Effects Of Ligustrazine On Activation Of Hepatic Stellate Cells Induced By Angiotensin ?

Objective:Hepatic fibrosis is a tissue repair process secondary to a variety of liver injuries and also a common pathogenesis for progressing to cirrhosis.In the injured liver,HSC activation and proliferation are closely associated with liver fibrogenesis.HSCs are critical target cell for prevention and management of chronic liver disease.Intrahepatic Ang ? was positively correlated with the degree of liver fibrosis.It can activate HSC via various pathways and make it difficult to reverse liver fibrosis.Many studies have demonstrated that the natural product ligustrazine has significant anti-oxidative,anti-inflammatory and anti-fibrotic properties both in vivo and in vitro.However,the underlying molecular mechanisms are poorly comprehended.We used Ang ? as an inducer of activation in HSC and aimed at investigating the following:1)Ang? effects on HSC proliferation and activation,and ligustrazine protection against injury in HSC;2)explore the molecular mechanism for ligustrazine affects PDGF-?R/MAPK and PI3K/AKT/mTOR signal transduction.These studies may provide effective approaches for prevention and treatment of liver fibrosis.Methods:This study mainly contains in vitro experiments using HSC,which is tightly linked to the pathogenesis of hepatic fibrosis.Isolation and culture of primary HSCs and assays of MTT,Hoechst staining,Immunofluorescence,Wound-healing assay,Transwell,flow cytometry,Western blot and Real-time PCR were used to determine:1)Ang ? effects on HSC proliferation,morphology,migration and expression of HSC activation marker ?-SMA,collagen ?1(?)and fibronectin;2)ligustrazine effects on HSC proliferation,a movemention,cell cycle,poptosis and protein and mRNA expressions of a-SMA,collagen ?1(?)and Fibronectin in HSC induced by Ang ?;3)ligustrazine effects on expression of key effectors in PDGF-?R and mTOR pathway.Results:The results demonstrated that Ang ? at 1?M can significantly induced HSC activation via promoting HSC proliferation and migration,changing HSC morphology,and upregulating protein expression of ?-SMA,collagen ?1(?)and fibronectin.Ligustrazine could remarkably inhibit Ang ?-induced HSC activation.Ligustrazine at 10?M could inhibite HSC proliferation(p<0.05),and at 50?M significantly inhibited HSC proliferation(p<0.01).And it also downregulating protein and mRNA expression of a-SMA,collagen ?1(?)and fibronectin.Ligustrazine at 20?M could significantly stimulate the expression of PPARy and reduce protein expression of TGF-?R?,TGF-?R?,NF-?B.Ligustrazine could arrest HSC in G2 phase and inhibited HSC proliferation via stimulating the expression of CDK2?Cyclin Aand reducing protein expression of CDK1?Cyclin B1.Ligustrazine could remarkably inhibit Ang ?-induced HSC migration and invasion and downregulat protein expression of p-FAK.Apoptosis analysis showed that HSC apoptosis could be induced by ligustrazine at 20?M(p<0.05)and significant effect was observed at 50?M(p<0.01).TMP have a pro-apoptotic effect by increasing the expression of c-caspase-3 and reducing the ratio of Bcl-2/Bax.Our mechanistic studies showed that ligustrazine's antifibrotic effects were mainly devoted to the inhibition of PDGF-?R and mTOR signaling and blockade of phosphorylation of ERK,p38 and JNK,and reduced the expression of phosphorylation of PI3K and AKT.Further use of U0126,Rapamycin and Imatinib,the specific inhibitors for ERK,mTOR and PDGF-?R respectively,demonstrate that PDGF-?R and mTOR signal transduction have interference.The results also suggested ligustrazine have synergistic effect with U0126 or Rapamycin or Imatinib thereby inhibiting protein expression of ?-SMA?collagen ?1(?)and Fibronectin,and inhibite HSC proliferation.Conclusion:Ang ? was a potent stimulator for HSC activation via increasing expression of a-SMA and collagen ?1(?)and Fibronectin at protein and mRNA levels.Ligustrazine could significantly reduced Ang ?-indcued HSC activation via inhibiting HSC proliferation?migration?cell cycle and promoting apoptosis and decreasing expression of key proteins relevant to HSC activation.These effects were mainly mediated by PDGF-?R and PI3K/AKT/mTOR signal transduction.And these two pathways have interference.