扶正化瘀方含药血清对肝星状细胞activinA/smad信号通路活化的影响

Study on the mechanism of Fuzheng Huayu prescription drug-containing serum affecting the activation of activinA/smad signaling pathway in hepatic stellate cells

目的探讨扶正化瘀方含药血清对肝星状细胞(HSC)激活素A(activinA)/smad信号通路活化的影响。方法20只SD大鼠按照随机数字表法分为对照组及扶正化瘀(Fzhy)-低、中、高剂量含药血清组,每组5只,分别以蒸馏水,0.75、1.5、3.0 g/kg扶正化瘀溶液(扶正化瘀胶囊药物粉末与蒸馏水配制)灌胃1次/d,连续3 d,取血制备空白血清(对照组)和含药血清。以大鼠HSC-T6细胞为研究对象,分别用5%、10%、20%体积分数的各组含药血清培养细胞。CCK-8检测细胞存活率,选取细胞存活率在50%左右的体积分数重新分为对照组及Fzhy-低、中、高剂量组。流式细胞术检测细胞周期及凋亡率、线粒体膜电位变化和活性氧(ROS)水平;实时荧光定量聚合酶链反应检测细胞中activinA、smad3、samd7、核因子(NF)-κB的mRNA水平;蛋白质印迹法检测细胞中activinⅡA受体(ActRⅡA)、smad3、NF-κB p65、胱天蛋白酶(caspase)-3的蛋白水平。结果各扶正化瘀含药血清组的细胞存活率均低于对照组(P<0.05),选择体积分数为10%的含药血清进行后续实验。对照组和各扶正化瘀方含药血清组细胞周期和凋亡率差异均无统计学意义。对照组及Fzhy-低、中、高剂量组细胞内ROS水平及线粒体膜电位水平降低比例逐次升高(P<0.05)。与对照组比较,Fzhy-中、高剂量组smad3 mRNA表达水平降低,smad7 mRNA升高,Fzhy-低、中、高剂量组NF-κB、activinA mRNA,smad3、NF-κB p65、ActRⅡA蛋白表达水平降低,Fzhy-低剂量组、中剂量组caspase-3蛋白表达水平升高(P<0.05);与Fzhy-低剂量组相比,Fzhy-中、高剂量组smad3 mRNA表达水平降低,Fzhy-中剂量组activinA及smad7 mRNA升高(P<0.05)。结论扶正化瘀方含药血清可通过影响HSC-T6细胞增殖、参与细胞的氧化应激以及调节细胞activinA/smad信号转导通路来实现抗纤维化作用。

Objective To investigate the effect of Fuzheng Huayu(Fzhy)Formula drug-containing serum on the activation of activinA/smad signaling pathway in hepatic stellate cells(HSC).Methods Twenty SD rats were divided into the control group and the Fzhy-low,medium and high dose drug-containing serum group according to the random number table method,5 rats in each group.Rats were gavaged with distilled water,0.75,1.5 and 3.0 g/kg Fzhy solution(drug powder of Fzhy capsule prepared with distilled water)once/d for 3 days,and blood samples were collected to prepare blank serum(the control group)and the drug-containing serum.HSC-T6 cells were cultured with 5%,10%and 20%of the drugcontaining serum in different volume fractions,respectively.Flow cytometry was used to detect cell cycle and apoptosis rate,mitochondrial membrane potential changes and reactive oxygen species(ROS)levels.Real-time fluorescence quantitative polymerase chain reaction was used to detect mRNA levels of activinA,smad3,samd7 and nuclear factor(NF)-κB in cells.Protein blotting was used to detect activinⅡA receptor(ActRⅡA),smad3,NF-κB p65,and cysteine aspartate protease(caspase)-3 protein levels.Results The cell survival rate of each Fzhy-containing serum group was lower than that of the control group(P<0.05),and the drug-containing serum with a volume fraction of 10%was selected for subsequent experiments.There were no significant differences in cell cycle and apoptosis rates between the control group and each Fzhycontaining serum group.The proportion of intracellular ROS level and mitochondrial membrane potential level reduction increased in the control group and the Fzhy-low,medium and high dose groups successively(P<0.05).Compared with the control group,smad3 mRNA expression level was decreased,and smad7 mRNA was increased in the Fzhy-medium and high dose groups.NF-κB and activinA mRNA,smad3,NF-κB p65 and ActRⅡA protein expression levels were decreased in the Fzhy-low and medium and high dose groups,and caspase-3 protein expression levels in the Fzhy-low and medium dose groups were increased(P<0.05).Compared with the Fzhy-low dose group,smad3 mRNA expression levels were decreased in the Fzhy-medium and high dose groups,and activinA and smad7 mRNA were increased in the Fzhy-medium dose group(P<0.05).Conclusion Fzhy containing serum can achieve antifibrotic effects by affecting the proliferation of HSC-T6 cells,participating in cellular oxidative stress,and regulating cellular activinA/smad signaling pathways.