, endotoxin, or performed experiments by cecal ligation and puncture (CLP). The results showed that after intraperitoneal injection of Escherichia coli, the occurrence of septic shock in TKT-KO-Mφ mice was different from that in TKT-WT-Mφ mice ( Figure 4 A in Figure 4 B in Figure 4 Consistent with this finding, we observed that the concentrations of IL-6, IL-1β, and TNF-α in the serum of TKT-KO-Mφ mice were significantly reduced ( Figure 4 C in Figure 4 In addition, the production of pro-inflammatory factors and chemokines in the lungs of TKT-KO-Mφ mice decreased, while the production of the anti-inflammatory cytokine IL-10 increased (

[0111] Subsequently, in Escherichia coli or CLP-induced septic shock, the survival rate of TKT-KO-Mφ mice was significantly higher than that of the WT littermate control group ( Figure 4 F in Figure 4 ). At 20 h after CLP surgery, the concentration of pro-inflammatory factors in the serum of TKT-KO-Mφ mice and their mRNA levels in multiple organs such as the lung, liver, kidney, and spleen were significantly lower than those of WT littermate mice ( Figure 4 G and Figure 4 H in

[0112] ). In addition, the results of H&E staining of the lungs showed that the degree of lung injury in TKT-KO-Mφ mice was reduced (

[0113] I in

[0114] ). Collectively, these results strongly suggest that myeloid TKT deficiency can inhibit the development of septic shock. Figure 5 Figure 5

[0115]

[0116] TLR4-induced NF-κB activation leads to the production of IL-1β, IL-6, and TNF-β. Subsequently, we focused on whether TKT affects NF-κB activation to enhance LPS-induced inflammatory responses.

[0114] Twenty minutes after LPS treatment, TKT knockdown decreased, while TKT overexpression increased the nuclear translocation of p65 ( Figure 5 A in Figure 5 ). In addition, knockdown of p65 also severely inhibited the upregulation of IL-6 and IL-1β expression induced by TKT overexpression ( Figure 5 B in Figure 5 ), indicating that NF-κB activation is crucial for the promoting effect of TKT on the production of these pro-inflammatory cytokines.

[0115]

[0116] Since MyD88, TRAF6, TIF, TAK1, TAB1, and IKKα / β are key regulators of NF-κB activation, we next determined which of these molecules function in concert with TKT by NF-κB luciferase reporter assays. For this purpose, expression vectors for MyD88, TRAF6, TRIF, TAK1, TAB1, IKKα / β, and p65 were co-transfected alone with NF-κB-driven luciferase in HEK 293T cells, and co-expressed with empty vectors or TKT expression vectors. Then, the NF-κB-driven luciferase activities were compared between cells infected with empty vectors and TKT expression vectors for each protein among these signaling molecules, respectively. Figure 5 Figure 5 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 5in D). In contrast, when TKT was co-expressed with TAK1, TAB1, IKKα / β, or p65, no significant increase was detected ( Figure 5 in D), suggesting that the node of TKT regulation of the signaling axis is upstream of TAK1 / TAB1 and downstream of TIF.

[0117] Since TAK1 is a key upstream molecule in the MAPK pathway and the IKKα / β-NF-κB pathway, we subsequently examined whether TKT regulates the phosphorylation of TAK1, IKKα / β, and MAPK kinases. Compared with WT macrophages, primary macrophages treated with OT and deficient in TKT showed reduced levels of p-TAK1, p-IKKα / β, p-p65, p-JNK, and p-P38 after LPS stimulation, but the level of p-ERK was not reduced ( Figure 5 in E and Figure 5 in F). However, overexpression of TKT promoted the phosphorylation of these pathway proteins ( Figure 5 in G). Taken together, we found that macrophage TKT promotes the expression of pro-inflammatory cytokines by inhibiting the TAK1-NF-κB / MAPK signaling pathway.

[0118] 2.5 Deletion of TKT reprograms macrophage metabolism and leads to increased levels of polyunsaturated fatty acids

[0119] Cell energy is a key factor in regulating immune-mediated inflammation. Therefore, we investigated whether TKT deficiency would alter macrophage energy metabolism. Seahorse showed that the extracellular acidification rate (ECAR) of PM in the KO group was lower than that of PM in the WT group at baseline and after LPS stimulation ( Figure 6 in A), indicating reduced glycolytic activity of PM in KO. Interestingly, PM deficient in TKT showed an increased oxygen consumption rate (OCR; Figure 6 in B). These results suggest that TKT deficiency leads to a shift in macrophages from glycolysis to oxidative phosphorylation. To further clarify the potential mechanism by which TKT promotes inflammation, we also performed metabolomic analysis on PM of TKT-WT and TKT-KO ( Figure 6 in C). TKT deficiency led to the accumulation of R5P and S7P in the non-oxidative PPP ( Figure 6 in D). In contrast, the levels of the glycolytic metabolites F6P and G3P increased. The level of pyruvate decreased, and the levels of the TCA cycle intermediates citrate, isocitrate, α-KG, fumarate, and malate did not change significantly. In addition, among the results of enrichment of differential metabolite pathways in metabolomics, enrichment revealed that TKT deficiency significantly altered the fatty acid metabolism pathway in macrophages, especially the α-linolenic acid / linolenic acid (ALA / LA) pathway ( Figure 6in E), while arachidonic acid (AA, 20:4 ω-6), eicosapentaenoic acid (EPA, 20:5 ω-3), and docosahexaenoic acid (DHA, 22:6 ω-3) among polyunsaturated fatty acids (PUFAs) were identified as the three fatty acids with the largest increase multiples ( Figure 6 in F).

[0120] 2.6 The increase in PUFAs caused by TKT knockout inhibited the interaction between TAK1 and TAB1, thereby inhibiting the activation of the TAK1-NF-κB / MAPK signaling pathway

[0121] Early 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 GPR, nuclear receptors, and other mechanisms. To study how AA and EPA / DHA affect the effect 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 production of pro-inflammatory cytokines induced by TKT overexpression and the increase in TAK1 signaling pathway activation ( Figure 7 in A and Figure 7 in B). In our previous results, it was found that the node where TKT plays a pro-inflammatory regulatory role is upstream of TAK1 / TAB1 and downstream of TRIF. It has been reported that DHA can inhibit the binding of TAK1 / TAB1. Therefore, we speculated that TKT might 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 in C and Figure 7 in D). Overall, these research results indicate that TKT promotes the binding of TAK1 to TAB1 downstream of TLR4 by regulating the metabolism of PUFAs, thereby affecting the activation of the TAK1-NF-κB / MAPK signaling pathway and ultimately altering the expression of pro-inflammatory factors.

[0122] 2.7 An AAV adenovirus vector targeting macrophage TKT improves the disease progression of E.coli septic shock induced in mice

[0123] To explore the therapeutic potential of targeting macrophage TKT in sepsis, we used an adeno-associated virus (AAV) delivery system to inject C57BL / 6 mice via the tail vein. An AAV8 vector carrying murine shTKT (AAV-shTKT) or an empty vector with a CD68 promoter (a promoter targeting macrophages) (AAV-EGFP-shNC) was injected into wild-type mice via the tail vein, and three weeks later, an intraperitoneal injection was given E.coli to establish a sepsis model. Twelve hours later, lung tissue cells of the mice were extracted, and macrophages were sorted by flow cytometry ( Figure 8 A in Figure 8 ), and the mRNA and protein levels of TKT were further detected. The results showed that the expression level of TKT in lung macrophages was significantly downregulated ( Il-6 B in Il-1β ). Meanwhile, qPCR detection found that the levels of inflammatory factors Tnf-α in the lung macrophages of mice injected with AAV-shTKT were downregulated, while the expression level of Il-10 inhibiting inflammation was upregulated ( Figure 8 C). In addition, the levels of inflammatory factors IL-6, IL-1β, TNF-α, and the liver and kidney injury indicators blood urea nitrogen, creatinine, AST, and ALT in the serum of mice injected with AAV-shTKT were significantly reduced ( Figure 8 D in Figure 8 - Figure 8 F), the wet / dry weight ratio of the lung tissue was downregulated ( Figure 8 G), and the H&E staining results showed that the degree of lung injury in mice injected with AAV-shTKT was significantly less than that in AAV-shNC mice ( Il-10 H). The production of pro-inflammatory factors and chemokines in the lungs of mice decreased, while the production of the anti-inflammatory cytokine Figure 8 I) increased. Meanwhile, in E.coli -induced septic shock, the survival rate of mice injected with AAV-shTKT was significantly higher than that of the control group ( Figure 8 J). In summary, these results strongly suggest that the AAV adenovirus vector targeting macrophage TKT can effectively inhibit the occurrence of septic shock in mice.

[0124] All in all, our research results indicate that TKT-mediated non-oxidative PPP integrates the regulation of cellular metabolism and signaling pathways to affect the inflammatory response.

[0125] The research results of the present invention show that the transketolase-mediated non-oxidative pentose phosphate pathway plays a key role in driving macrophage-mediated inflammatory responses through the regulation of integrated metabolism and signal transduction. Importantly, we designed an AAV-shTKT adenovirus vector targeting macrophage TKT in lung tissue and achieved good therapeutic effects in the treatment and prevention of murine sepsis, providing potential clinical applications for the management of acute inflammatory diseases.

[0126] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within 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, characterized in that: The transketolase inhibitor is hydroxythiamine.

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

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