Experimental Study Of MFG-E8 Gene Knockout Promoting Non-alcoholic Fatty Hepatitis In Mice

AIM:To investigate the effects of milk fat globule-epidermal growth factor-8(MFG-E8)and its molecular mechanisms in non-alcoholic steatohepatitis(NASH).Methods:24 wild type(WT)and MFG-E8 knock out(KO)C57BL/6 mice were randomly divided into 4 groups,CD-WT group,MCD-WT group,CD-KO group and MCD-KO group.The CD-WT group and the CD-KO group were given a control diet(CD),the MCD-WT group and the MCD-KO group were given a methionine and choline deficient diet(MCD).During the period,the body state of the mice was closely observed,and the body weight was measured and recorded at a fixed time every week.The blood and liver tissues were collected after 5 weeks.The kit detects the activity of alanine aminotransferase(AST)and aspartate aminotransferase(AST)in serum,and the content of triglyceride(TG)in liver tissue.Hematoxylin and eosin(HE)staining was used to evaluate liver pathological changes.Oil red O staining was used to evaluate liver steatosis.Sirius red and Masson trichrome staining were used to assess the degree of liver fibrosis.The recrutment and infiltration of macrophage(F4/80)and neutrophils(Ly6G)in the liver.Enzyme-lineked immunosorbent assay(ELISA)was used to detect the expression of cytokine TNF-? and IL-1? protein in liver tissue.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to detect the expression of TNF-?,IL-1?,IL-6,ICAM,CCL2,CXCL2 and TGF-? cytokine mRNA expression in liver tissues.Western blot(WB)was to detect the protein expression of TLR4,p-IRAK1,p-p38,p-p65 and p65 in liver tissue.Results:1.Compared with the CD-WT group,the MCD-WT group has a significantly increased in the activity levels of ALT and AST in the serum of mice,increased the TG content in liver tissue,aggravated liver pathological damage and steatosis,aggravated liver fibrosis,aggravated the infiltration of macrophages and neutrophils,increased the expression of cytokines TNF-? and IL-1? in liver tissues,and increases intracellular cytokines TNF-?,IL-1?,IL-6,ICAM,CCL2,CXCL2.And TGF-? mRNA expression,up-regulates the expression of TLR4,p-IRAK1,p-p38,p-p65 and p65 proteins in TLR4-related pathways.2.Compared with the MCD-WT group,the MCD-KO group significantly increased the activity levels of ALT and AST in the serum of mice,increased the TG content in liver tissue,aggravated liver pathological damage and steatosis,aggravated liver fibrosis,aggravated the infiltration of macrophages and neutrophils,increased the expression of cytokines TNF-? and IL-1? in liver tissues,and increases intracellular cytokines TNF-?,IL-1?,IL-6,ICAM,CCL2,CXCL2.And TGF-? mRNA expression,up-regulates the expression of TLR4,p-IRAK1,p-p38,p-p65 and p65 proteins in TLR4-related pathways.Conclusion: MFG-E8 gene knockout promotes the development of non-alcoholic steatohepatitis,which may be related to its increased release of inflammatory cytokines,increased infiltration of inflammatory cells,activation of TLR4 signal-related pathways,and expansion of inflammatory cascade reaction.