Effects Of Ligustrazine On Activation Of Hepatic Stellate Cells Under Oxidative Stress

Objective:Hepatic fibrosis is a tissue repair process secondary to a variety of liver injuries and also a common pathogenesis for progressing to cirrhosis. HSC activation and proliferation are closely associated with liver fibrogenesis. HSCs are critical target cell for prevention and management of chronic liver disease. Oxidative stress can activate HSC via autocrine and paracrine pathways and make it difficult to reverse liver fibrosis. Accumulating in vivo and in vitro studies have demonstrated that the natural product ligustrazine has significant anti-inflammatory, anti-oxidative and anti-fibrotic properties. However, the underlying molecular mechanisms are poorly understood. We herein used H2O2 as an inducer of oxidative injury in HSC and aimed at investigating the following:1) H2O2 effects on HSC proliferation and activation, and ligustrazine protection against oxidative injury in HSC; 2) explore the molecular basis for ligustrazine effects under oxidative stress focusing on the PDGF-βR/MAPK/NF-κB signal transduction. These studies may provide effective approaches for prevention and treatment of hepatic fibrosis.Methods:This study mainly contains in vitro experiments using HSC, which are tightly linked to the pathogenesis of hepatic fibrosis. Isolation and culture of primary HSCs and assays of MTT, Annexin-V/PI double staining. Western blot and Real-time PCR were used to determine:1) H2O2 effects on HSC proliferation, morphology, and expression of HSC activation marker a-SMA, and ECM components al(I)procollagen and fibronectin; 2) ligustrazine effects on HSC proliferation, apoptosis and protein and mRNA expressions of a-SMA, al(I)procollagen and fibronectin in the presence of H2O2; 3) ligustrazine effects on expression of key effectors in PDGF-(3R/MAPK pathway.Results:The results demonstrated that H2O2 at 5μM significantly induced HSC activation via promoting HSC proliferation, changing HSC morphology, and upregulating protein expression of a-SMA, al(I)procollagen and fibronectin. Ligustrazine could remarkably inhibit H2O2-induced HSC activation. It at 10μM could suppress HSC proliferation (p<0.05), and at 50μM significantly inhibited HSC proliferation (p<0.01). Apoptosis analysis showed that apparent HSC apoptosis could be induced by ligustrazine at 25μM (p<0.05) and significant effect was observed at 50μM (p<0.01).Mechanistic studies showed that ligustrazine’s antifibrotic effects were mainly attributed to the inhibition of PDGF-βR signaling and blockade of phosphorylation of ERK and p38, and thereby reduced NF-κB phosphorylation. Specifically, ligustrazine reduced expression of α-SMA, CTGF, and ECM components al(I)procollagen and fibronectin, but increased PPARy expression. These effects might be attributed to reduced NF-κB phosphorylation and TIMP-1 expression, and increased ratio between MMP-2/TMP-1. Further use of U0126 and SB203580, the specific inhibitors for ERK and p38 respectively, suggested that EKR inhibition was the most critical for ligustrazine inhibition of PDGF-βR/MAPK pathway implicated in antifibrosis.Conclusion:H2O2 was a potent stimulator for HSC activation via increasing expression of a-SMA and ECM components al(I)procollagen and fibronectin at protein and mRNA levels. Ligustrazine could significantly reduced H2O2-indcued HSC activation via inhibiting HSC proliferation and decreasing expression of key proteins relevant to HSC activation. These effects were mainly mediated by PDGF-βR/MAPK/NF-KB signal transduction.