Lipoxin A₄ Inhibited The Activation Of HSC-T6 Cells And The Possible Mechanisms

Background and objective:The deposition of extracellular matrix?ECM? is an intermediate process in which various chronic liver diseases progress to cirrhosis.Activated hepatic stellate cells?HSC? are the main source of ECM,and HSC activation is the central link in the formation of liver fibrosis.Lipoxin A₄?LXA₄? and its analog BML-111 have anti-fibrotic effects,but the specific mechanism is still unclear.This study was to investigate the effect of Lipoxin A₄ on HSC cell activation and its mechanism.Methods:1.Experimental group,rat HSC-T6 cells were purchased from Procell Company?Wuhan,China?.First,we set different concentrations of Lipoxin A₄?group 6? to investigate the effects of Lipoxin A₄ on LPS-induced HSC activation.HSC-T6 cells were treated with LPS?10 ng/ml? and different concentrations of Lipoxin A₄?0-400 ng/ml? for 24h.Since the solvent of Lipoxin A₄ is ethanol,in order to eliminate the influence of ethanol,an same amount of ethanol was added to the Control group and LPS group,respectively.Then,we randomly divided rat HSC-T6 cells into the following four groups:?1?.Control group:the cells were treated with PBS?LPS solvent? and 0.2% ethanol?Lipoxin A₄ solvent? and incubated for 24 h.?2?.LPS group:the cells were treated with LPS?10 ng/ml? and 0.2% ethanol and incubated for 24 h.?3?.Lipoxin A₄ group?Lipoxin A₄+LPS?:the cells were treated with Lipoxin A₄?200 ng/ml? 15 min prior to LPS?10 ng/ml? and incubated for 24 h.?4?.Lipoxin A₄ receptor blocker group?BOC-2+Lipoxin A₄+LPS?:the cells were treated with BOC-2?10?mol/L? 15 min prior to Lipoxin A₄?200 ng/ml?,followed by LPS?10 ng/ml? 15 min after Lipoxin A₄,and then incubated for 24 h.2.To investigate Lipoxin A₄ inhibited the activation of HSC-T6 cells and the mechanisms?1?.The levels of ECM were assessed by the hydroxyproline?Hyp?kit.?2?.The protein levels of?-SMA,Collagen I and III,MMP-2,MMP-9,TGF-?1,PDGF A and B,NF-?B P65,phosphorylated NF-?B P65?P-P65? and NF-?B inhibitor??I-?B?? were measured via western blot.?3?.The mRNA levels of MMP-2 and MMP-9 were observed by real-time PCR.?4?.The contents of TGF-?1 and PDGF were assessed by enzyme-linked immunosorbent assay?ELISA? kits.?5?.NF-?B nuclear transfer assay kit was used to assess the activation and translation of NF-?B.Result:1.LPS activated HSC-T6 cells and up-regulated the expression level of?-SMA,and Lipoxin A₄ significantly decreased LPS-induced?-SMA in HSC-T6 cells.2.Lipoxin A₄ inhibited LPS-induced Hyp,and down-regulated LPS-induced Collagen ?,Collagen ?,MMP-2,and MMP-9 in HSC-T6 cells.3.Lipoxin A₄ decreased LPS-induced TGF-?1 and PDGF in HSC-T6 cells.4.Lipoxin A₄ repressed LPS activated NF-?B signaling pathway,causing a reduction of I-?B?degradation,NF-?B phosphorylation,and NF-?B p65transposition in HSC-T6 cells.5.BOC-2,the blocker of Lipoxin A₄ receptor,inhibited all the effects of Lipoxin A4.Conclusion:Lipoxin A₄ inhibits HSC cell activation by reducing the profibrotic cytokines TGF-?1 and PDGF,as well as inhibiting the NF-?B signaling pathway.