Application of Caspase8 in preparation of medicine for treating non-alcoholic fatty liver disease

Abstract

The invention provides an application of Caspase8 in preparation of a medicine for treating non-alcoholic fatty liver disease. Overexpression of the Caspase8 inhibits development of the non-alcoholic fatty liver disease. The specific effect of Caspase8 in the non-alcoholic fatty liver disease is determined: expression of liver macrophage Caspase8 is reduced, so that a Cflip / ELK4 / Psap signal channel is activated, macrophage is polarized to M1 type, a Rac1 / cdc42 / JNK signal channel in liver cells is activated through a Gpr37I1 receptor on the surfaces of the liver cells, and a molecular mechanism that lipid deposition of the liver cells is increased is caused, so that the non-alcoholic fatty liver disease is inhibited. Caspase8 or small molecular analogues may bring dawn to 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 Caspase8 in the preparation of drugs for non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a liver metabolic disorder syndrome characterized by lipid deposition and fatty degeneration in hepatocytes, including 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 globally, affecting nearly 30% of adults. It is predicted that the incidence of NAFLD will increase to 33.5% by 2030, becoming the main cause of end-stage liver disease and liver transplantation. Therefore, discovering important pathogenic mechanisms and diagnostic and treatment targets involved in NAFLD and achieving effective intervention are major issues faced by clinical and basic research.

[0003] Disorder of the liver immune microenvironment plays an important role in the occurrence and development of NAFLD. The liver is rich in macrophages, including resident Kupffer cells in the liver and macrophages derived from blood monocytes. During the process of NAFLD, factors such as free fatty acids (FFA) produced by adipose tissue decomposition and lipopolysaccharide (LPS) produced by the intestine activate Kupffer cells and macrophages derived from monocytes to release inflammatory factors and promote liver inflammation. In addition, inflammatory cytokines and chemokines secreted by Kupffer cells and macrophages derived from monocytes play diverse functions in the process 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 to the liver; TGF-β stimulates hepatic stellate cells to activate into fibroblasts and promotes extracellular matrix deposition, thereby aggravating fibrosis. Therefore, macrophages participate in the progression of NAFLD through multiple mechanisms. Further exploring the regulatory mechanisms of macrophage phenotype and function during 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 drugs for non-alcoholic fatty liver disease.

[0005] To achieve the above object, the technical solution of the present invention is realized as follows: The present invention provides an application of Caspase8 in the preparation of drugs for non-alcoholic fatty liver disease, and overexpression of Caspase8 inhibits the development of non-alcoholic fatty liver disease.

[0006] Furthermore, knocking out Caspase8 promotes the development of non-alcoholic fatty liver disease; knocking out Caspase8 regulates the increase in the expression of adipogenic genes and the decrease in the expression of β-oxidation genes in hepatocytes.

[0007] Furthermore, knocking out Caspase8 induces the polarization of macrophages into M1 macrophages and lipid deposition in hepatocytes by inducing Psap; knocking out Caspase8 regulates the inflammatory response of macrophages and induces lipid metabolism disorders in hepatocytes by activating Psap.

[0008] Furthermore, knocking out Caspase8 regulates the transcription of Psap through cFLIP and ELK4.

[0009] Furthermore, knocking out Caspase8 inhibits the cleavage of cFLIP, and the uncleaved cFLIP binds to Elk4 to promote the transcription of Psap, thereby regulating the inflammatory response of macrophages and increasing lipid deposition in hepatocytes.

[0010] Furthermore, the Rac1 / Cdc42 / JNK signaling pathway is involved in Gpr37I1-mediated lipid deposition in hepatocytes.

[0011] Furthermore, knocking out Caspase8 promotes the elevation of Racl and Cdc4; knocking out Caspase8 and knocking down Grp37I1 promote the decrease of Racl and Cdc4; knocking down Racl and Cdc4 promotes the decrease in the phosphorylation level of JNK protein.

[0012] Furthermore, overexpression of Grp37I1 and knocking down Racl and Cdc4 induce a decrease in the expression of adipogenic genes and an increase in the expression of β-oxidation genes.

[0013] Furthermore, the dosage form of the drug is at least one of capsules, pills, tablets, granules or injections.

[0014] The present invention also provides an application of Caspase8 in the preparation of a reagent for detecting non-alcoholic fatty liver disease.

[0015] Compared with the prior art, the present invention has the following advantages: The present invention determines the specific role of Caspase8 in non-alcoholic fatty liver disease: the decrease in the expression of Caspase8 in hepatic macrophages activates the Cflip / ELK4 / Psap signaling pathway, leading to the polarization of macrophages into M1 type, and then activating the Rac1 / cdc42 / JNK signaling pathway in hepatocytes through the Gpr37I1 receptor on the surface of hepatocytes, resulting in the molecular mechanism of increased lipid deposition in hepatocytes. Caspase8 or small molecule analogs may bring hope for the treatment of steatohepatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Reduced caspase8 level in macrophages described in the embodiments of the present invention: A is the Western blot analysis of caspase8 protein in macrophages and the relative density ratio, B is the QPCR analysis of the expression of Caspase8 gene in macrophages of normal diet (ND) and high-fat diet HFD mice, C is the liver biopsy of ND and HFD mice, and immunofluorescence staining of Caspase8 (green) and CD68 (red) (gray arrow: Caspase8 + CD68 + cells); Figure 2 Reduced caspase8 level in macrophages described in the embodiments of the present invention: A is t-SNE from GSE129516, B is the relative expression level of Caspase8 mRNA in macrophages of NASH mice (GSE129516), C is the expression of Caspase8 in monocytes-macrophages 1 of the control group and NASH mice, D is the Log2 fold change of the most significantly differentially expressed genes between Caspase8 low-expression macrophages and other macrophages; Figure 3 Knockout of Caspase8 can exacerbate NASH described in the embodiments of the present invention: A is the expression level of Caspase8 mRNA in the liver tissues of Caspase8 Fl / Fl or Caspase8 M-KO mice fed a high-fat diet, B is the detection of Caspase8 protein level in the liver tissues of Caspase8 Fl / Fl and Caspase8 M-KO mice fed a high-fat diet by immunoblotting, C is the serum ALT level and liver TG content of Caspase8 Fl / Fl and Caspase8 M-KO mice fed a high-fat diet; N = 6 / group, D is the body weight and liver weight of Caspase8 Fl / Fl and Caspase8 M-KO mice fed a high-fat diet; 6 cases per group, E is the representative HE staining and Oil Red O staining images of the liver tissues of Caspase8 Fl / Fl and Caspase8 M-KO mice fed a high-fat diet; N = 6 / group. (Black arrow: macrovesicular steatosis; gray arrow: microvesicular steatosis; red arrow: inflammatory focus; blue arrow: ballooning degeneration of hepatocytes, CV, central vein; PV, portal vein); Figure 4 Overexpression of Caspase8 inhibits the development of steatohepatitis described in the embodiments of the present invention: A is the detection of the control group and Caspase8 by immunoblotting 过表达Caspase8 protein levels in liver tissue of mice after high-fat diet. B shows the control group and Caspase8 过表达 Serum ALT levels and liver TG content in mice after high-fat diet; N = 6 / group, control group fed with high-fat diet and Caspase8 过表达 Body weight and liver weight of mice; 6 cases in each group. C shows the control group fed with high-fat diet and Caspase8 过表达 Percentage of oil red area and NAFLD activity score in liver tissue of mice; N = 6 / group. D shows the control group and Caspase8 过表达 Gene expression of Tnf-α, il-6 and il-1β in liver tissue of mice after high-fat diet. N = 6 / group. E shows the statistical chart of mitochondrial length, diameter, percentage of damage and ATP content; Figure 5 Macrophage Casp8 knockout-induced M1 macrophage polarization and hepatocyte lipid accumulation as described in the examples of the present invention: A-B show the analysis of M1 (CD86 Fl / Fl and Casp8 M-KO ) and M2 (CD86 + CD206 - macrophage ratios in primary bone marrow-derived macrophages of mice fed with high-fat diet by flow cytometry. C-D show the analysis of M1 (CD86 - CD206 + and M2 (CD86 Fl / Fl and Casp8 M-KO macrophage ratios in primary liver macrophages of mice fed with high-fat diet by flow cytometry. C-D show the analysis of M1 (CD86 + CD206 - and M2 (CD86 - CD206 + macrophage ratios; Figure 6 Macrophage Casp8 knockout-induced M1 macrophage polarization and hepatocyte lipid accumulation as described in the examples of the present invention: A shows the representative oil red O staining of co-culture of primary hepatocytes and primary bone marrow-derived macrophages after 24 h of PA+OA treatment. B shows the representative immunofluorescence of phalloidine (red) and PLIN2 (green) in the co-culture of primary hepatocytes and primary bone marrow-derived macrophages after 24 h of PA+OA treatment. C shows the statistical chart of mitochondrial damage. D shows hepatocytes and Casp8 Fl / Fl and Casp8 M-KOAfter co - culturing mouse bone marrow - derived macrophages for 24 h, the mRNA levels of adipogenic genes SREBP1c, ChREBP, LEX - α, USF1, Fasn. E is in PA+OA medium, hepatocytes and Caspase8 Fl / Fl and Caspase8 M-KO Mouse bone marrow - derived macrophages were co - cultured for 24 hours, and the mRNA levels of β - oxidation genes Acox1, CPT1, PPAR - α; Figure 7 Knocking out Caspase8 induces M1 - type polarization of macrophages and lipid deposition in hepatocytes by inducing the secretion factor PSAP as described in the embodiments of the present invention: A is a differential peak volcano plot. Peaks with P < 0.05 and |change| greater than 1.5 - fold are considered differential peaks. B is a bubble plot of ligand - receptor connections between hepatocyte clusters with low Caspase8 expression and myeloid cells. The color intensity represents the expression level, and the dot size represents the gene expression frequency (percentage of cells expressing the gene). C is a Venn diagram showing the overlap of the top 5 ligand - receptors of monocyte_macroph1 and hepatocyte cellphoneBD data and Caspase8 - negatively correlated differential genes of ATAC - seq and sc - RNA data; Figure 8 Knocking out Caspase8 induces M1 - type polarization of macrophages and lipid deposition in hepatocytes by inducing the secretion factor PSAP as described in the embodiments of the present invention: A is from Caspase8 Fl / Fl and Caspase8 M-KO RNA in situ hybridization of Psap transcripts in PA / OAa - stimulated macrophages from Caspase8 Fl / Fl or Caspase8 M-KO mice (n = 6 samples / group). B is the mRNA expression level of Psap in Psap of Caspase8 Fl / Fl and Caspase8 M-KO mice fed a high - fat diet. C is Western blot detection of the effect of a high - fat diet on the Psap protein level in Caspase8 Figure 9Cflip / ELK4 involved in the transcriptional regulation of PSAP by casp8 in the embodiments of the present invention: A. Compared with the IgGIP control, ChIP-PCR verified the binding of Cflip and ELK4 to the promoter of the Pasp target gene, n = 3; B. Real-time quantitative PCR was used to detect the expression of Psap in RAW macrophages transfected with shCtrl, shELK4-3 or shzbtb7a-1; C. In RAW macrophages treated with PA / OA, Qpcr was used to observe the effect of knockdown of Cflip and ELK4 genes on Pasp transcription; D. In RAW macrophages treated with PA / OA, WB was used to observe the effect of knockdown of Cflip and ELK4 genes on the Pasp protein level; E. In RAW macrophages treated with PA / OA, WB was used to observe the effect of overexpression of Caspase8 on the cleavage of cFLIP protein; F. In RAW macrophages treated with PA / OA, WB was used to observe the effect of overexpression of Caspase8 and point mutation of Caspase8 D377A on the cleavage of cFLIP protein; G. In RAW macrophages treated with PA / OA, CoIP was used to observe the effect of overexpression of Caspase8, cFLIP and ELK4 on the interaction between exogenous cFLIP and ELK4 proteins; Figure 10 Cflip / ELK4 involved in the transcriptional regulation of PSAP by casp8 in the embodiments of the present invention: A. In RAW macrophages treated with PA / OA, CoIP was used to observe the effect of overexpression of Caspase8 and point mutation of Caspase8 D377A on the interaction between cFLIP and ELK4 proteins in the nucleus; B. In RAW macrophages treated with PA / OA, ELISA was used to observe the effect of overexpression of Caspase8 and point mutation of Caspase8 D377A on macrophage inflammation; C. In RAW macrophages treated with PA / OA, oil red staining of hepatocytes was used to observe the effect of overexpression of Caspase8 and point mutation of Caspase8 D377A on lipid deposition in hepatocytes; Figure 11 The Rac1 / cdc42 / JNK signaling pathway involved in Gpr37I1-mediated lipid deposition in hepatocytes: A. Oil red staining of hepatocytes was used to observe the effect of knockdown of Gpr37I1 in hepatocytes on lipid deposition in hepatocytes; B. Qpcr was used to observe the effect of knockdown of Gpr37I1 in hepatocytes on genes related to lipid synthesis and fatty acid β-oxidation in hepatocytes; C-D. WB was used to detect the changes in the protein levels of Rac1, Cdc42 and the phosphorylation level of JNK protein; E. WB was used to detect the changes in the phosphorylation level of JNK protein; Figure 12The Rac1 / cdc42 / JNK signaling pathway involved in Gpr37I1-mediated hepatocyte lipid deposition in the embodiments of the present invention: A shows the detection of hepatocyte lipid deposition by Oil Red and BODIPY staining, and B shows the effect of knocking down Gpr37I1 in hepatocytes on lipid synthesis and fatty acid β-oxidation-related genes in hepatocytes by Qpcr. Detailed implementation manners

[0017] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following embodiments are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0018] The present invention will be described in detail below with reference to embodiments.

[0019] Example 1 Materials and methods 1. C57 mice for experiments 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) were used to generate myeloid-specific knockout (Casp8 M-KO ) mice. Lyz2.Cre mice were used as the control group, and for simplicity, we will refer to them as wild-type (WT). All mice were C57BL / 6J mice.

[0020] 2. C57 mice for experiments Male Casp8 M-KO mice and Casp8 Fl / Fl littermate (7-8 weeks old) mice were randomly assigned to be fed with normal diet (NC), high-fat diet (HFD) for 16 weeks, high-fat diet (HFD) for 24 weeks, or methionine- and choline-deficient diet (MCD) for 4 weeks (n = 4 - 7 in each group). After the experiment, the mice were fasted, weighed, and the liver was quickly excised and weighed. All mice were housed in ventilated cages, under a 12-hour light / dark cycle, and were provided with an enriched environment, water, and free access to food. On the first day after 4 weeks, 8 weeks, 12 weeks, 16 weeks, and 20 weeks of high-fat diet, mice were intravenously injected via the tail vein with BMDMs transfected with negative control lentivirus (5*10 6 cell amount, control group), BMDMs positive for casp8 (5*10 6 cell amount, Casp8 过表达) Three days after the last injection (16 weeks of high-fat diet), the mice were humanely sacrificed for further analysis. Four weeks after the last injection (20th week of high-fat diet), the mice were humanely sacrificed for further analysis. All animal studies were approved by the Animal Experiment Ethics Committee of Tianjin Fifth Central Hospital and were conducted in accordance with the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences of the United States and published by the National Institutes of Health (NIH Publication 86-23, Revised Edition 1985).

[0021] 3. Adeno-associated virus The following adeno-associated viruses were used in this invention: AAV8-Control (AAV8-con) and AAV8-shPsap. AAV gene amplification was performed by Genechem Co., Ltd. (Shanghai). Six-week-old male Casp8 M-KO mice were randomly divided into the AAV8-CON group and the AAV8-shPsap group to establish a high-fat diet-induced NASH model. Then, AAV8-shPsap or AAV8-CON was injected via the tail vein at a dose of 1×10 9 infection units (IFU) / 200 μl / mouse. Two weeks later, all mice were placed on a high-fat diet and administered AAV virus once every 8 weeks. After 24 weeks of high-fat diet, all mice were sacrificed for further analysis.

[0022] 4. Histological examination The left liver lobe of each mouse was collected and fixed in 4% neutral buffered formalin. After dehydration, paraffin embedding and other procedures, sections of 4 µm were cut. The samples were scored according to the Kleiner scoring system, and the NAFLD activity score of each sample was calculated. Steatosis index mammary gland: (<5%), 1 (5% - 33%), 2 (>33% - 66%), 3 (>66%). Steatosis was classified as macrovesicular, microvesicular or both, and zonal distribution was visible. When hepatocytes enlarged to more than twice the size of adjacent cells, the cytoplasmic membrane changed from hexagonal in normal hepatocytes to round, and most of the cytoplasm was empty, it was hepatocyte ballooning degeneration. The score of hepatocyte ballooning degeneration was 1 point (1 - 5 ballooned cells) or 2 points (>5 ballooned cells). Inflammation score was 1 point (1 - 2 lesions), 2 points (2 - 4 lesions), 3 points (>4 lesions), and the size and zonal distribution of the inflammatory foci were determined. The fibrosis degree score was 1 point (simple perisinusoidal and perivenous fibrosis), 2 points (1 point + portal fibrosis), 3 points (bridging fibrosis) or 4 points (cirrhosis). Each specimen was scored and evaluated through 5 periportal and 5 pericentral fields at a magnification of 200 times.

[0023] 5. Extraction of liver macrophages After anesthetizing the mice, open the abdominal cavity, perfuse the EGTA buffer through the inferior vena cava, and then perfuse with the EGTA buffer containing 0.08 U / ml collagenase D at a flow rate of 3 ml / min for 15 min. Remove the liver, and further digest it with 0.5 mg / ml pronase E and 2% DNase-I at 37°C for 20 minutes to obtain a single cell suspension. Purify the liver non-parenchymal cells using 35% and 70% concentration of PercoⅡ gradients. Suspend the purified cells in Dulbecco's phosphate buffered saline (DPBS), and stain them using the Zombie Aqua Fixable Viability Kit. Wash the cells with sterile FACS staining buffer (DPBS supplemented with 2 mM EDTA and 0.5% BSA), then incubate with anti-f4 / 80 microspheres, and perform magnetic bead sorting to isolate macrophages. Seed the eluted cells in RPMI1640 supplemented with 10% FBS and 10 ng / ml MCSF, and wash away the non-adherent dead cells after 30 minutes.

[0024] 6. Isolation of primary hepatocytes After anesthetizing the mice, open the abdominal cavity, perfuse the liver with EGTA buffer through the inferior vena cava, and then perfuse with HBSS containing 10 ml 25 μg / ml Liberase TM at a flow rate of 3 ml / min. Take the liver and purify the viable hepatocytes using 50% concentration of Percoll. Resuspend the cells in DMEM low glucose medium supplemented with 5% FBS, and further seed the cells in a tissue culture dish coated with rat tail type I collagen. After the hepatocytes adhere for 4 h, change the medium to William's E medium containing 2 mM L-glutamine for maintenance or use a specific medium in selected experiments.

[0025] 7. Isolation of bone marrow macrophages Isolate bone marrow cells from the femurs and tibias of male Casp8 M-KO mice and Casp8 Fl / Fl mice. Take the bone marrow and rinse it with DMEM. Seed the bone marrow cells in DMEM medium containing 10% FBS and 10 ng / mL colony stimulating factor 1 and culture them to induce their differentiation into bone marrow-derived macrophages (BMDMs). On the 7th day, all adherent cells are mature macrophages.

[0026] 8. Isolation of bone marrow macrophages In co-culture studies, primary hepatocytes from male Casp8 Fl / Fl mice and macrophages from the liver or bone marrow of male Casp8 M-KO mice and Casp8 Fl / Fl mice are seeded in the co-culture chamber. The primary hepatocytes from male Casp8 Fl / Fl mice and male Casp8M-KO Mouse and Casp8 Fl / Fl Mouse peritoneal macrophages were co-cultured in PA+OA medium. After 24 h, hepatocyte lipid deposition and inflammatory responses were detected.

[0027] 9. In situ hybridization experiment (FISH) FISH detection of V2 on formalin-fixed paraffin-embedded slides was performed using RNAscope multiplex fluorescence. The slides of fresh sections were dewaxed with xylene and ethanol. Antigen retrieval was carried out in a pressure cooker and then treated with hydrogen peroxide and protease plus. The slides were incubated with Psap of mouse samples in a humidified incubator at 42 °C for 1 hour, followed by incubation with Amp 1, 2, and 3 for the amplified probes for 30 minutes each, and C1-HRP probe for 15 minutes. The slides were incubated with Opal 520 (green) at 1:500 in a humidified incubator at 42 °C for 30 minutes and then treated with HRP blocker. Nuclei were stained with DAPI.

[0028] 10. Biochemical detection The serum alanine aminotransferase (ALT) level was detected using a Catalyst One chemical analyzer; liver triglyceride was detected using a Wako E-test triglyceride kit.

[0029] 11. qRT-PCR Total RNA was reverse transcribed using a reverse transcription Master kit. qRT-PCR was performed using cDNA in a lightcycler 480 Real-Time PCR 6 system with SYBR Green Master Mix.

[0030] 12. Single-cell sequencing sample processing and data analysis (1) Sample processing and library construction Single-cell transcriptome sequencing experiments were performed on liver tissue samples using instruments such as the 10x Chromium system. The brief process is as follows: myocardial tissue was dissociated into single-cell suspensions and beads were prepared; droplets were obtained through a microfluidic system; droplets were lysed and beads were collected; reverse transcription and template conversion; exonuclease I treatment; PCR of the first-round full-length cDNA; fragmentation and tagging of the library by the transposase method; second-round PCR: selective amplification of the 3' end of cDNA and addition of sequencing adapters; sequencing on the machine.

[0031] (2) Data analysis Using the Cell ranger software, cell samples were clustered based on gene expression levels; gene differential expression analysis was performed according to the cell clustering results. By comparing the gene expression results of each subgroup with those of other subgroups, the differentially expressed genes of each subgroup were identified, and at the same time, the corresponding fold change and the corresponding P-value of each gene were obtained; GO and KEGG enrichment analyses were performed on the top 20 differentially expressed genes of each subgroup; pseudotime analysis was performed through the cytoTRACE and Monocle 2 algorithms; cell communication analysis was based on CellPhoneDB.

[0032] 13. Knockdown of Gpr37l1 in primary hepatocytes Three Gpr37l1 shRNA interference plasmid sequences and one control shRNA Ctrl sequence were synthesized by Shanghai GenePharma Co., Ltd. Primary hepatocytes were seeded in 6-well plates and cultured until 70 - 80% confluence. Then, the three interference sequences and the control sequence were transfected into primary hepatocytes using Lipofectamine 3000 respectively. After 36 h, the expression of green fluorescent protein was observed under a fluorescence microscope. After 48 h of culture, some cells were collected for RNA isolation and WB.

[0033] 14. Histological examination Tissues were fixed overnight with 10% formalin at room temperature and then embedded in paraffin. Sections were stained with hematoxylin-eosin (HE) for pathological analysis. Frozen liver sections embedded in OCT were stained with Oil Red O to further confirm the presence of steatosis. 4-μm sections cut with a cryostat were fixed in 4% (v / v) paraformaldehyde for 10 minutes and then stained with freshly prepared Oil Red O working solution.

[0034] 15. Electron microscopy examination Liver tissues or hepatocytes were fixed, dehydrated, and embedded in epoxy resin, cut into 60-nm thin sections, double-stained with uranium and lead for 15 min, dried overnight at room temperature, and observed and photographed using a transmission electron microscope for analysis.

[0035] 16. ATAC-seq analysis The quality control of the data uses Fast QC software. The raw data after sequencing is processed to remove adapters. The BWA software aligns the clean data to the reference genome hg38_genecode. The bam file obtained after alignment analysis is used as the input file, and the MACS2 software is used to call peaks with a screening threshold of q < 0.05. DNA sequences are extracted by extending 200 bp from both the 5' end and 3' end of each peak region in two directions, and the HOMER software is used to predict motifs. Subsequently, the predicted motifs are matched with the existing motif data in the databases (HOMER, JASPAR) to identify the corresponding known motifs and the corresponding transcription factors. The deeptools software is used for the analysis of the signal distribution map near genes. The DAVID database is used for gene ontology (GO) enrichment analysis of genes associated with chromatin open regions. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, pathway enrichment analysis of genes adjacent to peaks is performed.

[0036] 17. Western Blot Mouse liver samples were stored in liquid nitrogen and sent to Hangzhou NewBay Biotechnology Co., Ltd. The expression of proteins related to lipid metabolism in mouse liver tissues was analyzed by Western blot. Development and fixation were carried out using SuperSignal® WestDura Extended Duration Substrate, and the Image J 1.8.0 image processing software was used to analyze the optical density values of the bands. Each band was repeated 3 times, and the relative expression level of the target protein = [optical density value of the target protein / optical density value of the internal reference] × 10 n 。

[0037] 18. Co-Immunoprecipitation First, the cells are lysed and centrifuged to obtain the supernatant, and then non-specific binding proteins are pre-cleared. The target antibody is added for incubation to form an antigen-antibody complex, and then the complex is captured by protein A / G agarose beads. The beads are washed to remove non-specific binding, and finally the target protein is eluted. The eluted samples are separated by SDS-PAGE and the target protein is detected by Western Blot. The experiment needs to be carried out at low temperature to prevent protein degradation, and a negative control (such as IgG) is set to ensure specificity. Optimizing the antibody and bead dosage can improve the experimental efficiency.

[0038] 19. Flow Cytometry (FACS) Analysis Liver sections were collected, digested with type IV collagenase, and filtered through a 70-μm cell strainer. Erythrocytes were removed using ammonium chloride-potassium (ACK) lysis buffer to prepare a cell suspension. Subsequently, the cells were 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, BDBiosciences), PerCP anti-mouse CD11c (117325, BioLegend). Sample analysis was performed using a flow cytometer (BDBiosciences). Subsequent analysis was performed using FlowJo software (Tree Star Inc, San Carlos, CA). M1 and M2 macrophages were defined as F4 / 80 + CD11b + CD86 + CD206 - and F4 / 80 + CD11b + CD86 - CD206 + .

[0039] 20. Statistical analysis of data Data were expressed as mean ± standard deviation (x ± SD), analyzed using SPSS 20.0 software, and graphed using Origin2022. One-way ANOVA and Duncan's test were used for multiple comparisons, and P < 0.05 indicated a significant difference

[0040] Example 2 Experimental results and analysis 1. In the NASH mouse model, the level of caspase8 in macrophages decreases We verified the cellular localization of Caspase8 in mouse tissues. Immunohistochemistry and WB results showed that Caspase8 was highly expressed in myeloid cells in normal tissues, while its expression decreased in myeloid cells of NASH mice ( Figure 1 A - B). Qpcr also showed a decreased expression in the NASH group ( Figure 1 B), and we determined the co-localization of Caspase8 and CD68 by immunofluorescence ( Figure 1 C). These results confirmed that the expression of Caspase8 in macrophages decreased in the NASH model

[0041] We further subdivided the KDM cell clusters into subpopulations and identified 10 myeloid subpopulations. The main myeloid cell types were determined by the unique expression of known marker genes in the existing samples ( Figure 2 A). By analyzing the distribution of differential genes in different subpopulations, we identified macrophage clusters with differential expression of Caspase8, including Kupffer1, Kupffer2, and Mono_Macrophage1 (MONO_MAC1). Compared with the control group, the most significant decrease in the NSAH group was observed in the MONO_MAC1 subpopulation ( Figure 2 B-C).

[0042] To further understand the functional role of Caspase8 in macrophages, we re-analyzed the publicly available scRNAseq dataset of NASH mice (GSE129516). Inter-group differential analysis of the cells showed that compared with other macrophages, the MONO_MAC1 MFs group had increased M1-type pro-inflammatory markers (CD68, CD14), decreased M2-type macrophage marker (CD163 expression), and relatively lower gene levels related to other inflammation (Il1b, S100a10, S100a11, S100a8, S100a6) and fibrosis (Tgfbi, Tgfbrap1), while the gene expressions related to lipid metabolism (Lpl, Fabp5) and extracellular matrix remodeling (Mmp12) were increased ( Figure 2 D) 2. Knockout of Caspase8 in macrophages (Casp8 M-KO ) promotes the occurrence and development of NASH To understand whether Caspase8 in macrophages plays a protective or damaging role in the development of NASH, we constructed macrophage-specific Caspase8 KO mice (Casp8 M-KO ). qPCR analysis showed that Casp8 M-KO did not affect the expression of Caspase8 in the whole liver tissue, but decreased the expression of Caspase8 in isolated MFs ( Figure 3 A). WB results showed that under the NASH model, in MFs, Caspase8 in WT (Casp8 Fl / Fl ) mice was more highly expressed during NASH, while there was no expression in Cas M-KO ( Figure 3 B).

[0043] Casp8 M-KO and Casp8 Fl / Fl mice were fed a normal diet or an NASH-induced diet for 16 weeks. Different from the Casp8 Fl / Fl control group, Casp8 M-KOLevels of triglyceride (TG) in mouse liver tissue and serum alanine aminotransferase (ALT) were increased, showing higher body weight gain and liver-to-body weight ratio ( Figure 3 C-D), and Casp8 M-KO showed more severe steatosis, inflammation, and hepatocyte ballooning degeneration. Similarly, Oil Red O staining confirmed that Casp8 M-KO mice had more significant liver steatosis than Casp8 Fl / Fl mice ( Figure 3 E). These results confirmed that macrophage Caspase8 knockout led to lipid accumulation in hepatocytes.

[0044] 3. Overexpression of macrophage Caspase8 inhibits the development of NASH Bone marrow-derived macrophages (BMDMs) with a purity of approximately 95% were overexpressed with Caspase8 by lentiviral technology to construct Caspase8-positive BMDMs (Casp8 过表达 ), to explore the role of Casp8 in NASH. We fed mice a high-fat diet for 24 weeks. During the HFD feeding period, control or Casp8 过表达 (5*10 6 Casp8-positive BMDMs) were injected into the tail vein once every 4 weeks to further explore the role of macrophage Caspase8 in the progression of steatohepatitis. WB results showed that Caspase8 protein was highly expressed in Casp8 过表达 mice ( Figure 4 A), and the levels of triglyceride (TG) in liver tissue, serum alanine aminotransferase (ALT), body weight, and liver weight of Casp8 过表达 mice fed a high-fat diet were significantly lower than those of control mice fed the same diet ( Figure 4 B). Histological examination of liver sections showed that compared with the control group, steatosis, inflammation, and hepatocyte ballooning degeneration were reduced in Casp8 过表达 mice fed HFD for 26 weeks. Oil Red O staining confirmed a reduction in liver steatosis in Casp8 过表达 mice ( Figure 4 C). The expression of pro-inflammatory cytokine-related genes decreased ( Figure 4 D). Transmission electron microscopy (TEM) showed that compared with control mice, lipid accumulation in hepatocytes was significantly reduced, mitochondrial damage was alleviated, and ATP content increased in Casp8 过表达 mice fed HFD ( Figure 4 E). In summary, Casp8 in macrophages protected mice from the development of steatohepatitis.

[0045] 4. Macrophage Casp8 knockout induces M1 macrophage polarization and hepatocyte lipid accumulation Subsequently, we detected the effect of macrophage caspase8 knockout on macrophage subtypes in NASH pathogenesis. We detected Casp8 by flow cytometry Fl / Fl and Casp8 M-KO in mouse bone marrow macrophages and total macrophages derived from liver tissue (F4 / 80 + CD11b + ), M1 type (F4 / 80 + CD11b + CD86 + CD206 - ), and M2 type (F4 / 80 + CD11b + CD86 - CD206 + ). The results of the analysis of macrophages derived from mouse bone marrow and liver tissues showed that, compared with Casp8 Fl / Fl mice fed with HFD, the ratio of M1 macrophages to total macrophages increased, but the ratio of M2 macrophages to total macrophages decreased in Casp8 M-KO mice fed the same diet ( Figure 5 A-D). This indicates that under HFD feeding conditions, Casp8 M-KO mice showed M1 polarization.

[0046] To further explore the effect of macrophage Casp8 on macrophage subtypes and the lipid metabolism of hepatocytes in vitro. We co-cultured hepatocytes isolated from Caspase8 Fl / Fl mice with bone marrow macrophages isolated from Caspase8 Fl / Fl and Casp8 M-KO mice in a PA+OA medium. The results of oil red O staining and immunofluorescence showed that, compared with the control group, after PA+OA treatment, hepatocytes co-cultured with bone marrow and liver tissue macrophages derived from Casp8 M-KO had more severe lipid accumulation ( Figure 6 A-B). Transmission electron microscopy (TEM) showed that in the PA+OA medium, in the co-culture group of hepatocytes with bone marrow macrophages derived from Casp8 M-KO mice, mitochondrial damage in hepatocytes was more severe and lipid accumulation in hepatocytes increased ( Figure 6 C). Meanwhile, bone marrow macrophages from Casp8 M-KO mice in the PA+OA medium led to increased expression of adipogenic genes (SREBP1c, ChREBP, LEX-α, USF1, Fasn) and decreased expression of genes regulating β-oxidation (Acox1, CPT1, PPAR-α) in hepatocytes ( Figure 6D-E). These results indicate that macrophage Caspase8 knockout increases hepatic lipid accumulation by inducing M1 macrophage polarization.

[0047] 5. Macrophage-specific Casp8 knockout induces M1 macrophage polarization and hepatic lipid deposition via the secreted factor PSAP To investigate the mechanism of environmentally specific gene expression programs, we determined the accessible chromatin defined by ATAC-seq in caspase8 cells overexpressing AO+PO. Data analysis of ATAC-seq showed that compared with the control group, 64 and 3,068 differential peaks (DPs) were specifically opened and closed, respectively, in Csapase8 overexpression. In the promoter region, the numbers of upregulated DPs and downregulated DPs were 3 and 187, respectively, in Csapase8 overexpression (Table 1, Figure 7 A).

[0048] To further explore the mechanism by which Csapase8 knockout macrophages affect hepatic lipid deposition, we further clustered and subdivided hepatocytes into subpopulations and identified 4 hepatocyte subpopulations. The main hepatocyte types were determined using the unique expression of known marker genes in existing samples ( Figure 7 B). Receptor-ligand-mediated intercellular communication is crucial for coordinating multiple biological processes, including proliferation, differentiation, and stress. To investigate the communication network between Csapase8-low-expressing macrophages and hepatocytes, we performed a comprehensive and systematic analysis of intercellular communication molecules using CellPhoneDB. We found complex reciprocal regulatory effects between MONO_MAC1 and various cell types (Table 2). We focused on its regulation of hepatocytes, and the top five ligand-receptors scored included Apoe-TREM2_receptor, Psap-Gpr37l1, Ppia-Bsg, Trf-Tfr2, Cadm1-Cadm1 (Table 2). We further performed a cross-analysis of the top 5 macrophage ligands scored with the low-expressed genes of ATAC-seq and the high-expressed genes of 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 secreted protein and an integral membrane protein ( Figure 7 C), and studies have shown that this protein exhibits regulatory ability in sphingolipid metabolism.

[0049] Table 1 Differential peak statistics

[0050] Table 2 Ligand-receptor pairing analyzed by CellphoneBD

[0051] RNA in situ hybridization experiments showed that in response to PA / OA stimulation ( Figure 8 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 Compared with the control group, the expression of Psap in Casp8 M-KO group macrophages increased ( Figure 8 B-C).

[0052] We knocked down the expression of Psap in primary bone marrow-derived macrophages from Casp8 M-KO mice by interfering plasmids, and again observed the inflammatory response of Caspase8 low-expression macrophages and its effect on hepatocyte lipid accumulation under the condition of PA+OA. Knocking down Psap in Casp8 M-KO macrophages decreased the expression of inflammatory cytokines in macrophages ( Figure 8 D). Oil red O staining and BODIPY staining showed that knocking down Psap in Casp8 M-KO macrophages initiated by PA+OA alleviated adipogenesis in hepatocytes ( Figure 8 E). These data indicate that Casp8 knockout in macrophages regulates the inflammatory response of macrophages and induces disorder of hepatocyte lipid metabolism by activating Psap.

[0053] 6. Cflip / ELK4 is involved in the transcriptional regulation of PSAP by Casp8 Transcription factors bind to open chromatin regions in a pattern called motif. Therefore, we can use the chromatin accessibility data of ATAC-seq to predict the binding of transcription factors to the open chromatin regions of the Psap gene. To identify potential transcription factors regulating the Psap gene, we used HOMER to screen for transcription factors enriched in the open chromatin region of the Psap promoter in the default mode. The results showed that we found binding sites for ZBTB7A and ELK4 in the open chromatin sites of the Psap gene promoter region, indicating that ZBTB7A and ELK4 may be involved in the transcriptional regulation of Psap by Caspase8 under inflammatory conditions. We further verified by ChIP-PCR and found that the promoter of PSAP could be enriched by ZBTB7A and ELK4, and the enrichment effect of ELK4 was more obvious ( Figure 9 A). The qPCR results showed that shELK4-3 downregulated the expression of Psap, shZBTB7A-1 upregulated the expression of Psap, and moreover, the regulatory trend of shELK4-3 was more obvious ( Figure 9 B). WB and qPCR experiments showed that in response to PA / OA stimulation (Figure 9 In the reaction of C-D, the transcriptional expression of the target gene Psap increased in Caspase8 M-KO macrophages, but this effect was inhibited after knockdown of ELK4, and the inhibitory effect of ZBTB7A was not obvious. Therefore, in the following, we focused on how Caspase8 regulates the transcription of Psap by ELK4.

[0054] We further explored how the transcription of Psap is regulated by ELK4 after the decrease of Caspase8. Some studies have shown that Caspase8 participates in TNF-induced necrosis and apoptosis by regulating the cleavage of cFLIP. Therefore, we first observed the cleavage effect of Caspase8 on cFLIP under inflammatory conditions. The results showed that after treatment with PA+OA, the cleavage of cFLIP decreased under the condition of PA+OA. However, overexpression of Caspase8 promoted the cleavage of cFLIP( Figure 9 E). Research has shown that D377 is an important site for caspase 8 to cleave cFLIP. We constructed a D377A plasmid to observe the effect of the mutation of D377 to alanine on the cleavage of cFLIP by caspase8. The results showed that under the condition of PA+OA, the point mutation of D377 inhibited the cleavage of cFLIP by caspase8, and the expression of Psap increased( Figure 9 F). When cFLIP and ELK4 were overexpressed in RAW macrophages, the results of CoIP and immunofluorescence showed that after treatment with PA / OA, there was an interaction between cFLIP and ELK4( Figure 9 G).

[0055] Next, we further detected the interaction between endogenous cFLIP and ELK4 in bone marrow macrophages under inflammatory conditions by co-immunoprecipitation (Co-IP) and cFLIP point mutant plasmids. The results showed that compared with the PA+OA treatment group, overexpression of caspase8 inhibited the interaction between cFLIP and ELK4, while the point mutation of cFLIP D377 inhibited the effect of caspase8( Figure 10 A). We observed the inflammatory response of macrophages and its effect on lipid accumulation in hepatocytes in Caspase8-overexpressing macrophages under the condition of PA+OA by cFLIP point mutation. The point mutation of cFLIP D377 reversed the inhibitory effect of Caspase8 overexpression on macrophage inflammation( Figure 10 B), and Oil Red O staining and BODIPY staining showed that the point mutation of cFLIP D377 reversed the inhibitory effect of Caspase8 overexpression on lipid deposition in hepatocytes ( Figure 10C). These data indicate that knockout of Caspase8 in macrophages inhibits the cleavage of cFLIP. Uncleaved cFLIP enters the nucleus and binds to Elk4, promoting the transcription of Psap, thereby regulating macrophage inflammatory response and increasing hepatic lipid deposition.

[0056] 7. The Rac1 / cdc42 / JNK signaling pathway is involved in Gpr37I1-mediated hepatic lipid deposition We used overexpression lentivirus to overexpress Psap in bone marrow macrophages isolated from Caspase8 Fl / Fl mice, and transfected hepatocytes isolated from Caspase8 Fl / Fl mice with shRNA Ctrl and shRNA Gpr37l1. We co-cultured these two types of cells in a PA+OA medium. Oil red O staining results showed that compared with the control group, hepatocytes with Gpr37l1 knockdown had fewer lipid droplets, reduced lipidogenesis genes, and increased β-oxidation-related genes ( Figure 11 A-B). We further explored the molecular mechanism of Gpr37I1-mediated hepatic lipid deposition. Cell interaction analysis showed that MONO_MAC1 had the most obvious regulation on the hepatocyte 3 subset. Therefore, we focused on analyzing the functions of the upregulated differential genes in the hepatocyte 3 subset cells, and the results showed that the upregulated genes in the hepatocyte 3 subset were mainly related to cell function, biological process regulation, and metabolic regulation. Among them, the upregulated genes Racl and Cdc4 belong to the members of the small G protein superfamily and are involved in multiple cell signal transmissions. Some studies have shown that they play important roles in the occurrence and development of fatty liver. We detected whether Gpr37I1 regulates hepatic lipid deposition through Racl and Cdc4. Under the treatment of PA+OA, we co-cultured macrophages isolated from Casp8 M-KO mice with hepatocytes with Grp37I1 knockdown. The results showed that compared with the PA+OA group, after knockout of Caspase8 in macrophages and knockdown of Grp37I1 in hepatocytes compared with the shRNA Ctrl group, the levels of Racl and Cdc4 proteins in hepatocytes decreased ( Figure 11 C). At the same time, compared with the shRNA Ctrl group, after knockdown of Grp37I1 in hepatocytes, the phosphorylation level of JNK protein in hepatocytes decreased ( Figure 11 D). We further interfered with the transcription of Racl and Cdc4 genes by shRNA Rac1 and shRNA Cdc42 in hepatocytes to verify whether Grp37I1 affects the phosphorylation level of JNK protein through Rac1 / Cdc42. The results showed that under the treatment of PA+OA and overexpression of Grp37I1, knockdown of Racl and Cdc4 genes decreased the phosphorylation level of JNK protein ( Figure 11 E).

[0057] The results of Oil Red and DOPIDY staining showed that under the treatment of PA+OA and overexpression of Grp37I1, lipid deposition occurred in hepatocytes of Racl and Cdc4 genes ( Figure 12 A). Similarly, the results of Qpcr showed that under the treatment of PA+OA and overexpression of Grp37I1, the expression of adipogenic genes decreased and the expression of genes regulating β-oxidation increased in hepatocytes with knockdown of Racl and Cdc4 genes ( Figure 12 B).

[0058] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of Caspase8 in the preparation of a medicament for non-alcoholic fatty liver, characterized in that: Overexpression of Caspase8 inhibits the development of non-alcoholic fatty liver disease.

2. The application according to claim 1, wherein: Knockout of Caspase8 promotes the development of non-alcoholic fatty liver disease; knockout of Caspase8 regulates the increase in the expression of adipogenic genes and the decrease in the expression of β-oxidation genes in hepatocytes.

3. The application according to claim 2, wherein: Knockout of Caspase8 induces the polarization of macrophages into M1 macrophages and hepatocyte lipid deposition by inducing Psap; knockout of Caspase8 regulates the inflammatory response of macrophages and induces lipid metabolism disorders in hepatocytes by activating Psap.

4. The application according to claim 2, characterized in that: Knockout of Caspase8 regulates the transcription of Psap through cFLIP and ELK4.

5. The application according to claim 4, characterized in that: Knockout of Caspase8 inhibits the cleavage of cFLIP, and the uncleaved cFLIP binds to Elk4 to promote the transcription of Psap, thereby regulating the inflammatory response of macrophages and increasing lipid deposition in hepatocytes.

6. The application according to claim 2, characterized in that: The Rac1 / Cdc42 / JNK signaling pathway is involved in Gpr37I1-mediated hepatocyte lipid deposition.

7. The application according to claim 6, wherein: Knockout of Caspase8 promotes the elevation of Racl and Cdc4; knockout of Caspase8 and knockdown of Grp37I1 promote the decrease of Racl and Cdc4; knockdown of Racl and Cdc4 promotes the decrease in the phosphorylation level of JNK protein.

8. The application according to claim 6, wherein: Overexpression of Grp37I1 and knockdown of Racl and Cdc4 induce a decrease in the expression of adipogenic genes and an increase in the expression of β-oxidation genes.

9. The application according to claim 1, characterized in that: The dosage form of the drug is at least one of capsules, pills, tablets, granules or injections.

10. Application of Caspase8 in the preparation of reagents for detecting non-alcoholic fatty liver disease.

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