Application of Caspase 8 in the preparation of drugs for non-alcoholic fatty liver disease

Abstract

The present invention provides the use of Caspase8 in the preparation of a medicament for non-alcoholic fatty liver disease. Overexpression of Caspase8 inhibits the development of non-alcoholic fatty liver disease. The present invention identifies a specific role of Caspase8 in non-alcoholic fatty liver disease: decreased Caspase8 expression in hepatic macrophages activates the Cflip / ELK4 / Psap signaling pathway, leading to macrophage polarization toward the M1 type. This, in turn, activates the Rac1 / cdc42 / JNK signaling pathway within hepatocytes through the Gpr37I1 receptor on the hepatocyte surface, leading to increased lipid deposition in hepatocytes. Caspase8 or its small molecule analogs may offer hope for the treatment of steatohepatitis.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to the application of Caspase 8 in the preparation of medicines for non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a liver metabolic disorder characterized by lipid deposition and fatty degeneration in hepatocytes. It includes simple fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), and can progress to cirrhosis and even liver cancer. NAFLD is one of the most common chronic liver diseases worldwide, affecting nearly 30% of adults. It is predicted that the incidence of NAFLD will increase to 33.5% by 2030, becoming the leading cause of end-stage liver disease and liver transplantation. Therefore, discovering the important pathogenesis and diagnostic and therapeutic targets involved in NAFLD and achieving effective intervention are major challenges facing clinical and basic research.

[0003] Disruption of the liver's immune microenvironment plays a crucial role in the development and progression of NAFLD. The liver is rich in macrophages, including liver-resident Kupffer cells and blood monocyte-derived macrophages. During the progression of NAFLD, factors such as free fatty acids (FFA) produced from adipose tissue breakdown and lipopolysaccharide (LPS) produced in the intestine activate Kupffer cells and monocyte-derived macrophages to release inflammatory factors and promote hepatic inflammatory responses. Furthermore, inflammatory cytokines and chemokines secreted by Kupffer cells and monocyte-derived macrophages play diverse functions in the progression of NAFLD. For example, IL-1β can promote hepatocyte steatosis, apoptosis, and liver fibrosis; TNF-α increases liver cholesterol synthesis and inhibits excretion; IL-6 exacerbates insulin resistance; CCL2 recruits monocytes to migrate into the liver; and TGF-β stimulates hepatic stellate cells to activate into fibroblasts, promoting extracellular matrix deposition and thereby exacerbating fibrosis. Therefore, macrophages participate in the progression of NAFLD through multiple mechanisms. Further exploration of the regulatory mechanisms of macrophage phenotype and function in the process of NAFLD is expected to provide new targets for disease prevention and treatment. Summary of the Invention

[0004] In view of this, the present invention aims to overcome the defects in the prior art and proposes an application of Caspase8 in the preparation of a drug for non-alcoholic fatty liver disease.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] The present invention provides an application of Caspase8 in preparing a medicament for treating non-alcoholic fatty liver disease. Overexpression of Caspase8 inhibits the development of non-alcoholic fatty liver disease.

[0007] Furthermore, knockout of Caspase8 promoted the development of non-alcoholic fatty liver disease; knockout of Caspase8 regulated the increased expression of lipogenic genes and the decreased expression of β-oxidation genes in hepatocytes.

[0008] Furthermore, knockout of Caspase8 induces macrophage polarization to M1 macrophages and lipid deposition in hepatocytes by inducing Psap; knockout of Caspase8 regulates the inflammatory response of macrophages and induces lipid metabolism disorders in hepatocytes by activating Psap.

[0009] Furthermore, knockout of Caspase8 regulated the transcription of Psap through cFLIP and ELK4.

[0010] Furthermore, knocking out Caspase8 inhibited the cleavage of cFLIP. The uncleaved cFLIP bound to Elk4 and promoted the transcription of Psap, thereby regulating the inflammatory response of macrophages and increasing lipid deposition in hepatocytes.

[0011] Furthermore, the Rac1 / Cdc42 / JNK signaling pathway is involved in Gpr37I1-mediated hepatocyte lipid deposition.

[0012] Furthermore, knockout of Caspase8 promoted the increase of Racl and Cdc4; knockout of Caspase8 and knockdown of Grp37I1 promoted the decrease of Racl and Cdc4; knockdown of Racl and Cdc4 promoted the decrease of JNK protein phosphorylation level.

[0013] Furthermore, overexpression of Grp37I1 and knockdown of Racl and Cdc4 induced a decrease in the expression of adipogenic genes and an increase in the expression of β-oxidation genes.

[0014] Furthermore, the dosage form of the drug is at least one of capsules, pills, tablets, granules or injections.

[0015] The present invention also provides an application of Caspase 8 in preparing a reagent for detecting non-alcoholic fatty liver disease.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The present invention identifies the specific role of Caspase8 in non-alcoholic fatty liver disease: decreased expression of Caspase8 in liver macrophages activates the Cflip / ELK4 / Psap signaling pathway, leading to macrophage polarization toward the M1 type, and then activates the Rac1 / cdc42 / JNK signaling pathway in liver cells through the Gpr37I1 receptor on the liver cell surface, leading to a molecular mechanism of increased lipid deposition in liver cells. Caspase8 or its small molecule analogs may bring hope for the treatment of fatty liver hepatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The level of caspase8 in macrophages is reduced as described in the examples of the present invention: A is the Western blot analysis and relative density ratio of caspase8 in macrophages, B is the QPCR analysis of the expression of Caspase8 gene in macrophages of mice fed a normal diet (ND) and a high-fat diet (HFD), C is the liver biopsy of ND and HFD mice, immunofluorescence staining of Caspase8 (green) and CD68 (red) (gray arrow: Caspase8 + CD68 + cell);

[0019] Figure 2 The level of caspase8 in macrophages is reduced as described in the examples of the present invention: A is t-SNE from GSE129516, B is the relative expression of Caspase8 mRNA in macrophages of NASH mice (GSE129516), C is the expression of Caspase8 in monocytes and macrophages of the control group and NASH mice, and D is the Log2-fold change of the most significantly differentially expressed genes between macrophages with low Caspase8 expression and other macrophages;

[0020] Figure 3 Knockout of Caspase8 in the embodiment of the present invention can aggravate NASH: A is high-fat feeding Caspase8 Fl / Fl or Caspase8 M-KO Caspase8 mRNA expression level in mouse liver tissue. B is immunoblotting detection of Caspase8 in mice fed with a high-fat diet. Fl / Fl and Caspase8 M-KO Caspase8 protein levels in liver tissue of mice, C represents Caspase8 protein levels in mice fed with a high-fat diet Fl / Fl and Caspase8 M-KO Serum ALT levels and liver TG content of mice; N = 6 / group, D is Caspase8 in mice fed with a high-fat diet Fl / Fl and Caspase8 M-KO Body weight and liver weight of mice; 6 cases per group, E is Caspase8 Fl / Fl and Caspase8 M-KO Representative images of mouse liver tissue stained with hematoxylin and eosin (HE) and Oil Red O; N = 6 / group. (Black arrows: macrovesicular steatosis; gray arrows: microvesicular steatosis; red arrows: inflammatory foci; blue arrows: hepatocyte ballooning; CV, central vein; PV, portal vein).

[0021] Figure 4The overexpression of Caspase8 in the embodiment of the present invention inhibits the development of fatty liver disease: A is the immunoblotting detection control group and Caspase8 过表达 Caspase8 protein levels in liver tissue of mice after high-fat diet, B is the control group and Caspase8 过表达 Serum ALT levels and liver TG content in mice fed a high-fat diet; N = 6 / group, high-fat diet-fed control group and Caspase8 过表达 Body weight and liver weight of mice; 6 cases in each group, C is the control group fed with high-fat diet and Caspase8 过表达 The percentage of oil red area in liver tissue and NAFLD activity score; N = 6 / group, D is the control group and Caspase8 过表达 Gene expression of TNF-α, IL-6, and IL-1β in liver tissue of mice fed a high-fat diet. N = 6 / group. E is a statistical graph of mitochondrial length, diameter, percentage of damage, and ATP content.

[0022] Figure 5 The knockout of macrophage Casp8 induces M1 macrophage polarization and hepatocyte lipid accumulation as described in the embodiment of the present invention: AB is the analysis of Caspase8 by flow cytometry after high-fat diet feeding Fl / Fl and Caspase8 M-KO M1 (CD86) in primary mouse bone marrow-derived macrophages + CD206 - ) and M2 (CD86 - CD206 + ) The ratio of macrophages, CD was analyzed by flow cytometry for Caspase8 in high-fat diet-fed cells Fl / Fl and Caspase8 M-KO M1 (CD86) in primary mouse liver macrophages + CD206 - ) and M2 (CD86 - CD206 + ) macrophage ratio;

[0023] Figure 6 The knockout of Caspase8 in macrophages induced M1 macrophage polarization and lipid accumulation in hepatocytes as described in the examples of the present invention: A is a representative Oil Red O staining of primary hepatocytes co-cultured with primary bone marrow-derived macrophages after 24 h of PA+OA treatment, B is a representative immunofluorescence of phalloidine (red) and PLIN2 (green) in co-cultured primary hepatocytes and primary bone marrow-derived macrophages after 24 h of PA+OA treatment, C is a statistical graph of mitochondrial damage, and D is a statistical graph of hepatocytes and Caspase8 in PA+OA culture medium. Fl / Fland Caspase8 M-KO After 24 h of co-culture of mouse bone marrow-derived macrophages, the mRNA levels of lipogenic genes SREBP1c, ChREBP, LEX-α, USF1, and Fasn were observed. E is the mRNA levels of hepatocytes and Caspase8 in PA+OA medium. Fl / Fl and Caspase8 M-KO Mouse bone marrow-derived macrophages were co-cultured for 24 hours to measure the mRNA levels of β-oxidation genes Acox1, CPT1, and PPAR-α;

[0024] Figure 7 Knockout of Caspase8 in the examples of the present invention induces macrophage M1 polarization and hepatocyte lipid deposition by inducing the secreted factor PSAP: A is a volcano plot of differential peaks, where peaks with a P < 0.05 and a |change| greater than 1.5 times are considered differential peaks; B is a bubble plot of ligand-receptor connections between hepatocyte clusters with low Caspase8 expression and myeloid cells, where color intensity indicates expression level and dot size indicates gene expression frequency (the percentage of cells expressing the gene); C is a Venn diagram showing the overlap of the top 5 ligand receptors in monocyte-macrophage 1 and hepatocyte cellphone BD data and Caspase8 negatively correlated differentially expressed genes in ATAC-seq and sc-RNA data;

[0025] Figure 8 The knockout of Caspase8 in the embodiment of the present invention induces macrophage M1 polarization and hepatocyte lipid deposition by inducing the secretion factor PSAP: A is from Caspase8 Fl / Fl and Caspase8 M-KO RNA in situ hybridization for Psap transcripts in PA / OAa-stimulated macrophages of mice (n=6 samples / group). B is Caspase8 in macrophages fed a high-fat diet. Fl / Fl or Caspase8 M-KO The mRNA expression level of Psap in Psap of mice. C is the immunoblotting detection of the effect of high-fat diet on Caspase8 Fl / Fl and Caspase8 M-KO Effects of Psap protein levels in mouse BMDMs. D shows the ELISA levels of TNF-α, IL-6, and IL-1β in the supernatant of bone marrow-derived macrophages treated with PA / OA and transfected with control or shRNA-Psap; 6 cases per group. E shows representative Oil Red O staining and BODIPY analysis of primary hepatocytes co-cultured with primary bone marrow-derived macrophages 24 hours after addition of PA / OA.

[0026] Figure 9Cflip / ELK4 is involved in the transcriptional regulation of PSAP by casp8 as described in the examples of the present invention: A is ChIP-PCR verification of the binding of Cflip and ELK4 to the Pasp target gene promoter compared with the IgGIP control, n=3, B is real-time quantitative PCR detection of Psap expression after RAW macrophages transfected with shCtrl, shELK4-3 or shzbtb7a-1, C is the effect of Cflip and ELK4 gene knockdown on Pasp transcription in RAW macrophages treated with PA / OA, observed by QPCR, D is the effect of Cflip and ELK4 gene knockdown on Pasp protein level in RAW macrophages treated with PA / OA, observed by WB, E is the effect of Caspase8 overexpression on cFLIP protein cleavage in RAW macrophages treated with PA / OA, observed by WB, F is the effect of Caspase8 overexpression and Caspase8 in RAW macrophages treated with PA / OA, observed by WB. Effect of D377A point mutation on cFLIP protein cleavage. G shows the effect of Caspase8 overexpression, cFLIP and ELK4 overexpression on the interaction between exogenous cFLIP and ELK4 proteins in RAW macrophages treated with PA / OA, observed by CoIP.

[0027] Figure 10 Cflip / ELK4 is involved in the transcriptional regulation of PSAP by casp8 as described in the examples of the present invention: A shows the effects of Caspase8 overexpression and Caspase8 D377A point mutation on the interaction between cFLIP and ELK4 proteins in the nucleus of RAW macrophages treated with PA / OA, as observed by CoIP; B shows the effects of Caspase8 overexpression and Caspase8 D377A point mutation on macrophage inflammation, as observed by ELISA; C shows the effects of Caspase8 overexpression and Caspase8 D377A point mutation on lipid deposition in hepatocytes, as observed by oil red staining of hepatocytes, as treated with PA / OA.

[0028] Figure 11 The Rac1 / cdc42 / JNK signaling pathway described in the examples of the present invention is involved in Gpr37I1-mediated hepatocyte lipid deposition: A shows the effect of Gpr37I1 knockdown in hepatocytes on lipid deposition in hepatocytes by oil red staining; B shows the effect of Gpr37I1 knockdown in hepatocytes on lipid synthesis and fatty acid β-oxidation-related genes in hepatocytes by qPCR; CD shows changes in Rac1 and Cdc42 protein levels and JNK protein phosphorylation levels detected by western blot; E shows changes in JNK protein phosphorylation levels detected by western blot;

[0029] Figure 12 The Rac1 / cdc42 / JNK signaling pathway described in the examples of the present invention is involved in Gpr37I1-mediated hepatocyte lipid deposition: A is the detection of hepatocyte lipid deposition by Oil Red and BODIPY staining, and B is the effect of hepatocyte Gpr37I1 knockdown on lipid synthesis and fatty acid β-oxidation-related genes in hepatocytes observed by qPCR. DETAILED DESCRIPTION

[0030] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0031] The present invention will be described in detail below with reference to the embodiments.

[0032] Example 1 Materials and Methods

[0033] 1. C57 mice used in the experiment

[0034] Caspase8 FL / FL (Casp8 FL / FL ) mice (IMSR_JAX:027002; Jackson Laboratory) and Cre mice (transgenic mice expressing Cre recombinase driven by the Lysozyme-2 promoter) (JAX004781; Jackson Laboratory, Bar Harbor, ME) to generate myeloid-specific knockout (Casp8 M-KO Lyz2.Cre mice were used as a control group and are referred to as wild-type (WT) for simplicity. All mice were C57BL / 6J mice.

[0035] 2. C57 mice used in the experiment

[0036] Male Casp8 M-KO Mice and Casp8 Fl / Fl Mice from the same littermate (7-8 weeks old) were randomly assigned to be fed a normal diet (NC), a high-fat diet (HFD) for 16 weeks, a high-fat diet (HFD) for 24 weeks, or a methionine and choline-deficient diet (MCD) for 4 weeks (n=4-7 per group). After the experiment, the mice were fasted and weighed, and the liver was quickly removed and weighed. All mice were housed in ventilated cages under a 12-h light / dark cycle and had access to an enriched environment, water, and free access to food. On the first day after 4, 8, 12, 16, and 20 weeks of high-fat diet, mice were transfected with BMDMs (5*10 6cell number, control group), casp8-positive BMDMs (5*10 6 Cell amount, Casp8 过表达 Three days after the last injection (16 weeks on a high-fat diet), mice were humanely sacrificed for further analysis. Four weeks after the last injection (20 weeks on a high-fat diet), mice were humanely sacrificed for further analysis. All animal studies were approved by the Animal Experimental Ethics Committee of Tianjin Fifth Central Hospital and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication 86-23, revised 1985, prepared by the National Academy of Sciences and published by the National Institutes of Health).

[0037] 3. Adeno-associated virus

[0038] The present invention uses the following adeno-associated viruses: AAV8-Control (AAV8-con) and AAV8-shPsap. AAV gene amplification was performed by Genechem Co., Ltd. (Shanghai). M-KO Mice were randomly divided into AAV8-CON group and AAV8-shPsap group to establish a high-fat diet-induced NASH model. Then, AAV8-shPsap or AAV8-CON was injected through the tail vein at a dose of 1×10 9 Infectious units (IFU) per 200 μl per mouse. Two weeks later, all mice were placed on a high-fat diet and administered AAV virus every 8 weeks. After 24 weeks of high-fat diet, all mice were sacrificed for further analysis.

[0039] 4. Histological examination

[0040] The left liver lobe of each mouse was collected and fixed in 4% neutral buffered formalin. After dehydration, paraffin embedding, and other procedures, the liver was cut into 4µm sections. Samples were scored according to the Kleiner scoring system, and a NAFLD activity score was calculated for each sample. Steatosis index for the breast was: (<5%), 1 (5% - 33%), 2 (>33% - 66%), and 3 (>66%). Steatosis was categorized as macrovesicular, microvesicular, or both, with visible zonation. Hepatocyte ballooning occurs when hepatocytes enlarge to more than twice the size of adjacent cells, the cytoplasmic membrane changes from the hexagonal shape of normal hepatocytes to a rounded shape, and the cytoplasm is mostly empty. Hepatocyte ballooning is scored as 1 (1-5 balloon-like cells) or 2 (>5 balloon-like cells). Inflammation is scored as 1 (1-2 lesions), 2 (2-4 lesions), and 3 (>4 lesions), which determines the size and zonation of the inflammatory foci. The degree of fibrosis was scored as 1 (pure perisinusoidal and perivenous fibrosis), 2 (1 plus portal venous fibrosis), 3 (bridging fibrosis), or 4 (cirrhosis). Each specimen was scored and assessed at ×200 magnification using five periportal and five pericentral fields.

[0041] 5. Liver Macrophage Extraction

[0042] After anesthesia, the peritoneal cavity of mice was opened and perfused via the inferior vena cava with EGTA buffer, followed by EGTA buffer containing 0.08 U / ml collagenase D at a flow rate of 3 ml / min for 15 minutes. The liver was excised and further digested with 0.5 mg / ml pronase E and 2% DNase-I at 37°C for 20 minutes to obtain a single-cell suspension. Non-parenchymal hepatic cells were purified using a 35% and 70% Perco II gradient. The purified cells were suspended in Dulbecco's phosphate-buffered saline (DPBS) and stained using the Zombie Aqua Fixation and Viability Kit. Cells were washed with sterile FACS staining buffer (DPBS supplemented with 2 mM EDTA and 0.5% BSA), incubated with anti-f4 / 80 microspheres, and magnetic bead sorting was performed to isolate macrophages. The eluted cells were plated in RPMI-1640 medium supplemented with 10% FBS and 10 ng / ml MCSF for 30 minutes to remove unattached dead cells.

[0043] 6. Isolation of Primary Hepatocytes

[0044] After anesthetizing mice, the peritoneal cavity was opened and the liver was perfused via the inferior vena cava with EGTA buffer, followed by 10 ml of HBSS containing 25 µg / ml Liberase™ at a flow rate of 3 ml / min. The liver was removed and viable hepatocytes were purified using 50% Percoll. The cells were resuspended in DMEM low-glucose medium supplemented with 5% FBS and plated onto rat tail collagen type I-coated tissue culture dishes. After 4 hours of adherence, the medium was replaced with William's E medium supplemented with 2 mM l-glutamine for maintenance or, if desired, with specific culture medium.

[0045] 7. Bone Marrow Macrophage Isolation

[0046] From male Casp8 M-KO Mice and Casp8 Fl / Fl Bone marrow cells were isolated from the femur and tibia of mice. The bone marrow was removed and rinsed with Dulbecco's Modified Eagle's Medium (DMEM). The cells were then plated and cultured in DMEM supplemented with 10% FBS and 10 ng / mL colony-stimulating factor-1 to induce differentiation into bone marrow-derived macrophages (BMDMs). By day 7, all adherent cells were mature macrophages.

[0047] 8. Isolation of bone marrow macrophages

[0048] In co-culture studies, Casp8 Fl / Fl Primary hepatocytes from mice and from male Casp8 M-KO Mice and Casp8 Fl / Fl Mouse liver or bone marrow macrophages were inoculated into the co-culture chamber. Male Casp8 Fl / Fl Mouse primary hepatocytes and male Casp8 M-KO Mice and Casp8 Fl / Fl Mouse peritoneal macrophages were co-cultured in PA+OA medium. Hepatocyte fat deposition and inflammatory response were detected 24 hours later.

[0049] 9. FISH

[0050] FISH was performed on formalin-fixed, paraffin-embedded slides using RNAscope Multiplex Fluorescence Assay V2. Freshly sectioned slides were deparaffinized with xylene and ethanol. Antigen retrieval was performed in an autoclave followed by treatment with hydrogen peroxide and protease. Slides were incubated with Psap from mouse samples for 1 hour in a humidified 42°C incubator. Subsequently, slides were incubated with Amp 1, 2, and 3 amplification probes for 30 minutes each, and C1-HRP probe for 15 minutes. Slides were incubated with Opal 520 (green) at a 1:500 dilution in a humidified 42°C incubator for 30 minutes, followed by treatment with HRP blocking reagent. Nuclei were stained with DAPI.

[0051] 10. Biochemical testing

[0052] Serum alanine aminotransferase (ALT) levels were measured using a Catalyst One chemistry analyzer, and liver triglycerides were measured using a Wako E-test triglyceride kit.

[0053] 11. qRT-PCR

[0054] Total RNA was reverse transcribed using a Reverse Transcription Master Kit, and qRT-PCR was performed using cDNA using SYBR Green Master Mix in a Lightcycler 480 Real-Time PCR 6 system.

[0055] 12. Single-cell sequencing sample processing and data analysis

[0056] (1) Sample processing and database construction

[0057] Single-cell transcriptome sequencing experiments were performed on liver tissue samples using instruments such as the 10x Chromium system. The workflow is as follows: dissociation of myocardial tissue into a single-cell suspension and preparation of beads; harvesting droplets using a microfluidics system; lysis of droplets and collection of beads; reverse transcription and template switching; exonuclease I treatment; first-round PCR of full-length cDNA; transposase-based fragmentation and tagging of the library; second-round PCR: selective amplification of the cDNA 3' end and addition of sequencing adapters; and sequencing.

[0058] (2) Data Analysis

[0059] Cell ranger software was used to cluster cell samples based on gene expression levels. Differential gene expression analysis was performed based on the cell clustering results. By comparing the gene expression results of each subpopulation with those of other subpopulations, the differentially expressed genes in each subpopulation were identified, and the corresponding difference fold and P value for each gene were obtained. GO and KEGG enrichment analysis were performed on the top 20 differentially expressed genes in each subpopulation. Pseudo-time series analysis was performed using cytoTRACE and Monocle 2 algorithms. Cell communication analysis was performed based on CellPhoneDB.

[0060] 13. Knockdown of Gpr37l1 in primary hepatocytes

[0061] Three Gpr37l1 shRNA interference plasmid sequences and one control shRNA Ctrl sequence were synthesized by Shanghai Gene Biotechnology. Primary hepatocytes were seeded in 6-well plates and cultured to 70-80% confluency. The three interference sequences and the control sequence were then transfected into the primary hepatocytes using Lipofectamine 3000. Green fluorescent protein expression was observed under a fluorescence microscope 36 hours later. After 48 hours of culture, cells were harvested for RNA isolation and western blot analysis.

[0062] 14. Histological examination

[0063] Tissues were fixed in 10% formalin overnight at room temperature and then embedded in paraffin. Sections were stained with hematoxylin and eosin (HE) for pathological analysis. Oil Red O staining was performed on frozen OCT-embedded liver sections to further confirm the presence of steatosis. Cryostat-cut 4 μm sections were fixed in 4% (v / v) paraformaldehyde for 10 minutes and then stained with freshly prepared Oil Red O working solution.

[0064] 15. Electron microscopy

[0065] Liver tissue or hepatocytes were fixed, dehydrated, embedded in epoxy resin, cut into 60 nm thin sections, stained with uranium-lead double staining for 15 min, dried overnight at room temperature, and observed using a projection electron microscope and filmed for analysis.

[0066] 16. ATAC sequencing analysis

[0067] Data quality control was performed using Fast QC software. Off-line raw data were processed for adapter removal. Clean data were aligned to the hg38_genecode reference genome using BWA software. The resulting bam file was used as input for peak calling using MACS2 software, with a q threshold of <0.05. DNA sequences were extracted from each peak region, extending 200 bp from both the 5' and 3' ends. Motifs were predicted using HOMER software. The predicted motifs were then matched to existing motif databases (HOMER and JASPAR) to identify corresponding known motifs and transcription factors. Gene-proximal signal distribution maps were analyzed using deeptools software. Genes associated with open chromatin regions were enriched in the Gene Ontology (GO) using the DAVID database. Pathway enrichment analysis of peak-proximal genes was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

[0068] 17. Western blotting

[0069] Mouse liver samples were stored in liquid nitrogen and sent to Hangzhou NewBe Biotechnology Co., Ltd. Western blot analysis was performed to analyze the expression of lipid metabolism-related proteins in mouse liver tissue. Development and fixation were performed using SuperSignal® WestDura Extended Duration Substrate, and the optical density of the bands was analyzed using Image J 1.8.0 image processing software. Each band was analyzed in triplicate. The relative expression level of the target protein was calculated as [target protein (optical density) / internal reference (optical density)] × 10 n .

[0070] 18. Co-immunoprecipitation

[0071] First, cells are lysed and centrifuged to obtain the supernatant, which is then pre-cleared of nonspecifically bound proteins. The target antibody is then added and incubated to form an antigen-antibody complex, which is then captured using Protein A / G agarose beads. The beads are washed to remove nonspecific binding, and the target protein is eluted. The eluted sample is separated by SDS-PAGE and detected by Western blot. Experiments should be performed at low temperatures to prevent protein degradation, and negative controls (such as IgG) should be included to ensure specificity. Optimizing the amount of antibody and beads used can improve experimental efficiency.

[0072] 19. Flow cytometry (FACS) analysis

[0073] Liver sections were collected, digested with type 4 collagenase, and filtered through a 70 μM cell strainer. Red blood cells were removed using ammonium chloride-potassium (ACK) lysis buffer, and a cell suspension was prepared. Cells were then stained with fluorescently labeled monoclonal antibodies: Alexa Fluor® 700 anti-mouse CD86 (A17199A, Biolegend), PE anti-mouse F4 / 80 (123110, BioLegend, CA), APC anti-mouse CD206 (141708, BioLegend), FITC anti-mouse CD11b (557396, BD Biosciences), and PerCP anti-mouse CD11c (117325, BioLegend). Samples were analyzed using a flow cytometer (BD Biosciences). Subsequent analysis was performed using FlowJo software (Tree Star Inc, San Carlos, CA). M1 and M2 macrophages were F4 / 80, respectively. + CD11b + CD86 + CD206 - and F4 / 80 + CD11b + CD86 -CD206 + .

[0074] 20. Data Statistical Analysis

[0075] Data are expressed as mean ± standard deviation (x ± SD) and analyzed using SPSS 20.0 software, with graphics generated using Origin 2022. One-way analysis of variance and Duncan's test were used for multiple comparisons, with P < 0.05 indicating a significant difference.

[0076] Example 2 Experimental results and analysis

[0077] 1. In the NASH mouse model, caspase8 levels in macrophages are reduced

[0078] We verified the cellular localization of Caspase8 in mouse tissues. Immunohistochemistry and WB results showed that Caspase8 was highly expressed in normal tissues and myeloid cells, while the expression of Caspase8 in myeloid cells of NASH mice was decreased ( Figure 1 AB). QPCR also showed that the expression of NASH group was reduced ( Figure 1 B), we used immunofluorescence ( Figure 1 C) Confirmation of colocalization of Caspase 8 and CD68. These results confirm that Caspase 8 expression is decreased in macrophages in the NASH model.

[0079] We further subdivided the KDM cell clusters into 10 myeloid subpopulations. We used the unique expression of known marker genes in the available samples to identify the major myeloid cell types ( Figure 2 A). Analysis of the distribution of differentially expressed genes in different subpopulations revealed that the macrophage clusters with differential expression of Caspase8 included Kupffer1, Kupffer2, and monocyte-macrophage 1 (MONO_MAC1). Compared with the control group, the most significant decrease in the NSAH group was in the MONO_MAC1 subpopulation ( Figure 2 BC).

[0080] To further understand the functional role of Caspase8 in macrophages, we re-analyzed the public scRNAseq dataset of NASH mice (GSE129516). Intergroup difference analysis of cells showed that compared with other macrophages, the MONO_MAC1 MFs group had increased M1 pro-inflammatory markers (CD68, CD14), decreased M2 macrophage markers (CD163 expression), and relatively low levels of genes related to other inflammation (Il1b, S100a10, S100a11, S100a8, S100a6) and fibrosis (Tgfbi, Tgfbrap1), while the expression of genes related to lipid metabolism (Lpl, Fabp5) and extracellular matrix remodeling (Mmp12) increased ( Figure 2 D)

[0081] 2. Caspase8 knockout in macrophages (Casp8 M-KO ) Promote the occurrence and development of NASH

[0082] To understand whether Caspase8 plays a protective or damaging role in the development of NASH in macrophages, we constructed macrophage-specific Caspase8 KO mice (Casp8 M-KO ). qPCR analysis showed that Casp8 M-KO It did not affect the expression of Caspase8 in whole liver tissue, but reduced the expression of Caspase8 in isolated MFs ( Figure 3 A). WB results showed that in NASH model, WT (Casp8 Fl / Fl ) Caspase8 in mice is more expressed in NASH, and Cas M-KO There is no ( Figure 3 B).

[0083] Casp8 M-KO and Casp8 Fl / Fl Mice were fed a normal diet or a NASH-inducing diet for 16 weeks. Fl / Fl Different from the control group, Casp8 M-KO The levels of triglyceride (TG) and serum alanine aminotransferase (ALT) in the liver tissue of mice increased, and they showed higher weight gain and liver-to-body weight ratio ( Figure 3 CD), and Casp8 M-KO The patients showed more severe steatosis, inflammation, and hepatocyte spherical degeneration. Similarly, Oil Red O staining confirmed that Casp8 M-KO Mouse liver steatosis is more obvious than Casp8 Fl / Fl Mice showed a significant increase ( Figure 3E) These results confirm that macrophage Caspase8 knockout leads to lipid accumulation in hepatocytes.

[0084] 3. Overexpression of Caspase8 in macrophages inhibits the development of NASH

[0085] BMDMs with a purity of about 95% were overexpressed with Caspase8 by lentiviral technology to construct Caspase8-positive BMDMs (Casp8 过表达 ) to explore the role of Caspase8 in NASH. We fed mice with a high-fat diet for 24 weeks. During the HFD feeding period, control or Caspase8 were injected into the tail vein every 4 weeks. 过表达 (5*10 6 Casp8 positive BMDMs) to further explore the role of macrophage Caspase8 in the progression of fatty liver disease. WB results showed that Caspase8 protein was expressed in Casp8 过表达 Highly expressed in mice ( Figure 4 A) Casp8 cells fed with a high-fat diet 过表达 The liver tissue triglyceride (TG), serum alanine aminotransferase (ALT) levels, body weight and liver weight of mice were significantly lower than those of the control group mice fed the same diet ( Figure 4 B). Histological examination of liver sections showed that Casp8 过表达 The mice showed reduced steatosis, inflammation, and hepatocyte spheric degeneration, and Oil Red O staining confirmed the expression of Casp8 过表达 Reduced liver steatosis in mice ( Figure 4 C). Decreased expression of pro-inflammatory cytokine-related genes ( Figure 4 D) Transmission electron microscopy (TEM) showed that Casp8 过表达 In mice, lipid accumulation in hepatocytes was significantly reduced, mitochondrial damage was alleviated, and ATP content was increased ( Figure 4 E), In summary, Caspase 8 in macrophages protects mice from the development of steatohepatitis.

[0086] 4. Macrophage Casp8 knockout induces M1 macrophage polarization and hepatocyte lipid accumulation

[0087] Subsequently, we examined the effect of macrophage caspase8 knockout on macrophage subtypes involved in NASH pathogenesis. We detected Caspase8 by flow cytometry. Fl / Fl and Casp8 M-KO Mouse bone marrow macrophages and liver tissue-derived total macrophages (F4 / 80 + CD11b + )、M1 type(F4 / 80+ CD11b + CD86 + CD206 - ) and M2 (F4 / 80 + CD11b + CD86 - CD206 + The results of the analysis of macrophages from mouse bone marrow and liver tissue showed that the Casp8 Fl / Fl Compared with mice fed the same diet, Casp8 M-KO The ratio of M1 macrophages to total macrophages increased, but the ratio of M2 macrophages to total macrophages decreased ( Figure 5 AD). This shows that under HFD feeding conditions, Casp8 M-KO Mice showed M1 polarization.

[0088] In order to further explore the role of macrophage Caspase8 on macrophage subtypes and its effect on hepatocyte lipid metabolism in vitro, we will start from Caspase8 Fl / Fl Caspase 8 was expressed in isolated mouse hepatocytes Fl / Fl With Casp8 M-KO Bone marrow macrophages isolated from mice were co-cultured in PA+OA culture medium. The results of Oil Red O staining and immunofluorescence showed that compared with the control group, PA+OA treated macrophages were significantly inhibited by Casp8 M-KO Lipid accumulation in hepatocytes co-cultured with bone marrow and liver tissue macrophages was more severe ( Figure 6 AB), transmission electron microscopy (TEM) showed that in PA+OA culture medium, Casp8 M-KO In the mouse bone marrow macrophage co-culture group, mitochondrial damage of hepatocytes was more severe and lipid accumulation in hepatocytes increased ( Figure 6 C). Meanwhile, PA+OA medium contains Casp8 M-KO Mouse bone marrow macrophages lead to increased expression of adipogenic genes (SREBP1c, ChREBP, LEX-α, USF1, Fasn) in hepatocytes and decreased expression of genes regulating β-oxidation (Acox1, CPT1, PPAR-α). Figure 6 DE). These results indicate that macrophage Caspase8 knockout increases hepatocyte lipid accumulation by inducing M1 macrophage polarization.

[0089] 5. Macrophage-specific Casp8 knockout induces M1 macrophage polarization and hepatocyte lipid deposition by inducing the secretion factor PSAP

[0090] To investigate the mechanism of context-specific gene expression programs, we identified accessible chromatin defined by ATAC-seq in AO+PO overexpressing caspase8 cells. ATAC-seq data analysis revealed that 64 and 3068 differential peaks (DPs) were specifically turned on and off, respectively, in response to Csapase8 overexpression compared to the control group. In the promoter region, the number of upregulated DPs and downregulated DPs in Csapase8 overexpression was 3 and 187, respectively (Table 1, Figure 7 A).

[0091] To further explore the mechanism by which Csapase8 knockout in macrophages affects lipid deposition in hepatocytes, we further subdivided hepatocytes into subpopulations and identified four hepatocyte subpopulations. The main hepatocyte types were identified using the unique expression of known marker genes in existing samples ( Figure 7 B). Receptor-ligand mediated intercellular communication is crucial for coordinating a variety of biological processes including proliferation, differentiation, and stress. In order to study the communication network between Csapase8-low-expressing macrophages and hepatocytes, we used CellPhoneDB to conduct a comprehensive and systematic analysis of intercellular communication molecules. We found complex mutual regulatory effects between MONO_MAC1 and various cell types (Table 2). We focused on its regulation of hepatocytes, and the top five ligand receptors included Apoe-TREM2_receptor, Psap-Gpr37l1, Ppia-Bsg, Trf-Tfr2, and Cadm1-Cadm1 (Table 2). We further cross-analyzed the top five macrophage ligands with ATAC-seq low-expression genes and high-expression genes in the Csapase8-low-expressing macrophage subpopulation, namely the MONO_MAC1 subpopulation, and screened out Psap, which is mainly present in lysosomes and can also function as a secretory protein and integral membrane protein ( Figure 7 C) Studies have shown that this protein exhibits regulatory capabilities in sphingolipid metabolism.

[0092] Table 1 Difference peak statistics

[0093]

[0094] Table 2 Ligand-receptor pairing analysis by CellphoneBD

[0095]

[0096] RNA in situ hybridization experiments showed that after PA / OA stimulation ( Figure 8In the reaction of A), the transcriptional expression of the target gene Psap increased in Caspase8 knockout macrophages. We further verified by qPCR and WB that Casp8 Fl / Fl The results showed that compared with the Casp8 M-KO The expression of Psap in macrophages of group A increased ( Figure 8 BC).

[0097] We used an interference plasmid to M-KO The Psap expression in primary bone marrow-derived macrophages of mice was knocked down, and the inflammatory response of macrophages and its effect on lipid accumulation in hepatocytes under PA+OA conditions were observed again. M-KO Knockdown of Psap in macrophages reduced the expression of inflammatory cytokines in macrophages ( Figure 8 D). Oil Red O staining and BODIPY staining showed that Casp8 was activated in PA+OA. M-KO Knockdown of Psap in macrophages attenuated lipogenesis in hepatocytes ( Figure 8 E). These data indicate that macrophage Casp8 knockout regulates the inflammatory response of macrophages and induces lipid metabolism disorders in hepatocytes by activating Psap.

[0098] 6. Cflip / ELK4 is involved in the transcriptional regulation of PSAP by Casp8

[0099] Transcription factors bind to open chromatin regions in a pattern called a motif. Therefore, we can use chromatin accessibility data from ATAC-seq to predict the binding of transcription factors to open chromatin regions of the Psap gene. In order to identify potential transcription factors that regulate the Psap gene, we used HOMER in default mode to screen for transcription factors enriched in open chromatin regions of the Psap promoter. The results showed that we found ZBTB7A and ELK4 binding sites in the open chromatin sites in the promoter region of the Psap gene, indicating that ZBTB7A and ELK4 may be involved in the transcriptional regulation of Psap by Caspase8 under inflammatory conditions. We further verified this by ChIP-PCR and found that the PSAP promoter can be enriched by ZBTB7A and ELK4, and the ELK4 enrichment effect is more obvious ( Figure 9 A). qPCR results showed that shELK4-3 down-regulated the expression of Psap, while shZBTB7A-1 up-regulated the expression of Psap, and the regulatory trend of shELK4-3 was more obvious ( Figure 9 B). WB and qPCR experiments showed that after PA / OA stimulation ( Figure 9 In the CD) reaction, the target gene Psap is transcribed and expressed in Caspase8 M-KOThe expression of Caspase-8 in macrophages was increased, but this effect was inhibited by knockdown of ELK4, and the inhibitory effect of ZBTB7A was not obvious. Therefore, we will focus on how Caspase-8 regulates the transcription of Psap by ELK4.

[0100] We further explored how Caspase8 regulates Psap transcription through ELK4 after Caspase8 decreases. Studies have shown that Caspase8 participates in TNF-induced necrosis and apoptosis by regulating the cleavage of cFLIP. Therefore, we first observed the effect of Caspase8 on cFLIP cleavage under inflammatory conditions. The results showed that after PA+OA treatment, cFLIP cleavage decreased under PA+OA conditions, while Caspase8 overexpression promoted cFLIP cleavage ( Figure 9 E). Studies have shown that D377 is an important site for caspase 8 to cleave cFLIP. We constructed a D377A plasmid to observe the effect of D377 mutation to alanine on caspase 8 cleavage of cFLIP. The results showed that under PA+OA conditions, the D377 point mutation inhibited caspase 8 cleavage of cFLIP and increased Psap expression ( Figure 9 F). When RAW macrophages overexpress cFLIP and ELK4, CoIP and immunofluorescence results showed that cFLIP and ELK4 interacted after PA / OA treatment ( Figure 9 G).

[0101] Next, we further used co-immunoprecipitation (Co-IP) and cFLIP point mutation plasmids to detect the interaction between endogenous cFLIP and ELK4 in bone marrow macrophages under inflammatory conditions. The results showed that compared with the PA+OA treatment group, caspase8 overexpression inhibited the interaction between cFLIP and ELK4, while the cFLIP D377 point mutation inhibited the effect of caspase8 ( Figure 10 A). We observed the inflammatory response of Caspase8-overexpressing macrophages and its effect on hepatocyte lipid accumulation in PA+OA by cFLIP point mutation. cFLIP D377 reversed the inhibitory effect of Caspase8 overexpression on macrophage inflammation ( Figure 10 B), Oil red O staining and BODIPY staining showed that cFLIP D377 reversed the inhibitory effect of Caspase8 overexpression on hepatocyte lipid deposition ( Figure 10 C) These data indicate that Caspase8 knockout in macrophages inhibits cFLIP cleavage, allowing uncleaved cFLIP to enter the nucleus and bind to Elk4, promoting Psap transcription, thereby regulating macrophage inflammatory responses and increasing hepatocyte lipid deposition.

[0102] 7. Rac1 / cdc42 / JNK signaling pathway is involved in Gpr37I1-mediated hepatocyte lipid deposition

[0103] We used lentivirus to overexpress Caspase8 Fl / Fl Bone marrow macrophages isolated from mice overexpress Psap, which depletes Caspase8 Fl / Fl Hepatocytes isolated from mice were transfected with shRNA Ctrl and shRNA Gpr37l1. We co-cultured these two cells in PA+OA medium. Oil red O staining showed that compared with the control group, Gpr37l1 knockdown hepatocytes had fewer lipid droplets, decreased lipid production genes, and increased β-oxidation-related genes ( Figure 11 AB). We further explored the molecular mechanism of Gpr37I1-mediated lipid deposition in hepatocytes. Cell interaction analysis showed that MONO_MAC1 regulated the 3 subpopulations of hepatocytes most significantly. Therefore, we focused on analyzing the functions of differentially upregulated genes in the 3 subpopulations of hepatocytes, showing that the upregulated genes in the 3 subpopulations of hepatocytes were mainly related to cell function, biological process regulation, and metabolic regulation. Among them, the upregulated genes Racl and Cdc4 belong to the small G protein superfamily and are involved in a variety of cell signal transductions. Studies have shown that they play an important role in the occurrence and development of fatty liver. We detected whether Gpr37I1 regulates lipid deposition in hepatocytes through Racl and Cdc4. Under the condition of PA+OA treatment, Casp8 M-KO Macrophages isolated from mice were co-cultured with hepatocytes in which Grp37I1 was knocked down. The results showed that compared with the PA+OA group, after Caspase8 knockout in macrophages, and compared with the shRNA Ctrl group, after Grp37I1 knockdown in liver cells, the expression of Racl and Cdc4 proteins in liver cells decreased ( Figure 11 C) At the same time, compared with the shRNA Ctrl group, the phosphorylation level of JNK protein in liver cells decreased after Grp37I1 knockdown ( Figure 11 D). We further interfered with the transcription of Racl and Cdc4 genes in hepatocytes by shRNA Rac1 and shRNA Cdc42 to verify whether Grp37I1 affects the phosphorylation level of JNK protein through Rac1 / Cdc42. The results showed that under PA+OA treatment and Grp37I1 overexpression, the knockdown of Racl and Cdc4 genes reduced the phosphorylation level of JNK protein ( Figure 11 E).

[0104] Oil red and DOPIDY staining results showed that under PA+OA treatment and Grp37I1 overexpression, lipid deposition in hepatocytes expressing Racl and Cdc4 genes ( Figure 12A) The same qPCR results showed that under the conditions of PA+OA treatment and Grp37I1 overexpression, the expression of adipogenic genes in hepatocytes with Racl and Cdc4 gene knockdown was reduced, while the expression of genes regulating β-oxidation was increased ( Figure 12 B).

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Preparation of myeloid-specific knockout of Caspase8 in bone marrow-derived macrophages from Caspase8-overexpressing mice M-KO Application of the invention in medicine for treating non-alcoholic fatty liver disease in mice, characterized by: Overexpression of Caspase8 in mouse macrophages inhibits the development of non-alcoholic fatty liver disease; knockout of Caspase8 in mouse macrophages induces macrophage polarization to M1 macrophages and lipid deposition in hepatocytes by inducing Psap; knockout of Caspase8 regulates the inflammatory response of macrophages and induces lipid metabolism disorders in hepatocytes by activating Psap; knockout of Caspase8 in mouse macrophages regulates the transcription of Psap through cFLIP and ELK4.

2. The use according to claim 1, characterized in that: Knockout of Caspase8 in mouse macrophages inhibits the cleavage of cFLIP. The uncleaved cFLIP binds to Elk4 and promotes the transcription of Psap, thereby regulating the inflammatory response of macrophages and increasing lipid deposition in hepatocytes.

3. The use according to claim 1, characterized in that: The Rac1 / Cdc42 / JNK signaling pathway is involved in Gpr37I1-mediated hepatocyte lipid deposition.

4. The use according to claim 3, characterized in that: Knockout of Caspase8 in mouse macrophages promoted the increase of Racl and Cdc4; knockout of Caspase8 and knockdown of Grp37I1 promoted the decrease of Racl and Cdc4; knockdown of Racl and Cdc4 promoted the decrease of JNK protein phosphorylation level.

5. The use according to claim 3, characterized in that: Overexpression of Grp37I1 and knockdown of Racl and Cdc4 induced decreased expression of adipogenic genes and increased expression of β-oxidation genes.

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