Investigating The Inhibitory Effect Of Helenalin On Hepatic Stellate Cells Based On Nf-κb And Pi3k/akt Signaling Pathways

Objective:An ingredient was isolated from Herba Centipedae and identified as Helenalin in our previous study.In this study,the effects of Helenalin on HSCs activation were explored and its underlying mechanism was also investigated based on the NF-κB and PI3K/Akt signaling pathways.Method:Hepatic stellate cells(HSC-T6 and LX-2)and human liver cells(L02)were used in this study.HSC-T6 and LX-2 cells were divided into 6 groups including the normal control group,IL-1β stimulation group(20 ng/mL),positive control group(20 μmol/L LY294002),high-,medium-,and low-dosages of Helenalin-treated groups(2,1 and 0.5 μmol/L Helenalin).L02 cells were divided into 4 groups:the normal control group,high-,medium-,and low-dosages of Helenalin-treated groups(2,1 and 0.5 μmol/L Helenalin).HSCs were pre-treated with IL-1β for 30 min and then incubated with Helenalin for 24h.The following experiments were performed.(1)The effects of Helenalin on HSCs proliferation,migration and clonogenicityMTT assay was performed to assess the effect of Helenalin on HSCs proliferation.The levels of AST,ALT and LDH in the culture supernatant were measured by commercially available kits.Additionally,clonogenicity assay and wound-healing assay were performed to evaluate clonogenicity and cell migration ability,respectively.(2)The effects of Helenalin on inflammatory cytokinesThe levels of IL-6 and TNF-α were assessed by ELISA kits.Moreover,the protein expressions of IL-6,TGF-β1 and TNF-α were detected by Western blot.(3)The effects of Helenalin on HSCs apoptosisCell apoptosis was analyzed using flow cytometry and AO/EB staining assay.The expressions of apoptosis-related protein including Bax,Bcl-2 and Caspase-3 were detected by Western blot.(4)The effects of Helenalin on collagen accumulationTo investigate the effect of Helenalin on collagen accumulation,the protein expressions of a-SMA,Collagen-I,Collagen-III,MMP-9 and TIMP-1 were detected by Western blot.(5)The effect of Helenalin on the NF-κB signaling pathwayThe NF-κB signaling pathway related proteins including TLR4,MyD88,NF-κB p65,IKKa,IκBα,p-p65 and p-IKKa were detected by Western blot.Meanwhile,the mRNA expression of NF-κB p65 was analyzed by RT-PCR.(6)The effect of Helenalin on the PI3K/Akt signaling pathwayThe PI3K/Akt signaling pathway related protein including PI3K,Akt,mTOR,P70S6K,p-PI3K,p-Akt,p-mTOR,p-P70S6K and PTEN were detected by Western blot.Meanwhile,the mRNA expressions of PI3K,Akt,mTOR and P70S6K were analyzed by RT-PCR.(7)The effect of Helenalin on the expression of miR-200a/TLR4After incubated with IL-1β(20 ng/mL)for 24 h,the expression of miR-200a in HSC-T6 was determined by RT-PCR.Subsequently,the miR-200a mimics were transfected into HSC-T6 at different concentrations(20,40,60,80 and 100 μM)by using Lipofectamine 2000.And the highest transfection efficiency was selected for the following experiments.In the following study,the cells were divided into six groups:normal control group,negative control group(NC group,cells were transfected with miR-200a NC),miR-200a up group(cells were transfected with miR-200a mimics),high-,medium-,and low-dosages of Helenalin-treated groups(2,1 and 0.5 μmol/L Helenalin).And then,the TLR4 protein expression in miR-200a over-expressed HSC-T6 cells was detected by Western blot.Result:(1)Helenalin inhibited HSCs proliferation,migration and clonogenicityThe results showed that IL-1β markedly increased HSCs proliferation(P<0.01);however,Helenalin significantly inhibited the proliferation of HSCs in a dose-dependent manner(P<0.01);L02 cell viability was also inhibited by Helenalin at a high dose.Similarly,IL-1β stimulation markedly increased cell colony formation,migration and the levels of AST and ALT(P<0.01);However,treatment with Helenalin significantly decreased colony formation,migration and the levels of AST,ALT and LDH(P<0.05 or P<0.01).Furthermore,treatment with Helenalin did not affect the levels of AST,ALT and LDH in L02 cells.These results suggest that Helenalin can inhibit HSCs activation and proliferation.(2)Helenalin reduced inflammatory cytokinesELISA results showed that IL-1β stimulation led to a significant increase in the levels of IL-6 and TNF-α(P<0.01),while treatment with Helenalin markedly reversed the abnormal changes of these inflammatory cytokines(P<0.01).Moreover,Helenalin significantly reduced the protein expressions of IL-6,TGF-β1 and TNF-α(P<0.01).These results demonstrate that Helenalin can reduce inflammatory cytokines release.(3)Helenalin induced HSCs apoptosisAO/EB staining and flow cytometry results showed that Helenalin significantly promoted HSCs apoptosis in a dose-dependent manner.Furthermore,Helenalin significantly decreased Bcl-2 and Caspase-3 expression,whereas increased Bax and Cleaved-Caspase-3 expression in HSCs(P<0.05 or P<0.01).These results suggest that Helenalin induces HSCs apoptosis likely by regulating the expression of the apoptosis-related proteins.(4)Helenalin alleviated collagen accumulationWestern blot results showed that the protein expressions of a-SMA,Collagen-I and Collagen-III in the IL-1(3 stimulation group were increased(P<0.05 or P<0.01).Interestingly,the expressions of these liver fibrosis biomarkers were significantly decreased after treatment with Helenalin(P<0.05 or P<0.01).Moreover,the results showed that IL-1β stimulation significantly increased MMP-9 expression and decreased TIMP-1 expression compared to the normal control group.Helenalin treatment markedly reversed the abnormal expression of these proteins(P<0.01).These results indicate that Helenalin alleviates collagen accumulation by restoring the balance between MMPs and TIMPs.(5)Helenalin inhibited the NF-κB signaling pathwayWestern blot results showed that the expressions of TLR4 and MyD88,as well as the phosphorylations of NF-κB p65 and IKKα,were markedly increased in the IL-1β stimulation group(P<0.05 or P<0.01).However,Helenalin or PDTC(NF-κB inhibitor)treatment significantly decreased TLR4 and MyD88 expressions and the phosphorylations of NF-κB p65 and IKKα(P<0.01).Similarly,the mRNA expression of NF-κB p65 was decreased after treatment with Helenalin.Our results suggest that Helenalin can inhibit the activation of NF-κB signaling pathway.(6)Helenalin inhibited the PI3K/Akt signaling pathwayIn the present study,we found that the phosphorylations of PI3K,Akt,mTOR and P70S6K were significantly increased and the expression of PTEN was decreased in the IL-1β stimulation group(P<0.05 or P<0.01).However,treatment with Helenalin markedly reversed the abnormal expressions of these proteins induced by IL-1β stimulation nearly to the normal levels(P<0.05 or P<0.01).Similarly,IL-1β stimulation markedly increased the mRNA expressions of PI3K,Akt,mTOR and P70S6K(P<0.05 or P<0.01).Interestingly,treatment with Helenalin or LY294002(PI3K/Akt inhibitor)significantly decreased the mRNA expressions of these genes in activated HSCs(P<0.05 or P<0.01).These results suggest that Helenalin can inhibit the PI3K/Akt signaling pathway,ultimately inhibiting HSC activation.(7)Helenalin inhibited the mRNA expression of miR-200aRT-PCR results showed that the mRNA expression of miR-200a in the IL-1β stimulation group was markedly increased compared with the normal control group(P<0.01).However,treatment with Helenalin significantly decreased miR-200a expression(P<0.01).Moreover,our results showed that miR-200a mimics at a concentration of 100 μM had the highest transfection efficiency,which should be used in the following experiments.Western blot results showed that,compared with the NC group,the TLR4 protein expression was significantly decreased in the miR-200a up group(P<0.01).Interestingly,compared with the miR-200a up group,Helenalin treatment further decreased the protein expression of TLR4(P<0.01).These results demonstrated an inhibitory effect of Helenalin on the NF-κB and PI3K/Akt signaling pathways via up-regulating miR-200a expression.Conclusion:This study demonstrates that Helenalin significantly inhibits HSCs proliferation,migration and clonogenicity,induces HSCs apoptosis,reduces inflammatory cytokines release,restores the balance between MMPs and TIMPs,and promotes ECM degradation.The underlying mechanism may be related to the inhibition of NF-κB and PI3K/Akt signaling pathways via up-regulating miR-200a expression.