Regulation Of Autophagy On Hedgehog Signaling Pathway In Liver Fibrosis In Mice

All kinds of chronic liver diseases cause liver fibrosis.The activation of HSC is the main cause of liver fibrosis.Autophagy may be involved in the formation of liver fibrosis by promoting the activation of HSC.And the activation of HSC requires the participation of specific signaling pathways.Hedgehog signaling pathway plays an important role in the in hepatic fibrosis;The relationship between autophagy and Hedgehog signaling pathway in HSC is not clear.In the literature,the immunohistochemical analysis of the cancer tissues of the patients with gastric adenocarcinoma showed that the expression of Gli2 increased with the enhancement of autophagy.Therefore,we speculate whether autophagy can promote the formation of hepatic fibrosis through regulating the expression of Hedgehog signaling pathway.Through the solution of this scientific problem,we can further understand the mystery of HSC activation,and provide new ideas for the treatment of liver fibrosis.ObjectiveInvestigating the dynamic expression of autophagy in liver fibrosis induced by CCl₄ in mice.futhermore;Exploring the effect of autophagy on Hedgehog signaling pathway in primary M-HSC.Methods1、32 male C57BL/6J mice were randomly divided into blank group,CCl₄ 2w group,CCl₄ 3w group and CCl₄ 4w group.The blank group was intraperitoneally injected with olive oil according to the dose of 4ml/kg.In CCl₄ group,the mixture of CCl₄ and olive oil（1:3 ratio）was intraperitoneally injected at the dose of 4ml/kg.2times/week,replicating the model of liver fibrosis in mice;after 2 weeks,3 weeks and 4weeks,respectively extracted from the liver and serum of mice.liver index,ALT and AST were detected;HE,Masson and Sirius red staining to observe the pathological changes of hepatic fibrosis in mice;Western-blot detect the expression of LC3、p62.And the image analysis is carried out by Image J software.2、By density gradient centrifugation method to extract the primary M-HSC were observed,identified the purity activity.Second days after the cells were observed,identified the lipid droplets by the phase contrast microscope and oil red O staining.In the second and seventh days after extraction,the primary M-HSC was identified byα-SMA and vimentin by immunofluorescence.Adding TGF-β1 to stimulate M-HSC for0h、6h、12h、24h,the expression ofα-SMA、LC3、p62 were detected by Western-blot;and in the seventh day,the cells were respectively divided into four groups:The blank group,TGF-β1 group,TGF-β1+rapamycin group,TGF-β1+3-MA group,the expression of Gli1 and Gli2 in the Hedgehog signaling pathway were detected by Western-blot、immunofluorescence and PT-PCR.Results1、the liver specimens showed that the liver in the blank group was soft and smooth.After intraperitoneal injection of CCl₄ mixture,the surface of the liver was rough,the edge became sharp,and there was a clear sense of particle in the CCl₄4w.Compared with the blank group,CCl₄ groups were increased of liver index and ALT,AST,And they were increased with the time of intraperitoneal injection of CCl₄.it was statistically significant（p<0.05）;The liver pathological tissue staining showed that there was no abnormal liver in the blank group;a small amount of fatty degeneration and the deposition of collagen fibers in the liver of CCl₄ 2w group;the accumulation of collagen fibers and the formation of the interlobular septum in the liver of CCl₄ 3w group;in CCl₄ 4w week group,the infiltration of inflammatory cells,the increase of septum formation,and the increase of the collagen fibrils in the central and sink areas.Western-blot detection found that there was no autophagy in the liver of the blank group,and with the increase of the time of intraperitoneal injection of CCl₄ mixture,The expression of LC3-II increased,the expression of LC3-I decreased,and the ratio of LC3-II to LC3-I increased gradually,and the ratio of LC3-II to LC3-I was the largest in CCl₄ 4w group,and the ratio was greater than 1.At the same time,the expression of p62 gradually decreased with the increase of model time.Image J software carries out image analysis to get the same results,the difference was statistically significant（p<0.05）.2、Extraction of primary M-HSC,the purity can reach more than 90%,the activity of cells was good.extracted after second days,oil red O staining showed red lipid droplets,small circular transparent granules were observed under phase contrast microscope,immunofluorescence staining showed noα-SMA expression,but had some expression of vimentin;extracted after seventh days,immunofluorescence staining to detect the expression of a large number ofα-SMA and vimentin,and the expression was more than second days,It was proved that the extracted cells were primary M-HSC.TGF-β1 timulation primary M-HSC 0h、6h、12h、24h,the expression of LC3-I decreased,the expression of LC3-II increased gradually,the ratio of LC3-II to LC3-I increased gradually,and the expression level of LC3-II was the most after 24h,and the ratio of LC3-II to LC3-I was the largest,which was more than 1.The expression of p62 decreased with the prolongation of the time of TGF-β1.adding autophagy enhancers and inhibitors,western-blot showed that,compared with the blank group,the expression of Gli1,Gli2 increased in TGF-β1 group;and TGF-β1+rapamycin group increased markedly compared with TGF-β1 group;while TGF-β1+3-MA group inhibited the expression of Gli1,Gli2,and lower than TGF-β1 group and TGF-β1+rapamycin group;at the same time,immunofluorescence and RT-PCR get the same results.Conclusion1.In male mice by CCl₄ and olive oil according to the ratio of 1:3 configuration into mixture solution,you can successfully replicate mice liver fibrosis model.2.This study showed that less autophagy in normal mice liver.In liver fibrosis,autophagy occurs in liver。3.In the inflammatory microenvironment,Autophagy can regulate the expression of Gli1 and Gli2 in the Hedgehog signaling pathway of primary M-HSC,thereby promoting the activation of HSC and the formation of liver fibrosis.