【摘要】 目的研究活血消癥益气方对体外转化生长因子β1（TGF-β1）诱导的小鼠肝实质细胞AML12凋亡的影响及其可能的机制。方法利用TGF-β1(5 ng·mL^-1)诱导AML12细胞建立凋亡模型（诱导24 h）及氧化应激模型（诱导4 h）,设置空白对照组、模型对照组、不同剂量活血消癥益气方(0.1、0.2、0.5 g·L^-1)组。采用CCK-8法检测药物对细胞活力的影响,Hoechst 33342法及Annexin V-FITC法检测药物对凋亡的影响,DCFH-DA法检测药物对细胞内活性氧含量的影响,蛋白印迹法检测药物对Bcl-2、Bax、Caspase-3及Nrf2蛋白表达量的影响。结果与空白对照组比较,凋亡模型对照组细胞活力下降,凋亡细胞占比及出现核固缩细胞比例等明显增加（P<0.05）,抗凋亡蛋白Bcl-2明显下调,同时Bax及Capase-3等凋亡蛋白明显上调（P<0.05）;且氧化应激模型对照组细胞内活性氧含量明显升高（P<0.05）,细胞内Nrf2表达增加但核转位减少（P<0.05）。与凋亡模型组比较,活血消癥益气方各剂量组可明显恢复细胞活力,减少凋亡细胞占比及核固缩细胞比例（P<0.05）;同时上调Bcl-2表达,下调Bax及Capase-3的表达（P<0.05）。与氧化应激模型组比较,活血消癥益气方各剂量组可明显减少细胞内活性氧的含量（P<0.05）,增加Nrf2蛋白的表达及促进其核转位（P<0.05）。结论活血消癥益气方可逆转TGF-β1诱导的肝细胞凋亡,其机制可能与调控线粒体凋亡途径及Nrf2通路功能有关。更多还原

【Abstract】 Objective To investigate the effect of Huoxue Xiaozheng Yiqi recipe on TGF-β1-induced apoptosis of alpha mouse liver 12（AML12） cells and its possible mechanism. Methods AML12 cells were used to establish apoptotic model by incubating with TGF-β1(5 ng·mL^-1)for 24 h and oxidative stress model by incubating with the same concentration of TGF-β1 for 4 h. Blank control group, model control group and different doses of HXXZYQ(0.1,0.2 and 0.5 g·L^-1)groups were set up. CCK-8 method was used to detect cell viability. Hoechst33342 staining and Annexin V-FITC method were used to detect apoptosis. DCFH-DA method was performed to detect intracellular ROS. The protein expression of Bcl-2,Bax,Caspase-3 and Nrf2 was detected by Western Blot.Results Compared with the blank control group, the cell viability, the proportion of apoptotic cells and the proportion of pyknotic nuclei in the apoptotic model group were significantly increased（P<0.05）. Bcl-2 protein was significantly down-regulated,while Bax and Capase-3 were significantly up-regulated（P<0.05）. Moreover,the content of intracellular ROS was significantly increased in the oxidative stress model group,and the expression of intracellular Nrf2 was increased but nuclear translocation was decreased（P<0.05）. Compared with the apoptotic model group,different doses of Huoxue Xiaozheng Yiqi recipe could significantly restore the cell viability,reduce the proportion of apoptotic cells and pyknotic nuclei（P<0.05）,and up-regulate the expression of Bcl-2,while down-regulate the expression of Bax and caspase 3（P<0.05）. Compared with the oxidative stress model,different doses of HXXZYQ could significantly reduce the content of intracellular ROS（P<0.05）,increase the expression of Nrf2 protein and promote its nuclear translocation（P<0.05）. Conclusion Huoxue Xiaozheng Yiqi recipe can reversed TGF-β1-induced apoptosis of AML12 cells. The mechanism may be related to the regulation of mitochondrial apoptosis pathway and Nrf2 pathway.更多还原