The Role Of HMGB1 In Hypoxia-induced Ferroptosis In The Development Of Hepatic Stellate Cell Activation

Background and aims Liver fibrosis is the same pathological feature of most liver disease progression,and the activation,proliferation and secretion of extracellular matrix by hepatic stellate cells(HSCs)(extracellular matrix,ECM)are key factors that exacerbate hepatic fibrosis.Hypoxia is a common feature in the development of liver fibrosis.High-mobility group protein 1(HMGB1)is a highly conserved non-histone chromosomal binding protein and an important extracellular damage-related pattern(DAMP)molecule,a large number of studies have shown that HMGB1 is involved in the progression of multiple chronic liver diseases.Ferroptosis is an iron-dependent new form of cell death.HMGB1 expression increased in liver fibrosis mice and ferroptosis occurred in fibrosis mice.Studies have confirmed that HMGB1 is mainly secreted by hepatocytes,and conditional knockout HMGB1 in liver fibrosis mice can attenuate the degree of liver fibrosis.So can HMGB1 play a role by inhibiting the ferroptosis of HSC ? There is no report at present.This experiment intends to further study the relationship between HMGB1 in promoting liver fibrosis and ferroptosis of hepatic stellate cells.Method1.In vivo experiments: mice were injected intraperitoneally with CCl4 for hepatic fibrosis modelling,setting normal group,liver fibrosis model group,Adsh HMGB1 group,Adsh NC group.HE staining,MASSON staining,and Sirius scarlet staining to observe the pathological changes of liver tissue and collagen deposition;Western blot detection of liver fibrosis indicators α-SMA,COL1A1 and liver tissue HMGB1,ptgs2,GPX4 and other expression differences Adsorption test(ELISA)to detect the amount of HMGB1 expression in blood;AST and ALT to detect the degree of liver damage;electron microscopy and kits to detect lipid peroxidation products malondialdehyde(MDA),glutathione(GSH),iron content,mitochondria morphological assessment of the occurrence of iron death in liver tissue.2.In vitro experiment: The human hepatic stellate cell line LX-2 and the rat hepatic stellate cell line T6 were taken as the research objects.After hypoxia and rh HMGB1(recombinant human HMGB1,rh HMGB1)treatment of cells,western blot was used to detect hepatic stellate cell activation signs protein;CCK8 to detect HSC proliferation;transwell migration test and cell scratch test to detect HSC migration ability;mitochondrial ROS flow cytometry,electron microscopy and kit detection to assess HSC iron death.Result1.In vivo studies showed that compared with the normal group,the expression of HMGB1 in liver tissue of liver fibrosis model mice was significantly enhanced,the serum HMGB1 content was significantly higher.Electron microscopy showed that the mitochondrial cristae decreased and the peroxidation product MDA increased significantly,antioxidant GSH decreased,iron content increased,ptgs2 expression increased,GPX4 expression decreased.Compared with the Adsh NC group,Adsh HMGB1 group had significantly reduced liver tissue fibrous space and collagen deposition in the manifold area,and ALT and AST were significantly reduced;α-SMA and Collagrn1 expression decreased.2.In vitro studies have shown that rh HMGB1 showed a up-regulation of HSCs activation-related gene α-SMA and Collagrn1.CCK8 results showed that rh HMGB1 promoted the proliferation of HSCs.Transwell migration test resulted show that rh HMGB1 promoted the migration ability of HSCs.Cell scratch experiments confirmed the above results.After hypoxia treatment,α-SMA and COL1A1 expression decreased,MDA content increased,GSH content decreased,iron content increased,western blot showed ptgs2 expression increased,GPX4 expression decreased.After stimulation of rh HMGB1 with different concentration gradients of hypoxic HSC,MDA,GSH,and iron contents all showed negative changes related to the occurrence of iron death.At the same time,the results of CCK8 showed that after rh HMGB1,ferrostatin-1(iron death inhibitor),and liprostatin(iron death inhibitor)stimulate,marker content of ferroptosis-induced by Erastin is descend.Conclusion HMGB1 can promote hepatic fibrosis.Intrahepatic hypoxic microenvironment can induces the ferroptosis of HSCs.HMGB1 can inhibit the hypoxia-ferroptosis of HSCs to promote liver fibrosis and damage liver function.