Study On NAFL Hepatocyte Fibrosis Induced By HIF-1α/YAP/IL-1β In High Fat Microenvironment

Background and objectiveChronic liver disease has become the foremost source of non-alcoholic fatty liver disease and is a public health problem globally.Hepatic stellate cells,macrophages and hepatic sinusoidal endothelial cells may have a part in the fibrosis of NAFLD,However,the key role of hepatocytes in fibrosis has not been thoroughly studied.Study shows that hypoxia inducing factor 1α(HIF-1?)and Yes associated protein(YAP)molecules in the metabolism and proliferation of hepatocytes play an important role,Interleukin 1?(IL-1?)has a corresponding positive regulatory role in process of NAFLD to fibrosis.This research discusses mainly the high-fat microenvironment of hepatocytes mediating NAFL fibrosis and IL-1? expression regulation mechanism through HIF-1?/YAP signaling pathways,exploring HIF-1?/YAP interaction.Methods1.In animal experiments: Randomly dividing 24 male C57BL/6J mice aged4 weeks into two groups(the high-fat group total n=15 and the control group total n=9),they were fed in a single cage.The high-fat diet group was given a high-fat diet(HFD),while the control group was given chow diet(CD).After 1 month,2 months and 3 months(HFD,n = 5;CD,n= 3),the mice were sacrificed and the livers were extracted and weighed.Part of the livers were fixed at 10% neutral formalin and part was stored at-80 ℃.HE and oil red O staining revealed fatty degeneration of hepatocytes,to dentify NAFL stage.IHC was used to assess collagen Ⅲ deposition to dentify fibrotic lesions.Detect changes of free fatty acid(FFA),low density lipoprotein(LDL-C)to dentify high-fat microenvironment of NAFL.Western blotting and realtime quantitative q PCR(RT-q PCR)for the detection of HIF-1?,YAP,ACTA2,collagen Ⅰ,collagen Ⅲ,MMP2,IL-1?,TIMP1 expression.2.In cell experiments: the different concentrations of palmitic acid(PA)(Hep G2: 200,400,600 μM,Hepa 1-6: 100,200 μM)to culture liver cancer cell lines(Hep G2)and mice liver cancer cell lines(Hepa 1-6)to establish a cytological model of high fat microenvironment,DMEM complete culture-medium with 0.1% BSA for reference.Oil red O staining to observe cell lipid deposition,Western blotting and RT-q PCR detect the protein and m RNA changes of HIF-1α,YAP,ACTA2,Collagen Ⅰ,Collagen Ⅲ,IL-1β,TIMP1 expression.3.si RNA experiment: PA cultivate Hep G2 cells,HIF-1? knocked down using si RNA to determine the role of HIF-1? in high-fat microenvironment induced YAP/IL-1? expression.4.Co-IP experiment: PA cultivate Hep G2 cells,respectively using HIF-1?and YAP as IP to precipitate HIF-1α and YAP,determining the interaction of HIF-1?with YAP.Results1.HFD mice the quality of body were significantly increased in the 1,2,3months compared with the control group,the liver weight is the significant increase in3 months(P ? 0.05),it is manifested as higher levels of FFA and LDL-C(P ? 0.05).2.In the 2,3 months of feeding mice with HFD,the liver showed obvious steatosis compared with the control group,the accumulation of lipid droplets in hepatocyte.IHC showed that Collagen III was deposited in the periphery of hepatocytes in a grid pattern.3.In the 1,2,3 months of feeding mice with HFD,the m RNA level of Yap,Il1β,Timp1,Col1a1 and Col3a1 increased,the protein expression of YAP,COL1A1,COL3A1,MMP2 increased compared with the control group.The m RNA and protein expression level of HIF-1α increased in the 1,2 months,and decreased significantly in the 3 months(P ? 0.05),but Act?2 gene and protein expression is not have statistical significance.4.Hep G2 and Hepa 1-6 cells were induced by PA for 24 h,which significantly promoted the accumulation of lipid in the cells by a dose-dependent manner(P ? 0.05).The m RNA expression level of HIF1α,YAP,TIMP1,IL1β,COL1A1 and COL3A1 were increased in Hep G2 cells.As well as HIF-1α,YAP,COL1A1,COL3A1 have higher protein expression,the expressions of Hepa 1-6 consistent with Hep G2(P ? 0.05).Immunofluorescence staining showed that HIF-1α/YAP was colocalized in the nucleus of Hep G2.5.The expression of YAP protein decreased when HIF-1α was knocked down by si RNA under PA induction.Co-IP suggested that HIF-1α could bind to YAP under PA induction.Conclusions1.HFD induce NAFL fibrosis and that is unrelated to the activation of HSCs,but come from liver cells;HFD induce up-regulated expression of HIF-1α,YAP and IL-1β in the liver.NAFL have a high fatty acid microenvironment in the liver.2.A high-fat microenvironment induce the fibrosis of the liver cells and increase the expression of HIF-1α,YAP,IL-1?.3.The interaction between HIF-1α and YAP was confirmed,and HIF-1αregulate the expression of YAP.