Experimental Study Of The Roles And Mechanisms Of Extracellular Histones In The Development Of Hepatic Fibrosis

Liver fibrosis develops from chronic liver injury caused by various reasons and can progress to liver cirrhosis.Liver fibrosis is the process of self-repair of chronic liver injury and characterized by deposition of extracellular matrix（ECM）.The activation of hepatic stellate cells（HSCs）is considered to be the key factor responsible for liver fibrosis.Serious liver injury can cause cell apoptosis and necrosis.Then extracellular histones are released and aggravate liver inflammation with toll-like receptors 2,4,9/MyD88 signaling pathways.However,it is still not fully understood whether extracellular histones are involved in HSCs activation and liver fibrosis development.Objectives:To investigate the levels of circulating histones in CCl₄-induced mouse liver fibrosis model and the effect of non-anticoagulant heparin on the levels of circulating histones.To explore if extracellular histones induce collagen Ⅰ expression in LX2 cells.To investigate the effect of non-anticoagulant heparin on collagen synthesis in vitro and in vivo.To investigate if TLR/MyD88 signaling pathway is involved in histone-enhanced liver fibrosis in CCl₄–induced mouse liver fibrosis model.Our work has demonstrated a novel mechanism of liver fibrosis and new target for liver fibrosis.Methods:Part one:A mouse model of CCl₄liver fibrosis was established.Western blotting was used to detect the levels of circulating histones.The pathological changes of liver tissue was also detected by HE staining in mice.Part two:Different concentrations of extracellular histones were used to stimulate directly LX2,a human hepatic stellate cell（HSCs）line,or stimulate HL-7702,THP-1,HMEC-1 cell lines,then these supernatants from the cultures were collected and added to LX2 cells.Western blotting was used to detect the expression level of collagen Ⅰ in LX2 cells.Part three:LX2 cells were treated with extracellular histones in the presence of non-anticoagulant heparin.Western blotting was used to detect collagen Ⅰ in LX2 cells.Non-anticoagulant heparin was also used to treat mice for hepatic fibrosis model that induced by CCl4.H&E staining showing the pathological morphology,α-SMA staining showing activated HSCs,and Sirius red staining showing the degree of liver fibrosis in the liver sections from mice were performed.Part four:In vitro,we used TLR2/4 neutralizing antibodies to block these receptors on LX2 cells and HMEC-1 cells,and the changes in collagen Ⅰ expression were detected by Western blotting.In vivo,the CCl₄-induced mouse liver fibrosis model was generated in wild type（C57BL/6J）,TLR2,TLR4 and MyD88 knockout mice,H&E staining,α-SMA staining and Sirius red staining of liver sections were performed.Results:Part one:Plasma histone concentration in the CCl₄group peaked at 8 h,while that in the non-anticoagulant heparin+CCl₄group peaked at 24 h and both then decreased after 24h,but all significantly higher than that in control group and non-anticoagulant heparin alone group and The levels of circulating histones in non-anticoagulant heparin alone group showed no significant difference from control group,indicating that the non-anticoagulant heparin could not increase or reduce the levels of circulating histones.This is consistency with previous publication,indicating non-anticoagulant heparin only binds to circulating histones.Alanine aminotransferase（ALT）in circulation was elevated in CCl₄group but significantly reduced in CCl₄+non-anticoagulant heparin group,indicating non-anticoagulant heparin could reduce the cytotocicity of extracellular histones.HE staining showed that CCl₄-induced liver injury and inflammatory cells infiltration were also reduced in the CCl₄+non-anticoagulant heparin group.Part two:Extracellular histones could directly stimulate LX2 to increase collagen Ⅰ expression.Media from HL-7702,and HMEC-1,but not THP-1 cells incubated with extracellular histones could stimulate collagen Ⅰ production in LX2 cells.Part three:In vitro histone-enhanced collagen Ⅰ production was suppressed by non-anticoagulant heparin that neutralizes extracellular histones.In CCl4-induced mouse liver fibrosis model,fibrogenesis was significantly inhibited by non-anticoagulant heparin as indicated by the reduced levels of collagen Ⅰ/α-SMA staining in liver sections and hydroxyproline in liver tissues.Part four:TLR2/4 neutralizing antibodies to block these receptors on LX2 cells and HMEC-1 cells significantly inhibited histone-enhanced collagen Ⅰ production.In vivo,CCl₄-caused elevation of circulating histones in C57BL/6J wt mice and gene knockout mice were comparable to that in ICR mice treated with CCl₄.H&E staining showed that the degree of liver necrosis of TLR2/4 knockout mice was similar to that of wild-type mice,but the infiltration of inflammatory cells in TLR4 and MyD88knockout mice was significantly less than in other mice.Sirius red staining showed liver fibrosis was significantly reduced in TLR2,TLR4 and MyD88 knockout mice compared to that in wt mice.Conclusions:In the course of CCl₄-induced fibrogenesis,circulating histones significantly elevated.Non-anticoagulant heparin can bind to histones and neutralizing its toxicity to reduce liver injury,but cannot reduce the levels of circulating histones.Extracellular histones can activate HSCs directly or indirectly through activation of other cells in the liver.Non-anticoagulant heparin can reduce histone-enhanced collagen Ⅰ production in LX2 cells,and fibrogenesis in CCl₄-induced mouse liver fibrosis model.Extracellular histones may play a role in promoting fibrosis mainly by activating TLR/MYD88 signaling pathways.