| Objective: HSC activation is a central mechanism in the development of liver fibrosis.Many factors are associated with HSC activation,such as growth factors,inflammatory factors and free radicals.It has been reported that TGF-?1is a critical factor causing HSC activation and ECM accumulation,which leads to fibrosis.Thus,the proliferation and activation of HSCs are considered underlying mechanisms relevant to the development of anti-fibrotic agents.Acanthus ilicifolius is a traditional Chinese medicinal herb that has been used to treat inflammation,hepatitis and malignant tumors.The biologically active substances of Acanthus ilicifolius,4-hydroxy-2(3H)-benzoxazolone(HA),our previous study has been chemically synthesized the chemical construction.Although our previous study showed that HA exhibits potential hepatoprotective activity,its precise mechanisms remain unclear.To understand the underlying mechanism of HA hepatoprotective activity,this study assessed the effect of HA on fibrosis by investigating its precise molecular mechanisms,focusing mainly on the LX2 and T6 HSC lines in vitro.Method: HSCs(HSC-T6 and LX2)were treated with variousconcentrations of HA(0.3125,0.625,1.25,2.5,5,10 and 20 ?g/ml)for 12,24 or48 h.Cell viability was then detected using CCK8 kits.In addition,to observe the effect of HA on cell proliferation,cells were treated with TGF-?1(10 ng/ml)for 30 min followed by various concentrations of HA(10,5,or 2.5 ?g/ml)for 24 h.Cell proliferation was analyzed with a CCK8 kits.HSC-T6 and LX2 cells were divided into five groups,including normal control group,the TGF-?1group(cells were treated with 10 ng/ml TGF-?1),and the HA+ TGF-?1 group(cells were treated with 2.5 ?g/ml HA + 10 ng/ml TGF-?1,5 ?g/ml HA + 10ng/ml TGF-?1 or,10 ?g/ml HA + 10 ng/ml TGF-?1).To detect the effect of HA on cell apoptosis,the cells were stained with acridine orange(AO)and ethidium bromide(EB)and observed under a fluorescence microscope.In addition,the HSC-T6 and LX2 cells were analyzed using an Annexin V-FITC Apoptosis Detection Kit under flow cytometry.The JC-1 Detection Kit was used to detect potential HA-induced injury to the HSCs mitochondrial membrane(MMP),and the cells were analyzed using flow cytometry.The expressions of ?-SMA,and COL-I were observed by immunohistochemical staining.Moreover,the mRNA expressions of ?-SMA,COL-III and COL-I,T?RI,T?RII,Smad2,Smad7,TNF-?,IL-1? and IL-6 in the HSC-T6 and LX2 were determined by RT-qPCR.Apoptosis-related factors such as cytochrome c(Cyt c),Bcl-xl,Bcl-2,BAK,BAX,caspase-3 and caspase-9 were assessed by Western blot.Moreover,the protein expressions of peroxisome proliferator-activated receptor-?(PPAR-?),COL-I,COL-III,matrix metalloproteinases(MMPs)and their inhibitors(TIMPs)were detected by Western blot,as were the TGF-?/Smads and NF-?B pathways related protein.Results:1.The effects of HA on cell proliferation in TGF-?1-stimulated HSC-T6 and LX2 cells.The results showed HA inhibited T6 and LX2 cell proliferation in a distinct dose-and time-dependent manner,and the compound was optimized at concentrations of 2.5,5 and 10 ?g/ml for 24 h.Therefore,we examined the effect of HA on cell proliferation in T6 and LX2 cells stimulated with TGF-?1.HA significantly inhibited the proliferation of HSCs induced by TGF-?1(P<0.05 or P<0.01).2.The effects of HA on ?-SMA and collagen levels in TGF-?1-stimulated HSC-T6 and LX2 cells.As a marker of quiescent HSCs,the PPAR-? expression in T6 and LX2 cells was markedly increased by HA compared with the TGF-?1 group(P<0.05 or P<0.01).Indicating that HA inhibits T6 and LX2 activation.In addition,some proteins and genes involved with HSCs activation were also analyzed in T6 and LX2 cells.The protein and gene expression of ?-SMA,COL-I and COL-III were markedly down-regulated by HA compared with the TGF-?1 group(P<0.05 or P<0.01).3.The effects of HA on cell apoptosis in TGF-?1-stimulated HSC-T6 and LX2 cells.HA induced the apoptosis of HSC-T6 and LX2 cells based on acridine orange and ethidium bromide(AO/EB)fluorescent staining and a flow cytometry assay.MMP changes were analyzed by flow cytometry using a JC-1kit.HA significantly decreased the MMP in the TGF-?1 group,indicating that HA treatment caused HSCs apoptosis by disrupting MMP.In addition,the Cyt c level was analyzed by western blot,and Cyt c release from the mitochondria to the cytoplasm was obvious in the HA-treated groups.Furthermore,we examined apoptosis-related proteins and found that HA up-regulated Bcl-xl and Bcl-2 anddown-regulated BAK,BAX,caspase-3 and caspase-9 more than the TGF-?1group(P<0.05 or P<0.01).4.The effects of HA on MMPs and TIMPs in TGF-?1-stimulated HSC-T6 and LX2 cells.As described above,HA inhibits ?-SMA expression and collagen level in TGF-?1-stimulated T6 and LX2 cells.We examined the effect of HA on MMP-2,MMP-9,MMP-1,MMP-13 and TIMP-1 expression.HA markedly increased the levels of MMP-1 and MMP-13 and decreased the levels of MMP-9,MMP-2 and TIMP-1 compared with the TGF-?1 group,indicating that HA inhibits ECM accumulation by regulating the levels of MMPs and TIMPs(P<0.05 or P<0.01).5.HA affects the TGF-?/Smad signaling pathway in TGF-?1-stimulated HSC-T6 and LX2 cells.Smad2/3,TGF? type I receptor(T?R-I)and TGF? type II receptor(T?R-II)protein expression was significantly increased in the TGF-?1-stimulated T6 and LX2 cells,but Samd7 expression was down-regulated compared to the control group,and these changes were reversed in HA-treated cells compared to the TGF-?1-stimulated group.Consistent with the western blot analysis,RT-PCR showed that HA significantly inhibited Smad2,Smad3,T?R-I and T?R-II mRNA expression,but down-regulated Smad7 mRNA expression in TGF-?1-stimulated HSC-T6 and LX2 cells(P<0.05 or P<0.01).6.HA attenuates NF-?B signaling pathway and inflammation-associated cytokine expressionOur results showed that the protein levels of COX2 and NF-?B were significantly decreased by HA treatment compared with the TGF-?1-stimulated group,and the I?B? level was increased.Furthermore,the results showed that TNF-?,IL-1? and IL-6 mRNA expression was markedly reduced in HA-treatedHSCs compared with the TGF-?1-stimulated group.Conclusion: our study revealed that HA inhibited HSC-T6 and LX2 cell activation and induced HSCs apoptosis.HA promoted ECM degradation by regulating the expression levels of MMPs and TIMPs.Moreover,pro-inflammatory cytokine production was inhibited by HA treatment.The TGF-?1/Smad and NF-?B pathways were inhibited by HA treatment.These findings were the first to identify the underlying anti-liver fibrosis mechanisms of HA.This research provides novel insights into the mechanisms of HA as a potent anti-fibrotic agent. |
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