Application of macrophage PHGDH in the preparation of TAK1 inhibitors

Abstract

The present invention provides the use of macrophage PHGDH in the preparation of TAK1 inhibitors, and belongs to the field of biomedicine technology. The present invention finds that knocking down PHGDH in macrophages can promote PA-induced inflammation. In addition, macrophage-specific PHGDH binds to TAK1, inhibiting its interaction with TAB1, thereby inhibiting the activation of TAK1 and downstream NF-κB and MAPK signaling. Inhibiting TAK1 activation can slow the development of metabolic dysfunction-related fatty hepatitis MASH caused by myeloid PHGDH knockout. Importantly, adeno-associated virus-mediated overexpression of PHGDH in liver macrophages alleviates MAFLD. The present invention finds that macrophage PHGDH is a promising drug for the treatment of MAFLD.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of macrophage PHGDH in the preparation of TAK1 inhibitors. Background Art

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD) is a complex multifactorial metabolic disease. 20% of MAFLD patients develop from simple fatty degeneration of metabolic dysfunction-associated fatty liver disease (MAFLD) to metabolic dysfunction-associated steatohepatitis (MASH). MASH is often accompanied by metabolic disorders such as obesity, type 2 diabetes, insulin resistance (IR), and dyslipidemia. Its pathological characteristics are excessive accumulation of lipids in the liver, leading to fat toxicity and inflammatory response, resulting in liver tissue damage and fibrosis. If not controlled and treated, MASH can develop into cirrhosis and liver cancer.

[0003] Although some drugs have shown efficacy in MAFLD, including peroxisome proliferator-activated receptor (PPAR) agonists, farnesone X receptor (FXR) agonists, insulin sensitizers, glucagon-like peptide 1 (GLP-751) receptor agonists, and sodium-glucose cotransporter-2 (SGLT-2) inhibitors, they have not been approved for clinical treatment of MAFLD until recently. The first drug approved for the treatment of MASH, REZDIFFRA (active ingredient resmerol), is a thyroid hormone receptor β (THR-β) agonist that activates this receptor in the liver and reduces liver fat accumulation in patients. Therefore, there is an urgent need to study the mechanism of MAFLD progression to explore its potential molecular targets.

[0004] Hepatic macrophages account for approximately 15% of the total number of hepatocytes. The hepatic macrophage population shows significant heterogeneity, mainly composed of intrahepatic macrophages (KCs) and infiltrating macrophages (mainly monocyte-derived macrophages MDMs). Hepatic macrophages are involved in regulating liver immune homeostasis and the occurrence of various liver diseases such as MAFLD, chronic viral hepatitis, and liver cancer. During the progression of MAFLD, macrophages detect saturated fatty acids such as palmitic acid (PA) as danger signals, activate Toll-like receptor-mediated signaling, and secrete proinflammatory cytokines such as interleukins (IL-6 and IL-1β). This leads to neutrophil recruitment, IR, and altered hepatocyte metabolism by increasing lipogenesis, gluconeogenesis, and cell proliferation. Increased lipotoxicity further stimulates macrophage activation. These findings demonstrate the importance of the innate immune system, especially macrophages, in the pathophysiology of MAFLD. Therefore, elucidating the regulatory mechanisms of macrophage inflammatory activation is of great significance for understanding the pathogenesis of MAFLD and developing therapeutic strategies for the disease.

[0005] Transforming growth factor-β-activated kinase 1 (TAK1) is a key mediator of the Toll-like receptor signaling pathway and is involved in the activation of proinflammatory cytokines. Metabolic stress overactivates TAK1 by inducing its post-translational modification, namely ubiquitination and subsequent autophosphorylation, leading to the activation of downstream nuclear factor-κB (NF-κB) and N-terminal kinase / p38 (JNK / p38) signaling pathways, which further promote the development of metabolic disorders and steatohepatitis. Inhibition of TAK1 overactivation by deubiquitination or dephosphorylation may be a feasible strategy for the treatment of MALFD. An increasing number of studies have reported that different proteins can affect the progression of MALFD by regulating TAK1 activity. TNF-α-induced protein 3 (TNFAIP3)-interacting protein 3 (TNIP3) inhibits TAK1 activity by inhibiting E3 ubiquitin ligase tripartite motif 8 (TRIM8)-mediated TAK1 ubiquitination, thereby inhibiting the occurrence of MASH. E3 ubiquitin ligase tripartite motif 16 (TRIM16) can improve MASH by catalyzing K48-linked p-TAK1 ubiquitination to target its degradation, thereby inhibiting the JNK / p38 signaling pathway. Switch-associated protein 70 (SWAP70) slows down the progression of MASH by inhibiting the interaction between TAK1-binding protein 1 (TAB1) and TAK1, thereby inhibiting the activation of TAK1 and downstream pathways.

[0006] Serine is a non-essential amino acid that is a key link in glycolysis, one-carbon metabolism, lipid metabolism, and purine and glutathione synthesis. Cells can not only obtain serine from the surrounding environment through amino acid transporters, but also synthesize serine from glucose through the serine synthesis pathway, in which the key first step is catalyzed by phosphoglycerate dehydrogenase (PHGDH). A genome-scale hepatocyte metabolic model revealed that MAFLD patients are serine deficient. Downregulation of hepatic PHGDH and reduced serine levels contribute to the pathogenesis of MALD in mice. Endogenous or exogenous serine deficiency induced by PHGDH inhibition impairs lipid homeostasis and accumulation in the liver through multiple mechanisms, including by affecting NADPH or ceramide synthesis, the gut-liver axis, and oxidative stress. Notably, supplementation with exogenous serine can inhibit the progression of MAFLD in mice. These observations suggest that hepatic PHGDH and serine metabolism play an important role in the progression of MAFLD. However, the role of macrophage PHGDH in lipotoxicity-induced inflammatory responses and the progression of MAFLD remains unclear and needs further exploration. Summary of the invention

[0007] The present invention provides the use of macrophage PHGDH in the preparation of TAK1 inhibitors. The present invention found that knocking down PHGDH in macrophages can promote PA-induced inflammation. In addition, macrophage-specific PHGDH binds to TAK1, inhibiting its interaction with TAB1, thereby inhibiting TAK1 activation and downstream NF-κB and MAPK signaling (see Figure 1 ). Inhibition of TAK1 activation slows the development of metabolic dysfunction-associated fatty liver disease (MASH) caused by myeloid PHGDH knockout. Importantly, adeno-associated virus-mediated overexpression of PHGDH in liver macrophages alleviates MAFLD. The present invention finds that macrophage PHGDH is a promising drug for the treatment of MAFLD.

[0008] The technical solution of the present invention includes:

[0009] In a first aspect, the present invention provides the use of macrophage-specific PHGDH in the preparation of a TAK1 inhibitor.

[0010] Specifically, the macrophage-specific PHGDH includes monomeric PHGDH, dimeric PHGDH or tetrameric PHGDH.

[0011] Preferably, the macrophage-specific PHGDH is a tetrameric PHGDH.

[0012] Preferably, the TAK1 inhibitor includes: small molecule inhibitors, antibody inhibitors, small molecule compounds or high molecular weight compounds.

[0013] Specifically, the TAK1 inhibitor is used for the prevention, treatment and / or auxiliary treatment of MAFLD and MAFLD complications.

[0014] Preferably, the MAFLD includes: simple fatty liver, metabolic dysfunction-related fatty liver hepatitis, liver fibrosis and cirrhosis, acute liver injury or liver cancer.

[0015] Preferably, the complications of MAFLD include: hepatocellular carcinoma, obesity, cardiovascular disease, diabetes or chronic kidney disease.

[0016] Preferably, the TAK1 inhibitor exerts its effect by inhibiting the activation of the TAK1-NF-κB / MAPK signaling pathway.

[0017] Specifically, the TAK1 inhibitor further includes a pharmaceutically acceptable excipient.

[0018] Preferably, the pharmaceutically acceptable excipients include: wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, solutes that make the preparation isotonic with the recipient's blood, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors, or a combination of two or more thereof.

[0019] Specifically, the dosage forms of the TAK1 inhibitor include: tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays or injections.

[0020] The beneficial effects of the present invention are:

[0021] The present invention found that knocking down PHGDH in macrophages can promote PA-induced inflammation. In addition, macrophage-specific PHGDH binds to TAK1 and inhibits its interaction with TAB1, thereby inhibiting TAK1 activation and downstream NF-κB and MAPK signaling. Inhibition of TAK1 activation can slow the development of metabolic dysfunction-related fatty hepatitis MASH caused by myeloid PHGDH knockout. Importantly, adeno-associated virus-mediated overexpression of PHGDH in liver macrophages alleviated MAFLD. The present invention found that macrophage PHGDH is a promising drug for the treatment of MAFLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Flow chart for the treatment of MAFLD with macrophage PHGDH.

[0023] Figure 2 PHGDH negatively regulates PA-induced proinflammatory cytokine production in macrophages; A in the figure is the analysis of PHGDH expression in BMDM stimulated by different concentrations of PA for 12 hours; B is the analysis of PHGDH expression in BMDM stimulated by 0.2 mM PA at different time points; C is the PHGDH protein expression and proinflammatory cytokine mRNA expression levels after RAW264.7 cells were transfected with siCtrl or siPHGDH; D is the PHGDH protein expression and proinflammatory cytokine mRNA expression levels in BMDM isolated and differentiated from PHGDH-WT-MΦ and PHGDH-KO-MΦ mice after being stimulated with PA for 12 hours; E is the HA (PHGDH) protein expression and proinflammatory cytokine mRNA expression levels in RAW264.7 cells transfected with Ctrl-HA or PHGDH-HA and stimulated with 0.2 mM PA for 12 hours; * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0024] Figure 3Figure 1 shows that bone marrow-specific PHGDH deficiency exacerbates the progression of high-fat diet (HFD)-induced MAFL. A is the animal model of HFD-induced MAFL and the experimental flow chart. B is the ratio of liver weight to body weight. C is the liver H&E staining image, with a scale bar of 100 μm. D is the liver Oil Red O staining image, with a scale bar of 50 μm. E is the qPCR analysis of mRNA expression of genes related to fatty acid metabolism. F is the content of total cholesterol and triglycerides in the liver. G is the immunofluorescence image of liver sections, with a scale bar of 50 μm. H is the qPCR analysis of mRNA expression of pro-inflammatory / anti-inflammatory genes and chemokines. I is the picrosirius red staining image, with a scale bar of 100 μm. J is the qPCR analysis of mRNA expression of fibrosis-related genes. K is the serum AST concentration. NS represents no significant difference (p ≥ 0.05). * represents p<0.05. ** represents p<0.01. *** represents p<0.001.

[0025] Figure 4 Bone marrow-specific PHGDH deficiency aggravates the progression of HFD-induced MAFL; A in the figure is the change in body weight of mice; B is the change in liver weight of mice; C is the glucose tolerance test and its area under the curve; D is the insulin tolerance test and its area under the curve; E is a representative image of H&E staining of white fat, with a scale bar of 100 μm; NS represents no significant difference (p≥ 0.05); * represents p<0.05; ** represents p<0.01; *** represents p<0.001.

[0026] Figure 5 Figure 1 shows that bone marrow-specific PHGDH deficiency exacerbates the progression of MASH induced by HFHC diet. A is the animal model of MASH induced by HFHC diet and the experimental flow chart. B is the ratio of liver weight to body weight. C is the H&E staining image of the liver, with a scale bar of 100 μm. D is the Oil Red O staining image of the liver, with a scale bar of 50 μm. E is the qPCR analysis of the mRNA expression of genes related to fatty acid metabolism. F is the content of total cholesterol and triglycerides in the liver. G is the immunofluorescence image of liver sections, with a scale bar of 50 μm. H is the qPCR analysis of the mRNA expression of pro-inflammatory / anti-inflammatory genes and chemokines. I is the Masson staining image of the liver, with a scale bar of 150 μm. J is the qPCR analysis of the mRNA expression of fibrosis-related genes. K is the serum AST and ALT concentrations. NS represents no significant difference (p ≥ 0.05). * represents p<0.05. ** represents p<0.01. *** represents p<0.001.

[0027] Figure 6Bone marrow-specific PHGDH deficiency aggravates the MASH process induced by HFHC diet; A in the figure is the change in body weight of mice; B is the change in liver weight of mice; C is the glucose tolerance test and its area under the curve; D is the insulin tolerance test and its area under the curve; E is a representative image of H&E staining of white fat, with a scale bar of 100 μm; NS represents no significant difference (p ≥ 0.05); * represents p<0.05; ** represents p<0.01; *** represents p<0.001.

[0028] Figure 7 PHGDH inhibits PA-mediated TAK1–NF-κB / MAPK signaling pathway; A is the Western blot analysis results of different antibodies; B is the nuclear p65 Western blot analysis results; C is the qPCR analysis of NF-κB target gene expression; D is the Western blot analysis results of TAK1-NF-κB / MAPK signaling pathway; E is the qPCR analysis of pro-inflammatory cytokine expression; * represents p<0.05; ** represents p<0.01; *** represents p<0.001.

[0029] Figure 8 Figure 2 is the interaction between PHGDH tetramer and TAK1; A in the figure is after RAW264.7 was transfected with the specified plasmid, PA was treated or not treated, and the exogenous PHGDH-HA was immunoprecipitated using anti-HA agarose, and immunoblot analysis was performed using different antibodies; B is after RAW264.7 was transfected with the specified plasmid, endogenous TAK1 was immunoprecipitated using anti-TAK1 antibody, and immunoblot analysis was performed using different antibodies; C is Ctrl-HA or PHGDH-HA and TAK1-Flag were expressed in 293T cells, and PA was used to stimulate at the specified time points, and the exogenous PHGDH-HA was immunoprecipitated using anti-HA agarose, and immunoblot analysis was performed using different antibodies; D is F lag-TAK1 and full-length or truncated PHGDH, the binding sites of PHGDH and TAK1 were explored by immunoblotting analysis; E is the use of HA-PHGDH and full-length or truncated TAK, the binding sites of TAK1 and PHGDH were explored by immunoblotting analysis; F is the GST pull-down experiment showing the direct binding of PHGDH and TAK1; G is the His pull-down experiment showing the direct binding of PHGDH and TAK1; H is the level of proteins related to the TAK1-NF-κB / MAPK signaling pathway; I is the determination of pro-inflammatory cytokines by qPCR in RAW264.7 cells transfected with siCtrl or siPHGDH and / or different plasmids and stimulated with PA for 12 hours Il-6 and Il-1βJ is the expression level of PHGDH by western blot analysis after PMs were pretreated with 15 μM CBR-5884 for 6 hours and cross-linked with 0.25 mM DSS; K is the effect of PHGDH inhibitor CBR-5884 on the interaction between PHGDH and TAK1; L is the protein level of p-TAK1 (Thr184 / 187) and TAK1 by western blot analysis; M is the determination of pro-inflammatory cytokines by qPCR after PA stimulation for 12 hours Il-6 and Il-1β mRNA levels of pro-inflammatory cytokines in RAW264.7 cells transfected with siCtrl or siTAK1 and / or different plasmids and pretreated with 15 μM CBR-5884 for 6 hours and stimulated with PA for 12 hours. Il-6 and Il-1β expression levels; * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

[0030] Fig. 9 Figure 3 is the interaction between PHGDH tetramer and TAK1; A in the figure is a schematic diagram of the truncated construction of PHGDH; B is a schematic diagram of the truncated construction of TAK1; C is the detection of PHGDH polymers by western blot; D is the effect of PHGDH enzyme active site mutation on the interaction between PHGDH and TAK1; E is the detection of phosphorylated and total TAK1 protein levels by western blot; F is the measurement of pro-inflammatory cytokine mRNA expression levels by qPCR; NS represents no significant difference (p ≥ 0.05); * represents p<0.05; ** represents p<0.01; *** represents p<0.001.

[0031] Fig.10 PHGDH regulates PA-induced inflammation by inhibiting the interaction between TAB1 and TAK1; AB in the figure is 293T cells transfected with the specified plasmids and treated with PA for 30 minutes, and the ubiquitination of TAK1 was detected by immunoprecipitation experiment; C is the interaction between TAB1 and TAK1; D is the phosphorylated and total TAK1, IKKβ, JNK, P38 and ERK protein levels; E is qPCR determination of pro-inflammatory cytokines Il-6 and Il-1β expression levels; * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

[0032] Fig.11Inhibition of TAK1 can eliminate the progression of MASH caused by myeloid PHGDH deficiency; A is the 5Z-7-ox -MASH animal model and experimental flow chart; B is a representative western blot image of phosphorylation and total protein levels; C is the ratio of liver weight to body weight of mice; D is an image of liver H&E staining, with a scale bar of 100 μm; E is an image of liver Oil Red O staining, with a scale bar of 50 μm; F is qPCR analysis of mRNA expression of genes related to fatty acid metabolism; G is the content of total cholesterol and triglycerides in the liver; H is qPCR analysis of mRNA expression of proinflammatory genes and chemokines; I is an image of liver Masson staining, with a scale bar of 150 μm; J is qPCR analysis of mRNA expression of fibrosis-related genes; K is serum AST and ALT concentrations; NS represents no significant difference (p ≥ 0.05); * represents p<0.05; ** represents p<0.01; *** represents p<0.001.

[0033] Fig.12 Inhibition of TAK1 eliminated the promotion of MASH progression caused by myeloid-specific PHGDH deficiency; A in the figure is the change in body weight of mice; B is the change in liver weight of mice; C is the glucose tolerance test and its area under the curve; D is the insulin tolerance test and its area under the curve; E is a representative image of H&E staining of white fat, with a scale bar of 100 μm; * represents the comparison between vehicle-WT-Mφ group and vehicle-KO-Mφ group; # represents the comparison between vehicle-KO-Mφ group and 5Z-7-KO-Mφ group; NS represents no significant difference (p ≥ 0.05); * represents p<0.05; ** represents p<0.01; *** or ### represents p<0.001.

[0034] Fig.13 Adenovirus-mediated overexpression of PHGDH in liver macrophages alleviated the progression of MASH induced by HFHC diet; Figure A is an evaluation of PHGDH in liver macrophages (AAV- Phhd) Animal model and experimental flow chart of the treatment effect; B is a representative image of immunofluorescence staining, with a scale bar of 50 μm; C is the ratio of liver weight to body weight of mice; D is an image of liver H&E staining, with a scale bar of 100 μm; E is an image of liver Oil Red O staining, with a scale bar of 50 μm; F is qPCR analysis of mRNA expression of genes related to fatty acid metabolism; G is the content of total cholesterol and triglycerides in the liver; H is qPCR analysis of mRNA expression of proinflammatory genes and chemokines; I is a representative image of liver picrosirius red staining, with a scale bar of 100 μm; J is qPCR analysis of mRNA expression of genes related to fibrosis; K is the concentration of AST and ALT in serum; NS represents no significant difference (p ≥ 0.05); * represents p<0.05; ** represents p<0.01; *** represents p<0.001.

[0035] Fig.14 Adeno-associated virus-mediated overexpression of PHGDH in liver macrophages alleviated the progression of MASH caused by HFHC diet; A in the figure is the mouse body weight curve; B is the change of mouse liver weight; C is the glucose tolerance test and its area under the curve; D is the insulin tolerance test and its area under the curve; E is a representative image of H&E staining of white fat, with a scale bar of 100 μm; NS represents no significant difference (p ≥ 0.05); * represents p<0.05; ** represents p<0.01; *** represents p<0.001. DETAILED DESCRIPTION

[0036] The present invention is described below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention so that the technical solution of the present invention is easier to understand and grasp. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0037] Generated by CRISPR-Cas9 gene editing technology Phhd fl / fl Mice were purchased from Saiye Bioscience Co., Ltd. (Guangzhou, China). To generate myeloid-specific PHGDH knockout mice and their offspring, Phhd fl / fl Mice and Dr. Xiaoyue Tan from Nankai University, China Lyz2- Cre mice were hybridized to obtain myeloid-specific PHGDH knockout mice ( Phhd fl / fl Lyz2 -Cre + , PHGDH-KO-Mφ) and littermate control mice Phhd fl / fl Lyz2 -Cre -(PHGDH-WT-Mφ).

[0038] The experimental methods involved in the following embodiments are as follows:

[0039] Mouse metabolic and liver function tests: The body weight of mice was assessed every two weeks. After feeding, the intact liver tissue was extracted and weighed, and the ratio of liver weight to body weight was calculated. For glucose tolerance test (GTT) and insulin tolerance test (ITT), mice were fasted for 14 h and 6 h, respectively, and blood glucose levels were measured at designated time points after tail vein injection of 1.0 g / kg glucose or 1.0 IU / kg insulin. Then, the area under the curve was calculated using the conventional trapezoidal method. In the high-fat diet (HFD) model, GTT and ITT assays were performed at 18 and 20 weeks, and in the high-fat and high-concentration (HFHC) model, they were performed at 10 and 12 weeks. Liver total cholesterol (TC) and triglyceride (TG) concentrations were determined according to the manufacturer's instructions (Nanjing Jiancheng). Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assays were also performed according to the manufacturer's instructions (Nanjing Jiancheng).

[0040] Histological Analysis: For histological analysis, liver sections were prepared using paraffin or Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan), and epididymal white adipose tissue sections were prepared using paraffin. After dewaxing and rehydration, paraffin sections were stained with H&E to observe the distribution of lipid accumulation. Oil Red O staining was used to observe fatty degeneration in frozen liver sections (8–10 μm). Liver paraffin sections were stained with Sirius red or Masson staining to visualize fibrotic deposits. Digital images were acquired by light microscopy.

[0041] Immunofluorescence staining: To detect the colocalization of F4 / 80 with IL-1β or EGFP, 5 μm thick liver cryosections were immunofluorescently stained using specific antibodies against F4 / 80 (1:200; #ab16911, Abcam) or IL-1β (1:200; #9722, Abcam). Subsequently, the primary antibodies were detected using secondary antibodies labeled with Alexa Fluor 594 or Alexa Fluor 488. Nuclei in all images were stained with DAPI (S36939; Invitrogen; Carlsbad, CA, USA). Images were acquired by fluorescence microscopy.

[0042] Protein extraction, immunoprecipitation, and Western blot: For immunoblotting experiments, cells were lysed using cold RIPA lysis buffer containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholic acid, 0.1% SDS, and a protease inhibitor cocktail. Nuclear extracts were prepared using a nuclear and cytoplasmic protein extraction kit (Beyotime) according to the manufacturer's instructions. For immunoprecipitation, proteins were extracted from cultured cells using cold NETN buffer containing 0.2% NP-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, and a protease inhibitor cocktail, followed by incubation with Flag / HA agarose beads or PHGDH / TAK1 antibodies. Protein expression and interaction were detected by Western blot. Proteins were separated by SDS-PAGE, transferred to PVDF membranes (Bio-Rad), and probed with the indicated antibodies. Visualization was performed using the ECL chemiluminescence kit (Millipore) and the chemiluminescence imaging system (Tanon).

[0043] Recombinant protein purification and GST pull-down assay: Purified PHGDH, PHGDH-(1-285), PHGDH-(286-533), or PHGDH(V425M) were obtained from BL21(DE3) competent E. coli transformed with the relevant plasmid (tagged with pet28a-His tag). The culture was grown at 37°C to an OD 600 nm of 0.6 and then induced with 1 mM IPTG for 16 h and continued at 25°C. The cell pellet was collected and lysed by sonication in a lysis buffer containing 20 mM Tris (pH 7.5), 0.1% Triton X-100, 1 mg / mL lysozyme, and 1 mg / mL DNase. Protein enrichment was performed using TALON metal affinity resin (Takara, 635503) and eluted with 100 mM imidazole. Eluted proteins were concentrated into 25 mM Tris-Cl, 0.4 M NaCl (pH 8.0) buffer. Purified TAK1, TAK1-(1-300) or GST-TAK1-(301-579) were produced from BL21(DE3) competent E. coli transformed with the relevant plasmid (tagged with pGEX-4T-1-GST tag) and induced with 1 mM IPTG for 4 h at 37°C. Cell pellets were lysed as described above. Protein enrichment was performed using GST-Sefinose resin (Sangon, C600031). GST fusion proteins were eluted with 50 mM Tris (pH 8.0) containing 10 mM reduced glutathione. Eluted proteins were dialyzed overnight in PBS for subsequent pull-down experiments. Purified proteins were mixed and incubated according to the indicated conditions and then pulled down using TALON metal affinity resin or GST-Sefinose resin, respectively. The eluted proteins were analyzed by SDS-PAGE.

[0044] Cross-linking: PMs were collected after the designated treatments were completed, and 0.25 mM disuccinimidyl DSS (ThermoScientific Pierce) was added and incubated at room temperature for 10 minutes for cross-linking. Then 1 M Tris (pH 7.5) was added to a final concentration of 20 mM and reacted at room temperature for 15 minutes to terminate cross-linking. Lysates were analyzed by Western Blot.

[0045] Flow cytometry: To isolate hepatic tissue macrophages derived from monocytes, approximately 100 mg of liver tissue was removed from 5Z-7-ox-HFHC model mice and ground into single-cell suspension after digestion with collagenase IV at 37°C for 30 minutes. Hepatic macrophages were further purified from the single-cell suspension by density gradient centrifugation using 30% Percoll (#P8370; Solarbio). The collected cells were washed with FACS buffer (PBS containing 2% BSA and 2 mM EDTA) and diluted to 1 × 10^6 cells / mL. 1 mL of the cell suspension was centrifuged and blocked with FACS buffer for 15 minutes at room temperature. Then, staining was performed with Zombie NIR™ Fixable Viability APC-Cy7 (1:1000; #423105; Biolegend) in PBS for 15-30 minutes at room temperature. Then, the cells were stained with fluorescently labeled antibodies CD45 PE (1:100; #103105; Biolegend), Ly6G FITC (1:100; #127605; Biolegend), F4 / 80 PE-Cy7 (1:100; #123113; Biolegend), and CD11b APC (1:100; #101211; Biolegend) for 30 min in the dark on ice. Monocyte-derived macrophages (CD45+; Ly6G-; F4 / 80int+ CD11b hi) were sorted using a FACS Aria Fusion cell sorter (BD Biosciences, San Jose, CA, USA), and the data were analyzed using FlowJo software.

[0046] Example 1 Macrophage PHGDH negatively regulates PA-induced pro-inflammatory cytokine production

[0047] Bone marrow-derived macrophages (BMDMs) were stimulated with 0, 0.2 mM, or 0.4 mM PA for 12 h, and the expression of PHGDH was detected by qPCR and western blotting (WB).

[0048] RAW264.7 cells were transfected with siCtrl or siPHGDH plasmids and stimulated with 0.2 mM PA for 12 h. The expression of PHGDH protein was detected by WB, and pro-inflammatory cytokines were detected by qPCR. Il-6 and Il-1β mRNA expression levels.

[0049] From PHGDH fl / fl Lyz2 -Cre -(PHGDH-WT-MΦ) and PHGDH fl / fl Lyz2 -Cre + BMDM isolated from PHGDH-KO-MΦ mice were stimulated with PA for 12 h, and then the expression of PHGDH was analyzed by WB, and the expression of pro-inflammatory cytokines was detected by qPCR.

[0050] RAW264.7 cells were transfected with Ctrl-HA or PHGDH-HA and stimulated with 0.2 mM PA for 12 h. The expression of HA (PHGDH) was detected by WB assay and the expression of other related genes was analyzed by qPCR.

[0051] Increased macrophage-mediated inflammation contributes to the development of NAFLD. To investigate whether macrophage PHGDH is involved in lipotoxicity-induced inflammatory responses and the pathogenesis of MAFLD, the expression of PHGDH in BMDM after PA stimulation was evaluated. After PA stimulation, both PHGDH mRNA and protein levels were significantly increased ( Figure 2 Depletion of PHGDH with siPHGDH significantly increased pro-inflammatory cytokines in PA-treated RAW264.7 cells. Il-6 and Il-1β The expression ( Figure 2 C). Using myeloid-specific PHGDH knockout mice ( Phhd fl / fl Lyz2 -Cre + , PHGDH-KO-Mφ mice) and littermate control mice ( Phhd fl / fl Lyz2 -Cre - , PHGDH-WT-Mφ mice), similar upregulation of multiple proinflammatory cytokines was observed in PHGDH knockout BMDMs ( Figure 2 In contrast, PHGDH overexpression reduced the expression of these pro-inflammatory cytokines ( Figure 2 These results suggest that PHGDH inhibits the expression of proinflammatory cytokines induced by PA and plays an anti-inflammatory role in macrophage-mediated inflammation.

[0052] Example 2 Myeloid-specific PHGDH deficiency exacerbates HFD-induced MAFL progression

[0053] Construction of high-fat diet (HFD)-induced MAFL model: 8-10 week-old male PHGDH-WT-MΦ mice and PHGDH-KO-MΦ mice were selected and fed with a high-fat diet (HFD) or a common diet (CD), respectively.

[0054] Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed at 18 and 20 weeks of feeding; then, the relevant indices were analyzed after 24 weeks of feeding. The ratio of liver weight to body weight was measured in PHGDH-WT-MΦ mice and PHGDH-KO-MΦ mice;

[0055] The inflammatory infiltration, fatty degeneration and fibrosis in the mouse liver were analyzed by H&E, Oil Red O and Sirius Red staining. In addition, the mouse liver sections were stained with F4 / 80 and IL-1β and their immunofluorescence images were observed to determine the proportion of IL-1β positive macrophages;

[0056] qPCR was used to detect the mRNA expression of genes related to fatty acid metabolism, pro-inflammatory / anti-inflammatory genes and chemokines, and fibrosis-related genes in the livers of mice in different groups;

[0057] Commercial kits were used to detect the contents of total cholesterol (TC) and triglyceride (TG) in the liver and the concentration of AST in the serum of mice in different groups.

[0058] To further elucidate the role of macrophage PHGDH in the pathogenesis of MAFL in vivo, MAFL was induced in PHGDH-KO-Mφ mice and PHGDH-WT-Mφ (control) mice by feeding them with a high-fat diet (HFD) or a control diet (CD). Figure 3 After 24 weeks of HFD diet, there was no significant difference in body weight between PHGDH-KO-MΦ and PHGDH-WT-MΦ mice ( Figure 4 However, the liver weight and liver weight / body weight ratio of PHGDH-KO-MΦ mice were higher than those of control mice ( Figure 4 Medium B and Figure 3 Glucose tolerance test (GTT) and insulin tolerance test (ITT) showed that PHGDH-KO-MΦ mice also exhibited higher blood glucose and more severe IR ( Figure 4 C and D). Epididymal white adipose tissue of PHGDH-KO-MΦ mice fed HFD ( Figure 4 Middle E) and liver ( Figure 3 Severe lipid accumulation was observed in C and D), accompanied by increased expression of genes related to lipid synthesis ( Figure 3 E in the liver), total cholesterol (TC) and triglyceride (TG) concentrations increased ( Figure 3 F). Immunofluorescence staining and RT-PCR results showed that the proportion of IL-1β positive macrophages in the liver tissue of PHGDH-KO-MΦ mice was significantly higher than that of PHGDH-WT-MΦ mice ( Figure 3G) and the expression of pro-inflammatory cytokines and chemokines were significantly increased ( Figure 3 Fibrosis was rare in MAFL, but increased collagen deposition was also observed in PHGDH-KO-MΦ mice ( Figure 3 I) and the expression of genes related to fibrosis ( Figure 3 In addition, the livers of PHGDH-KO-MΦ mice showed more severe damage than those of HFD-fed mice, and the aspartate aminotransferase (AST) level in the PHGDH-KO-MΦ group was higher than that in the control group ( Figure 3 K in Figure 5). These findings suggest that myeloid-specific genetic deletion of PHGDH exacerbates HFD-induced MAFL progression.

[0059] Example 3 Myeloid-specific PHGDH deficiency exacerbates HFD-induced MAFH progression

[0060] Construction of high cholesterol (HFHC) diet-induced MASH model: 8-10 week old male PHGDH-WT-MΦ mice and PHGDH-KO-MΦ mice were selected and fed with high fat, high cholesterol (HFHC) or normal diet (CD), respectively.

[0061] Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed at 10 and 12 weeks of feeding, and then the relevant indices were analyzed after 16 weeks. The ratio of liver weight to body weight was measured in PHGDH-WT-MΦ mice and PHGDH-KO-MΦ mice;

[0062] The inflammatory infiltration, fatty degeneration and fibrosis in the mouse liver were analyzed by H&E, Oil Red O and Masson staining. In addition, the mouse liver sections were stained with F4 / 80 and IL-1β and their immunofluorescence images were observed to determine the proportion of IL-1β positive macrophages;

[0063] qPCR was used to detect the mRNA expression of genes related to fatty acid metabolism, pro-inflammatory / anti-inflammatory genes and chemokines, and fibrosis-related genes in the livers of mice in different groups;

[0064] Commercial kits were used to detect the contents of total cholesterol (TC) and triglyceride (TG) in the liver and the concentrations of AST and ALT in the serum of mice in different groups.

[0065] Consumption of a high-fat (HFC) / high-cholesterol (HFHC) diet can accelerate the progression of MAFLD and promote the transition from simple steatosis to MASH.

[0066] This example establishes a HFHC diet-induced mouse model to mimic the pathophysiology of human MASH ( Figure 5 After 16 weeks of HFHC feeding, the body weight of PHGDH-KO-MΦ mice was not significantly different from that of PHGDH-WT-MΦ mice, but the liver weight and liver-to-body weight ratio were significantly increased ( Figure 6 A and B in Figure 5 GTT and ITT results showed that PHGDH-KO-MΦ mice exhibited higher blood glucose and severe IR compared with PHGDH-WT-MΦ mice ( Figure 6 In addition, the livers of PHGDH-KO-MΦ mice fed with HFHC diet ( Figure 5 C and D) and white fat ( Figure 6 Severe lipid accumulation was observed in E), accompanied by increased expression of genes related to lipid synthesis ( Figure 5 E in the figure), the levels of TC and TG in the liver increased ( Figure 5 F). Immunofluorescence staining and RT-PCR showed that the proportion of IL-1β positive macrophages in the liver tissue of PHGDH-KO-MΦ mice ( Figure 5 G) and the expression of pro-inflammatory cytokines and chemokines was significantly increased ( Figure 5 H in the figure). The collagen deposition and fibrosis gene expression in the PHGDH-KO-MΦ group were also higher than those in the control group ( Figure 5 In addition, the livers of PHGDH-KO-MΦ mice showed more severe damage upon HFHC diet feeding, as demonstrated by higher levels of AST and alanine aminotransferase (ALT) in PHGDH-KO-MΦ mice than in control mice ( Figure 5 K in Figure 5). These findings suggest that myeloid-specific PHGDH genetic deletion exacerbates HFHC diet-induced MASH progression.

[0067] Example 4 PHGDH inhibits PA-mediated TAK1-NF-κB / MAPK signaling pathway

[0068] BMDM were extracted from PHGDH-WT and PHGDH-KO mice, and the cells were stimulated with palmitic acid (PA) for 0, 15, and 30 minutes, respectively. The expression of related proteins was analyzed by WB, and p65 in the nuclei of WT and KO mouse BMDM was analyzed after PA stimulation for 15 minutes.

[0069] qPCR analysis of the expression of NF-κB target genes in BMDM of WT and KO mice after PA stimulation for 12 h.

[0070] BMDM cells from WT and KO mice were pretreated with the TAK1 inhibitor 5Z-7-ox for 30 min, and then stimulated with PA for 30 min or 12 h, followed by western blotting analysis and qPCR to detect the expression of proinflammatory cytokines.

[0071] To investigate the potential regulatory mechanism of PHGDH in MAFLD, this example investigated the effect of macrophage PHGDH on the PA-mediated TLR4 inflammatory signaling pathway. WB analysis showed that under PA stimulation, the activation of TAK1, IKKα / β, JNK, and p38 (but not ERK) in PHGDH-KO-MΦ BMDMs was increased ( Figure 7 In Figure 2A). Upon PA stimulation, PHGDH knockout increased p65 nuclear translocation and the expression of NF-κB target genes, including Ccl5 , ler3 , Lt and Bcl2 ( Figure 7 B and C in Figure ). Previous studies have shown that TAK1 is a key upstream regulator of the convergence of NF-κB and JNK / p38 signaling pathways, leading to the expression of proinflammatory factors by activating downstream pathways. It is speculated that PHGDH may inhibit the inflammatory response of macrophages by regulating TAK1. Interestingly, inhibition of TAK1 by 5Z-7-ox reversed the activation of the TAK1-NF-κB / MAPK signaling pathway caused by PHGDH deficiency after PA treatment, as well as the increased expression levels of inflammatory factors ( Figure 7 Taken together, these results suggest that PHGDH can inhibit the PA-mediated TAK1-NF-κB / MAPK signaling pathway and suppress macrophage inflammatory responses through TAK1.

[0072] Example 5 PHGDH interacts with TAK1

[0073] RAW264.7 cells were transfected with Ctrl-HA, PHGDH-HA, and TAK1-Flag plasmids, and their proteins were extracted after stimulation with or without PA. Exogenous PHGDH-HA and endogenous TAK1 were immunoprecipitated with anti-HA agarose and anti-TAK1 antibodies, respectively, and then analyzed by immunoblotting with the indicated antibodies.

[0074] Ctrl-HA or PHGDH-HA and TAK1-Flag were expressed in 293T cells and cells were stimulated with PA at the indicated time points. Exogenous PHGDH-HA was then immunoprecipitated with anti-HA agarose and analyzed by immunoblotting with the indicated antibodies.

[0075] To explore the binding site of PHGDH and TAK1, immunoblotting analysis was performed using Flag-TAK1 and full-length PHGDH or PHGDH truncation; immunoblotting analysis was also performed using HA-PHGDH and full-length or TAK1 truncation.

[0076] RAW264.7 cells were transfected with si Ctrl or si PHGDH and HA-Ctrl, HA-PHGDH, HA-PHGDH aa 1-285, or HA-PHGDHHaa 285-533 plasmids and stimulated with PA for 30 min, and then the protein levels of p-TAK1 (T184 / 187), TAK1, p-IKKα / β (S176 / 180), IKKα / β, p-JNK (T183 / Y185), JNK, p-p38 (T180 / Y182), p38, p-ERK (T202 / Y204), and ERK were measured.

[0077] RAW264.7 cells were transfected with si Ctrl or si PHGDH and HA-Ctrl, HA-PHGDH, HA-PHGDH aa 1-285, or HA-PHGDH aa 285-533 plasmids and stimulated with PA for 12 h, and pro-inflammatory cytokines were detected by qPCR Il- 6 and Il-1β expression level.

[0078] In addition to metabolic functions, metabolic enzymes can also play non-metabolic functions by mediating post-translational modification of proteins, acting as transcription factors or regulators, or directly binding to and affecting related functional proteins to regulate cell signaling. In order to further explore how PHGDH affects macrophage-mediated inflammatory signaling through TAK1, this example performed immunoprecipitation experiments on exogenously and endogenously expressed proteins and found that PHGDH interacted with TAK1, and this interaction was inhibited after PA stimulation of RAW264.7 cells for 30 minutes ( Figure 8 Time course analysis showed that the interaction between PHGDH and TAK1 was significantly reduced 15-60 min after PA treatment ( Figure 8 C)

[0079] In addition, this example explores the key domains of the interaction between PHGDH and TAK1. PHGDH protein contains two substrate binding domains (aa 9-111 and aa 112-285), a nucleotide binding domain (aa 286-317) and a regulatory domain (aa 322-533) ( Fig. 9TAK1 protein contains a kinase domain (aa 43-284) and a TAB1 binding domain (aa 77-303) at the N-terminus and a TAB2 / TAB3 binding domain (aa 479-533) at the C-terminus ( Fig. 9 Immunoprecipitation experiments further demonstrated that both HA-tagged wild-type (WT) PHGDH and HA-PHGDH (286-533) mutants interacted with Flag-tagged TAK1 ( Figure 8 D), while Flag-tagged wild-type TAK1 and Flag-TAK1 (1-300) and (1-480) mutants interacted with HA-tagged PHGDH ( Figure 8 E), indicating that the aa 286-533 domain of PHGDH interacts with the aa 1-300 domain of TAK1. Consistent with this finding, purified recombinant PHGDH and PHGDH(286-533) could interact with wild-type TAK1 ( Figure 8 In contrast, TAK1 and TAK1(1-300) proteins can interact with wild-type PHGDH ( Figure 8 In addition, overexpression of wild-type PHGDH or PHGDH (286-533) mutant reversed the activation of TAK1-NF-κB / MAPK signaling pathway and the increased expression of inflammatory factors caused by PHGDH deficiency after PA treatment ( Figure 8 These results suggest that the aa 286-533 domain of PHGDH plays an important role in regulating the inflammatory response of macrophages.

[0080] Example 6 The tetrameric form of PHGDH plays a key role in regulating the inflammatory response of macrophages by interacting with and inhibiting TAK1

[0081] PMs were pretreated with 15 μM CBR-5884 for 6 h, and the cells were lysed with non-denaturing lysis buffer. 0.25 mM DSS was added for cross-linking, and the multimerization of PHGDH was detected by WB.

[0082] PHGDH-HA, EV-Flag, and TAK1-Flag plasmids were transfected into 293T cells. The cells were pretreated with CBR-5884 for 6 hours and stimulated with PA for 30 minutes. The effect of PHGDH inhibitor CBR-5884 on the interaction between PHGDH and TAK1 was observed by immunoprecipitation experiments.

[0083] PMs cells were pretreated with 15 μM CBR-5884 for 6 h, and the protein levels of p-TAK1 (Thr184 / 187) and TAK1 were analyzed by immunoprecipitation assay after PA stimulation for 30 min. Meanwhile, pro-inflammatory cytokines were detected by qPCR after PA stimulation for 12 h. Il-6 and Il-1β mRNA levels.

[0084] RAW264.7 cells were transfected with si Ctrl or si TAK1 and / or Flag-Ctrl, Flag-TAK1, Flag-TAK1-T184 / 187A, or Flag-TAK1-T184 / 187D plasmids, pretreated with 15 μM CBR-5884 for 6 h, and stimulated with PA for 12 h before the pro-inflammatory cytokines were measured by qPCR Il-6 and Il-1β expression level.

[0085] PHGDH exists in solution as a dynamic mixture of monomers, dimers, and tetramers. Therefore, this example aims to determine the form of PHGDH that binds to TAK1. The PHGDH inhibitor CBR-5884 can convert PHGDH from a tetramer to a dimer ( Figure 8 J in Figure 3) and weakened the interaction between TAK1 and PHGDH, indicating that the tetrameric form of PHGDH interacts with TAK1 ( Figure 8 CBR-5884 was also found to increase PA-induced TAK1 activation and expression of pro-inflammatory cytokines ( Figure 8 L and M in the diagram).

[0086] To further explore whether PHGDH tetramers inhibit macrophage inflammation through TAK1, TAK1-T184 / 187a and TAK1-T184 / 187d mutants were constructed, in which the phosphorylation sites T184 and T187 were replaced by alanine or aspartic acid, respectively. RT-qPCR results showed that knockdown of TAK1 inhibited the increase in the expression of inflammatory factors caused by CBR-5884 treatment, and this effect could be reversed by overexpression of wild-type TAK1 or TAK1-T184 / 187D mutant (which is constitutively activated), while overexpression of TAK1-T184 / 187A mutant (which cannot be phosphorylated) could not reverse this effect ( Figure 8 N in ). This indicates that PHGDH tetramer inhibits the inflammatory response of macrophages through TAK1.

[0087] The active site V425 of PHGDH is located in the aa 286-533 domain of PHGDH that binds to TAK1. However, there is no literature report on whether the mutation of this residue will affect the oligomerization state or change the conformation of PHGDH. The present invention found that the mutation of the V425M active site in PHGDH reduced the tetramerization of PHGDH and increased the dimerization of PHGDH ( Fig. 9 C in Figure 3). Subsequent experiments showed that, relative to wild-type PHGDH, the PHGDH(V425M) mutant inhibited PHGDH-TAK1 binding and greatly weakened the interaction with the PHGDH molecular chaperone DNAJA1, which has been reported to interact with PHGDH and promote the tetramerization of PHGDH ( Fig. 9 In addition, unlike wild-type PHGDH, PHGDH(V425M) could not restore the activation of TAK1 or the elevated expression of proinflammatory cytokines induced by PHGDH knockdown in PA-stimulated RAW264.7 cells ( Fig. 9 These results further suggest that the tetrameric form of PHGDH plays a key role in regulating the inflammatory response of macrophages by interacting with and inhibiting TAK1.

[0088] Example 7 PHGDH regulates PA-induced inflammation by inhibiting the interaction between TAB1 and TAK1

[0089] Ub-Myc, TAK1-Flag, and PHGDH-HA plasmids were transfected into 293T cells and stimulated with PA for 30 minutes. Immunoprecipitation assay was used to detect ubiquitination in the cells.

[0090] 293T cells were transfected with Ub-Myc, TAK1-Flag, TRAF6-GST, and PHGDH-HA plasmids and stimulated with PA for 30 minutes. Immunoprecipitation experiments were used to detect the interaction between TAB1 and TAK1 in 293T cells.

[0091] RAW264.7 cells were transfected with HA-Ctrl, HA-TAB or HA-PHGDH plasmids, and the phosphorylated and total TAK1, IKKβ, JNK, P38 and ERK protein levels were detected by western blotting after stimulation with PA for 30 min.

[0092] Pro-inflammatory cytokines were detected by qPCR after RAW264.7 cells were transfected with HA-Ctrl, HA-TAB1, or HA-PHGDH, HA-PHGDH aa 1-285, or HA-PHGDH aa 285-533 plasmids and stimulated with PA for 12 h. Il-6 and Il-1βThe expression level

[0093] In order to further explore the specific mechanism of PHGDH and TAK1 interaction in regulating macrophage inflammatory response, this example used immunoprecipitation to detect the effect of PHGDH on TAK1 ubiquitination and autophosphorylation. Fig.10 As shown in A and B, PHGDH had no significant effect on the total TAK1 ubiquitination level and TRAF6-mediated TAK1 ubiquitination level. However, PHGDH inhibited the interaction between TAB1 and TAK1 and reduced the phosphorylation of TAK1 ( Fig.10 In addition, overexpression of full-length PHGDH or the PHGDH(286-533) mutant (which consists of the TAK1-binding domain), but not overexpression of PHGDH(1-285) or PHGDH(V425M) mutant, inhibited TAB1-mediated activation of the TAK1-NF-κB / MAPK signaling pathway and expression of proinflammatory cytokines after PA stimulation ( Fig.10 In summary, PHGDH regulates PA-induced macrophage inflammation by inhibiting the interaction between TAB1 and TAK1.

[0094] Example 8 Inhibition of TAK1 can slow the progression of MASH caused by myeloid-specific PHGDH deficiency

[0095] HFHC diet-induced MASH model mice (PHGDH-WT-MΦ mice and PHGDH-KO-MΦ mice) were intraperitoneally injected with 5Z-7-ox (once every three days for 16 weeks) and fed with a HFHC diet for 16 weeks. GTT and ITT tests were performed at weeks 10 and 12, and then the relevant indices were analyzed at 16 weeks.

[0096] Peripheral blood monocyte-derived macrophages (highly expressing CD11b and medium expressing F4 / 80) were extracted from the livers of the above mice, and the phosphorylation and total protein levels of p-TAK1 (Thr184 / 187), TAK1, p-IKKα / β (S176 / 180), IKKα / β, p-JNK (T183 / Y185), JNK, p-p38 (T180 / Y182), p38, p-ERK (T202 / Y204) and ERK were detected by western blotting.

[0097] After 16 weeks of HFHC diet feeding, the liver weight of mice was weighed and the ratio of body weight was calculated. In addition, liver sections of mice in each group were taken for H&E staining, Oil Red O staining, and Masson staining to analyze the inflammatory infiltration, fatty degeneration, and fibrosis in the liver of mice.

[0098] qPCR was used to analyze the mRNA expression of genes related to fatty acid metabolism, proinflammatory factors and chemokines, and fibrosis-related genes in the livers of mice in each group.

[0099] Commercial kits were used to detect the total cholesterol (TC) and triglyceride (TG) contents in the liver of each group of mice and the AST and ALT concentrations in the serum of mice.

[0100] To further explore the role of TAK1 in the development of PHGDH-mediated MASH, 6- to 8-week-old WT and PHGDH-KO-MΦ male mice were intraperitoneally injected with the TAK1 inhibitor 5Z-7-ox (5 mg / kg) or control every three days for 16 weeks and fed only a HFHC diet for 16 weeks ( Fig.11 Western blot analysis of mouse liver MDMs showed that 5Z-7-ox inhibited the activation of TAK1-NF-κB / MAPK signaling pathway caused by PHGDH deficiency ( Fig.11 5Z-7-ox attenuated the increase in liver weight and liver-to-body weight ratio and alleviated IR caused by PHGDH deficiency ( Fig.12 AD and Fig.11 In addition, 5Z-7-ox ameliorates HFHC diet-induced lipid accumulation in PHGDH-KO-MΦ mice ( Fig.11 DG and Fig.12 E in), inflammation ( Fig.11 H in), fibrosis ( Fig.11 I and J in) and liver damage ( Fig.11 These findings further confirmed that PHGDH slows the progression of MASH by inhibiting TAK1.

[0101] Example 9 Adeno-associated virus-mediated PHGDH overexpression in hepatic macrophages alleviates HFHC diet-induced MASH progression

[0102] C57B / L male mice aged 6-8 weeks were fed a HFHC diet for 8 weeks and then injected with 100 μL AAV8- Phhd or AAV8-EGFP virus (5×10^11 vector genomes). Subsequently, mice continued to be fed with HFHC diet for 8 weeks. GTT and ITT tests were performed at the 10th and 12th weeks of feeding, and then the relevant indicators were analyzed at the 16th week.

[0103] Liver sections of mice in each group were taken and immunofluorescence stained with CD68 (red) and EGFP (green) (nuclei were stained with DAPI (blue)) to observe the expression of PHGDH in mouse liver macrophages.

[0104] After 16 weeks of HFHC diet feeding, AAV-GFP injected mice and AAV- Phhd Ratio of liver weight to body weight of injected mice.

[0105] Liver sections of mice in each group were taken for H&E staining, Oil Red O staining, and Sirius red staining to analyze the inflammatory infiltration, fatty degeneration, and fibrosis in the mouse liver.

[0106] qPCR was used to analyze the mRNA expression of genes related to fatty acid metabolism, pro-inflammatory / anti-inflammatory genes and chemokines, as well as genes related to fibrosis in the liver of each group of mice.

[0107] Commercial kits were used to detect the total cholesterol (TC) and triglyceride (TG) contents in the liver of each group of mice, as well as the AST and ALT concentrations in the serum of each group of mice.

[0108] This example examined the therapeutic effect of PHGDH in hepatic macrophages on MASH. PHGDH was overexpressed in mouse hepatic macrophages using a recombinant adeno-associated virus serotype 8 (AAV8) delivery system. An empty AAV8 vector (AAV-CD68-EGFP, referred to as AAV-EGFP) carrying a CD68 promoter (a promoter targeting macrophages) or an AAV8 vector carrying a mouse PHGDH gene (AAV-CD68- Phhd , referred to as AAV- Phhd ) were injected via the tail vein into mice fed a HFHC diet for 8 weeks and then fed a HFHC diet for another 8 weeks ( Fig.13 Immunofluorescence staining results confirmed that the injected AAV- Phhd PHGDH was successfully overexpressed in mouse liver macrophages ( Fig.13 (B) Compared with mice injected with AAV-EGFP, mice injected with AAV- Phhd Mice fed the HFHC diet for 16 weeks showed significant decreases in liver weight and liver-to-body weight ratio, with minimal changes in body weight ( Fig.14 AB and Fig.13 In addition, AAV-mediated overexpression of PHGDH significantly attenuated IR in the liver of HFHC diet-fed mice ( Fig.14 C and D in), lipid accumulation ( Fig.14 E and Fig.13 DG in), inflammation ( Fig.13 H in), fibrosis ( Fig.13 I and J) and injury ( Fig.13 in K).

[0109] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. Use of macrophage-specific phosphoglycerate dehydrogenase PHGDH in the preparation of a drug for preventing, treating and / or assisting in the treatment of metabolic dysfunction-associated fatty liver disease MAFLD, characterized in that: The macrophage-specific phosphoglycerate dehydrogenase PHGDH is a tetrameric PHGDH.

2. The use according to claim 1, characterized in that: The MAFLD includes simple fatty liver or metabolic dysfunction-related fatty liver disease.

3. The use according to claim 1, characterized in that: The drug exerts its effect by inhibiting the activation of TAK1-NF-κB / MAPK signaling pathway.

4. The use according to claim 1, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that: The pharmaceutically acceptable excipients include: one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, diluents, lubricants, solutes that make the preparation isotonic with the recipient's blood, suspending agents, solubilizers, thickeners, stabilizers, sweeteners and flavors.

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