Hypoxia Inducible Factor 1 Alpha Mediates Liver Fibrosis By Directly Driving IL-6 Expression In Hepatic Stellate Cells

BackgroundLiver cirrhosis is an end-stage liver disease characterized by tissue fibrosis.Liver fibrosis occurs when the liver is subjected to persistent damage factors,such as alcohol consumption,viral infection,genetic diseases,fatty liver,and autoimmune hepatitis.When these factors affect the continuous activation of hepatic stellate cells（HSCs）,eventually,cirrhosis can occur.Due to the irreversibility of liver cirrhosis,the focus of current research is still to prevent or reverse the progression of liver fibrosis,but treatment strategies have not been established.In terms of treatment,it is mainly aimed at the causes of liver fibrosis,including taking antiviral drugs,quitting alcohol,and reducing fat.Excessive deposition of extracellular matrix（ECM）produced by activated HSCs during chronic liver injury is the main reason for the development of liver fibrosis.Senescence,apoptosis,or inhibition of autophagy of HSCs can limit the progression of liver fibrosis or promote the regression of liver fibrosis.Therefore,the study of the specific mechanism of HSCs in liver fibrosis is the key to reverse or subside liver fibrosis.In recent years,hypoxia inducible factor 1α（HIF-1α）has gradually come into view as an important transcription factor driving the development of liver fibrosis.Liver fibrosis leads to increased blood flow resistance,together with sinus capillarization and sinus obstruction syndrome,leading to tissue hypoxia.Under hypoxic conditions,HIF-1αis significantly up-regulated.As an active transcription factor of the body in response to hypoxia,HIF-1αcan regulate a variety of genes in response to the decrease of oxygen content in tissues,and plays an important regulatory role in the hypoxia environment caused by liver fibrosis.Studies have shown that many immune cells are involved in the process of liver fibrosis.It has been found that the up-regulation of Th17/Treg balance plays an important role in the development of chronic inflammatory diseases,and the increase of Th17 is an important cause of inflammation and fibrosis.ObjectiveBased on the above background,the objectives of this thesis are as follows:first,whether HSCs up-regulate the expression of HIF-1αin fibrotic liver;Second,up-regulated HIF-1αin HSCs mainly regulates which genes play a role in fibrosis;Third,whether the upregulation of HIF-1αin HSCs under hypoxia will affect the differentiation of na?ve CD4 T cells to Th17 and further promote the development of liver fibrosis.MethodsFirst,Sirius Red staining,q PCR,and Western blot（WB）were used to detect the degree of liver fibrosis and the gene and protein expression levels of collagen I in tissue sections of patients with liver fibrosis.Then immunofluorescence,q PCR and WB were used to detect the co-localization of HIF-1αandα-smooth muscle actin（α-SMA）,as well as their expression at the gene and protein levels.The degree of liver fibrosis,the m RNA and protein expression levels of collagen I,the colocalization of HIF-1αandα-SMA,and the m RNA and protein expression levels of HIF-1αandα-SMA were detected by the same method in the liver fibrosis tissues of mice induced by carbon tetrachloride（CCl₄）.DMOG and YC-1 were used to induce and inhibit the expression of HIF-1αin a mouse model of liver fibrosis separately,and the above indicators were detected by the same method.Second,HIF-1αexpression was induced or inhibited in mouse HSCs with DMOG and YC-1,respectively.q PCR was used to detect the expression of liver fibrosis-related genes in response to up-regulation or down-regulation of HIF-1α.si RNA was used to knock down the target gene in human hepatic stellate cell line LX2 or the blocker or antagonist of the target gene was used to verify the effect of the target gene on liver fibrosis in mice.Immunofluorescence,q PCR and WB were used to detect the co-localization of HIF-1αand target gene in the tissue sections of liver fibrosis patients and mice with liver fibrosis model,as well as the expression of HIF-1αat the gene and protein levels.DMOG and hypoxia or YC-1 and normoxia were used to induce or inhibit the expression of HIF-1αin human and mouse primary HSCs and LX2,respectively.q PCR,WB and Elisa kits were used to detect the expression of the target gene.The lentiviral CRISPR/Cas9 system was used to knock down the HIF1A gene in LX2 cells,and the expression of the target gene was detected.The hypoxia response element（HRE）point mutation vector of the target gene promoter was constructed,and the binding site of HIF-1αin the target gene was identified by dual luciferase reporter gene assay.The results were verified by chromatin immunoprecipitation assay（Ch IP）.Third,na?ve CD4 T cells from healthy volunteers were cultured with cell supernatants from HIF1A^WTLX2 cultured under hypoxic or normoxic conditions for 1week with TGF-βand IL-2 as complementary regulators in addition to CD3/28stimulating beads.Flow cytometry was used to detect the expression of Foxp3 and IL-17A in CD4 T cells,and the ratio of IL-17A/Foxp3 was determined.This experiment was then repeated with HIF1A^KDLX2 supernatant under hypoxic or normoxic conditions to investigate whether HIF-1αaffected the IL-17A/Foxp3 ratio by regulating target genes.Finally,to investigate the role of IL-17A in HSCs and target genes,LX2 were cultured in conditioned medium（CM）containing the supernatant of CD4 T cells cultured in HIF1A^WTLX2 under hypoxic conditions.IL-17A（20ng/ml）was used as a positive control with or without IL-17A inhibitor Secukinumab（20 n M）.ResultsFirst,the gene and protein expression levels of type I collagen were significantly increased in the liver tissues of patients with liver fibrosis compared with normal liver tissues.The co-expression ofα-SMA and HIF-1αwas observed in the same cells,and they were significantly increased at the gene and protein levels.Similar results were observed in the CCl₄-induced liver fibrosis mouse model.DMOG could promote the expression of collagen type I,α-SMA and HIF-1α,while YC-1 could inhibit the expression of collagen type I,α-SMA and HIF-1α.Second,we found that the expression of Rarb,Il6,Cyp26a1,and Acta2 genes was elevated after stabilization of HIF-1αwith DMOG in mouse HSCs,and by dual luciferase reporter we found that only IL-6 expression was directly triggered by HIF-1α.By knocking down IL6 in LX2 with si RNA,we found that the expression ofα-SMA and type I collagen was down-regulated compared to the vector control LX2.Furthermore,inhibition of IL-6 signaling by Tocilizumab or LMT28 in a mouse model of liver fibrosis with or without DMOG treatment showed that the addition of Tocilizumab or LMT28 significantly reduced the protein and m RNA levels ofα-SMA and type I collagen,and alleviated liver fibrosis.Double immunofluorescence staining of HIF-1αand IL-6 in liver fibrosis tissues and normal liver tissues suggested that HIF-1αand IL-6 were co-localized in liver fibrosis tissues.Compared with normal liver tissues,the protein and m RNA levels of IL-6 in liver tissues of patients with liver fibrosis were significantly increased.Similarly,elevated IL-6 was also observed in the liver fibrosis tissues of mice,and HIF-1αand IL-6 were co-localized.Compared with the fibrosis control group,the co-localization area of HIF-1αand IL-6 was increased in the DMOG fibrosis group but decreased in the YC-1 fibrosis group.Promoting the expression of HIF-1αin human and mouse primary HSCs and LX2 increased the expression of IL-6,while inhibiting HIF-1αat the protein level or knocking down HIF1A at the gene level could down-regulate the expression of IL-6.Using dual luciferase reporter gene and Ch IP technology,we found that HIF-1αpromoted IL-6expression by directly binding to the hypoxia response element（HRE）region of the human HSC IL6（-771-CATCACGTTC-760）or mouse HSC Il6（-350-GCGCGTGC-343）promoter.Third,culturing na?ve CD4 T cells with supernatants of LX2 overexpressing HIF-1αpromoted IL-17A expression and increased IL-17A/Foxp3 ratio,which was abolished by blocking the IL-6 receptor with Tocilizumab or by culturing with supernatants from HIF1A^KDLX2,the ratio of IL-17A/Foxp3 was not significantly increased.The expression of IL-17A and IL-17RA（the receptor of IL-17A）is elevated in human fibrotic tissues.In the mouse liver fibrosis model,compared with the fibrotic control group,the expression of IL-17A and IL-17RA in the fibrotic liver of DMOG-treated mice was increased,and the expression of IL-17A and IL-17RA in the fibrotic liver of YC-1 treated mice was decreased.In addition,the up-regulation of IL-17A and IL-17RA was inhibited in both DMOG and control groups by blocking IL-6signaling with LMT28 or neutralizing IL-6R with Tocilizumab.When LX2 cells were cultured with conditioned medium（CM）containing the supernatant of CD4 T cells cultured with HIF1A^WTLX2 cells under hypoxic condition,the secretion of IL-6 by LX2 cells was increased,that is,the supernatant rich in IL-17A could in turn induce the secretion of IL-6 by HSCs,and this process was partially reversed by the IL-17A inhibitor Secukinumab.ConclusionHIF-1αdirectly binds to the HRE sequence on the promoter of IL6 to up-regulate the expression of IL-6 in HSCs to promote liver fibrosis,and can induce the secretion of IL-17A to promote liver fibrosis by promoting the expression of IL-6.The up-regulated IL-17A can continue to promote the secretion of IL-6 in HSCs,and continuously promote the progress of liver fibrosis.Targeting the HIF-1α/IL-6/IL-17A axis have value and application prospects in the treatment of liver fibrosis.