Effect Of Methyl Helicterate On Platelet-Derived Growth Factor-BB-Induced ERK1/2 Signal Pathway Of Hepatic Stellate Cells

Objective: Extracellular signal-regulated kinase(ERK)that is a protein kinase with translocating to the nucleus and activating transcription factor,which are one of the many signaling pathways act as a "bottleneck" and pooling point.It has been shown that ERK signaling pathway can affect the formation of liver fibrosis in many ways.So,gene intervention and drug treatment is a ideal choice for the treatment of hepatic fibrosis.In this study,to explore the molecular mechanism of methyl helicterate(MH)on liver fibrosis,the effects of MH on the expression of Raf and ERK1 / 2 and their phosphorylation level,and the expression of transcription factors c-fos,c-jun,c-myc and Ets-1 were examined.Methods: The hepatic stellate cells(HSC-T6,LX2 and 7702)cells werecultured in vitro in this experiment.With different concentrations of MH on cells,then,the cell toxicity was detected by CCK-8 kit method.Treatment with 10ng/m L PDGF-BB,50 ?M PD98059 and MH on cells,the effects of MH on cell proliferation,contens of LDH,migration,clonogenicity,cell cycle and apoptosis were analyzed by CCK-8 kit method,LDH kit method,wound-healing assay,clonogenicity observation,AO-EB and Annexin ?-FITC/PI staining,respectively.The m RNA expressions of ERK1,ERK2,Raf,c-fos,c-jun,c-myc and Ets-1 were observed by RT-PCR.The protein expressions of ERK1/2,phospho-ERK1/2,Raf and phospho-Raf were analyzed by Western blot.And the expressions of Col-I,Col-?,c-fos,c-jun,c-myc and Ets-1 were assessed by immunohistochemical staining.Results:1 The effects of MH on PDGF-BB induced cell proliferation,LDH activity,migration,clonogenicity,apoptosis and cell cycleThe results showed,compared to the normal control group,PDGF-BB significantly increased hepatic stellate cell proliferation at 24 h.And 12.5 ?g/m L,25 ?g/m L,50 ?g/m L MH group significatnly inhibited cell proliferation in a dose-dependent manner.Compared to the normal control group,PDGF-BB markedly increased the proliferation,migration and clonogenicity.But,PDGF-BB stimulation did not affect the morphology,apoptotic rate and cell cycle.Treatment with PD98059 or MH obviously increased the proliferation,migration and clonogenicity.In addition,MH treatment induced cell cycle arrest in G2 phase,led to apparent change of morphology and obviously increased the cell apoptotic rate.These results suggest that MH can significantly inhibithepatic stellate cells proliferation,migration and clonogenicity and promote its apoptosis.Thus,it was confirmed that the mechanism of MH anti-hepatic fibrosis is associated with the inhibition of hepatic stellate cells growth,which provides experimental basis and theoretical support for the treatment of liver fibrosis.2 The effects of MH on PDGF-BB induced cell collagenThe immunocytochemical staining showed that the protein expression of Col-I and Col-? in PDGF-BB-stimulated group were significantly increased compared to the normal control group.Treatment with PD98059 or MH obviously decreased the content of Col-I and inhibited the synthesis and secretion of Col-?.These results suggest that the mechanism of MH anti-hepatic fibrosis is closely related to MH inhibited the synthesis and deposition of hepatic stellate cell collagen.3 The effect of MH on the expression of ERK1/2 signaling pathwayThe results of RT-PCR showed that PDGF-BB obviously increased the m RNA expression of ERK1/2,Raf.Western blot results revealed that PDGF-BB markedly increased the expression of ERK1/2,p-ERK1/2,Raf,p-Raf protein.Treatment with PD98059 or MH significantly decreased the m RNA expressions of ERK1/2 and Raf.And decreased the protein expression of ERK1/2,Raf and their phosphorylation.These results indicate that MH obviously inhibits the levels of ERK1/2,Raf and their phosphorylation,then,decreases and inhibites ERK pathway activation.4 The effect of MH on the expression of c-fos,c-jun,c-myc,Ets-1Compared to the the normal control,the RT-PCR and immunocytochemical staining results showed that PDGF-BB stimulation obviously increased the m RNA expression of c-fos,c-jun,c-myc,Ets-1 and the percentage of c-fos,c-jun,c-myc,Ets-1 positive staining cells,respectively.Treatment with PD98059 or MH outstandingly decreased the m RNA expression of c-fos,c-jun,c-myc,Ets-1 and the c-fos,c-jun,c-myc,Ets-1 positive staining cells.These data indicate that MH inhibits the m RNA and protein expression of c-fos,c-jun,c-myc,Ets-1 are related to its inhibition ERK pathway activation.Conclusions: MH obviously inhibit the proliferation and LDH attivity of PDGF-BB induced hepatic stellate cells,and the protein expression of Col-I and Col-?,then,induce cell cycle arrest at G2 phase and ptomote cell apoptosis.In addition,MH regulate the levels of Raf,ERK1/2 and its phosphorylation.Moreover,MH inhibit the expression of c-fos,c-jun,c-myc and Ets-1.The above results indicate that MH significantly inhibit the activation of hepatic stellate cells,so,its mechanism may is associated with inhibition of ERK1/2signaling pathway.