柚皮素通过激活凋亡信号抑制肝星状细胞活化

Inhibitory effects of naringenin on the activation of hepatic stellate cells through activating the apoptosis signal

目的研究柚皮素对肝星状细胞活化的体外抑制作用及可能机制。方法以人正常肝细胞LO2为参照,在MTT法筛选药物干预浓度的基础上,考察柚皮素(Western blot实验和台盼蓝染色实验浓度为10、20、40μmol/L,免疫荧光实验浓度为40μmol/L)对人肝星状细胞LX2肝纤维化标志物蛋白[胶原纤维Ⅰ(collagenⅠ)、α-平滑肌肌动蛋白(α-SMA)]及mRNA(collagenⅠ前体α1-pro collagenⅠ、α-SMA)表达、细胞凋亡及凋亡相关蛋白[Bcl-2、Bax、剪切的胱天蛋白酶3(cleaved caspase-3)]表达的影响;选择凋亡抑制剂(Z-VAD-FMK,FMK)、铁死亡通路抑制剂ferrostatin-1、程序性死亡通路抑制剂necrostatin-1对上述作用机制进行验证。结果柚皮素可显著下调LX2细胞中collagenⅠ(柚皮素10μmol/L除外)、α-SMA蛋白及α1-pro collagenⅠ(柚皮素10μmol/L除外)、α-SMA mRNA的表达(P<0.05),可诱导LX2细胞凋亡并提高其凋亡比例,可显著下调Bcl-2蛋白的表达并上调Bax(柚皮素10μmol/L除外)、cleaved caspase-3(柚皮素10μmol/L除外)蛋白的表达;FMK可逆转柚皮素对LX2细胞的上述作用(P<0.05)。结论柚皮素可通过激活肝星状细胞LX2的凋亡信号来抑制其活化,从而发挥对肝纤维化的改善作用。

OBJECTIVE To study the inhibitory effects and possible mechanism of naringenin on the activation of hepatic stellate cells.METHODS Using human hepatocytes LO2 as reference,based on drug intervention concentration screened by MTT assay,the effects of naringenin(Western blot assay and trypan blue staining test in 10,20,40μmol/L,immunofluorescence assay in 40μmol/L)on the expressions of liver fibrosis markers protein(collagenⅠ,α-SMA)and mRNA(α1-pro collagenⅠ,α-SMA)in human hepatic stellate cells LX2,and the expressions of cell apoptosis and apoptosis-related proteins(Bcl-2,Bax,cleaved caspase-3)were investigated.The apoptosis agents(Z-VAD-FMK,FMK),ferroptosis pathway inhibitor ferrostatin-1,and programmed death pathway inhibitor necrostatin-1 were used to verify the mechanism of the above effects.RESULTS The naringenin could significantly down-regulate protein expressions of collagenⅠ(except for naringenin 10μmol/L)andα-SMA,mRNA expressions ofα1-pro collagenⅠ(except for naringenin 10μmol/L)andα-SMA(P<0.05);it also induced LX2 cell apoptosis and increased its apoptotic ratio,down-regulated the protein expression of Bcl-2 while up-regulated the protein expressions of Bax(except for naringenin 10μmol/L)and cleaved caspase-3(except for naringenin 10μmol/L).FMK could reverse above effects of naringenin on LX2 cells(P<0.05).CONCLUSIONS Naringenin can inhibit the activation of hepatic stellate cells LX2 through activating the cell apoptosis signal,which plays ameliorative role in liver fibrosis.