| Objective: Hepatic fibrosis,a common pathological process resulted from various chronic hepatic injuries,is characterized by the excessive accumulation of extracellular matrix(ECM).Many studies have shown that the activation of hepatic stellate cells(HSCs)is a major cellular source of intrahepatic extracellular matrix,and plays a key role in the development of hepatic fibrosis.Therefore,inhibiting the activation of HSCs and reducing the synthesis of extracellular matrix are the important strategy for the treatment of liver fibrosis.The present study has isolated a triterpene from Helicteres angustifolia,and identified it as methyl helicterate(MH).Based on our previous works,this study will investigate the effect of MH on HSCs activation and its mechanism against hepatic fibrosis through targeting the TGF-?1/Smads signalling pathway.Method: Rat hepatic stellate cells(HSC-T6),human hepatic stellate cells(LX2)and human liver cells(HL-7702)were used in this study.Hepatic stellate cells including HSC-T6 and LX2 were divided into six groups: the normal control group,the TGF-?1-stimulation group,the low-,medium-and high-dosages of MH(12.5,25 and 50 ?g/mL)treatment groups,and the positive control group(LY364947 group).HL-7702 cells were divided into four groups including the normal control group,the low-,medium-and high-dosages of MH(12.5,25 and 50 ?g/mL)treatment groups.To assess the cytotoxic effect of MH,cells were treated with various concentrations of MH and then the proliferation of HSCs was detected by using MTT colorimetric assay.Additionally,cells were pre-treated with TGF-?1 for 30 min and incubated with 12.5,25 or 50 ?g/mL MH for further 24 h.Sequently,the lactate dehydrogenase(LDH)level was detected by using commercially-available kit;the cell migration was observed by Wound healing assay;and the cell apoptosis and cycle were evaluated by AO-EB staining and flow cytometry,respectively.Moreover,the expressions of T?RI,Col-I and Col-III were observed by immunohistochemical staining;the mRNA expressions of T?RI,T?RII,Smad2,Smad3,Smad7 and PAI-1 were determined by RT-qPCR;and the protein expressions of ?-SMA,Col-I,Col-III,T?RI,T?RII and Smad2/3 were detected by Western blot.Result:1.The effects of MH on cell proliferation and lactate dehydrogenase level.Our results showed that MH at the concentration from 6.25 to 200 ?g/mL significantly inhibited the proliferation of HSC-T6,LX2 and 7702 cells in a dose-dependent manner(P<0.05 or P<0.01);in constrast,TGF-?1 stimulation obviously increased HSCs proliferation.Meanwhile,MH also inhibited the proliferation of HSCs induced by TGF-?1(P<0.05 or P<0.01).In addition,TGF-?1 had little effect on the lactate dehydrogenase(LDH)level,while treatment with MH(concentration > 12.5 ?g/mL)for 24 h markedly promoted the release of LDH.These results suggest that MH has moderate toxicity to the hepatic stellate cells.2.The effects of MH on cell migration,apoptosis and cell cycleCompared to the normal control group,TGF-?1 markedly accelerated HSC-T6 cells migration.However,TGF-?1 stimulation did not influence the cell morphology,apoptotic rate and cycle.Treatment with MH(12.5,25 and 50 ?g/mL)or LY364947 for 24 h visibly reduced the migration and led to eyeable change of morphology.Moreover,MH notably increased cell apoptosis rate in a dose-dependent manner(P<0.05 or P<0.01).Furthermore,MH treatment induced cell cycles arrest in G2 phase(P<0.05 or P<0.01).These results indicate that MH alleviates hepatic fibrosis maybe throgh inhibiting the activation of HSCs.3.The effects of MH on ?-SMA and collagenImmunohistochemistry results showed that the expressions of Col-I and Col-III in the TGF-?1-stimulated group were higher than those of the normal control group(P<0.01);however,MH treatment led to signicant decrease in the expressions of Col-I and Col-III.Similarly,the Western blot results showed that TGF-?1 promoted the expressions of ?-SMA,Col-I and Col-III,but MH or LY364947 treatment significantly reversed these proteins expression.These results indicate that MH evidently inhibits the synthesis of collagen and reduces the excessive accumulation of ECM,which may be the pharmacological foundation of its anti-hepatic fibrosis.4.The effects of MH on the TGF-?1/Smads signaling pathwayThe RT-qPCR results showed that,compared to the normal control group,the mRNA expressions of T?RI,T?R?,Smad2,Smad3 and PAI-1 in the TGF-?1-stimulated group were significantly increased(P<0.05 or P<0.01);however,these genes levels were notably decreased in the MH and LY364947 treatment groups.In addition,the Western blot results showed that TGF-?1 significantly increased the expressions of T?RI,T?RII and Smad2/3 protein(P<0.05 or P<0.01),interestingly,treatment with MH or LY364947 significantly reversed these proteins expressions induced by TGF-?1(P<0.05 or P<0.01).These results suggest that MH has evident inhibitory effect on hepatic stellate cells maybe due to the inhibition of the TGF-?1/Smads signaling pathway.Conclusion: Our study indicates that MH significantly inhibits HSCs proliferation,promotes HSCs apoptosis and reduces the excessive accumulation of collagen,which may be related to the inhibition of TGF-?1/Smads signaling pathway. |
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