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研究天然抗氧化剂右旋硫辛酸对人肝癌HepG2细胞生长、增殖的影响及其相关机制。采用MTT检测细胞增殖情况,活性氧试剂盒分析细胞内ROS水平。流式细胞术和Hoechst 33258染色观察细胞凋亡和形态变化。Western blot检测凋亡、自噬以及相关通路蛋白表达,包括Bax、Bcl-2、caspase 3、PARP、ATG5、ATG7、LC3、Beclin1、mTOR、P70S6K、P38、P53、ERK、Akt、MEK等。结果表明,经不同浓度不同时间药物处理,右旋硫辛酸可抑制HepG2细胞增殖,提高细胞内ROS水平,并呈时间和剂量依赖性。右旋硫辛酸通过上调促凋亡蛋白Bax,激活caspase家族,从而激活凋亡效应蛋白caspase 3和PARP,同时右旋硫辛酸还可上调自噬相关蛋白ATG5、ATG7、Beclin1、LC3水平,抑制磷酸化mTOR和P70S6K,激活自噬。通路研究表明:右旋硫辛酸可上调磷酸化的P38和JNK促凋亡通路促进凋亡,抑制磷酸化Akt、ERK的表达。添加自噬抑制剂3-甲基腺嘌呤后,明显抑制自噬发生。因此,右旋硫辛酸可能通过调控P38/AMPK-JNK,PI3K/Akt和Ras/Raf/MEK/ERK途径激活自噬,诱导细胞凋亡。
To study the effect and mechanism of natural anti-oxidant dextran on human hepatoma HepG2 cell growth and proliferation. MTT assay was used to detect cell proliferation, and ROS assay was used to analyze intracellular ROS levels. Flow cytometry and Hoechst 33258 staining were used to observe apoptosis and morphological changes. Western blot was used to detect the expression of apoptosis, autophagy and related pathways, including Bax, Bcl-2, caspase 3, PARP, ATG5, ATG7, LC3, Beclin1, mTOR, P70S6K, P38, P53, ERK, Akt, MEK. The results showed that D-lipoic acid could inhibit the proliferation of HepG2 cells and increase the intracellular ROS levels in a time-and dose-dependent manner. D-lipoic acid can activate the caspase 3 and PARP by up-regulating the pro-apoptotic protein Bax and activate the apoptotic effector proteins caspase 3 and PARP. At the same time, D-lipoic acid can up-regulate autophagy-related proteins ATG5, ATG7, Beclin1 and LC3, Activation of autophagy by mTOR and P70S6K. Pathway studies show that: D-lipoic acid can up-regulate the phosphorylation of P38 and JNK pro-apoptotic pathway to promote apoptosis and inhibit phosphorylation of Akt, ERK expression. After addition of autophagy inhibitor 3-methyladenine, autophagy was significantly inhibited. Therefore, D-lipoic acid may induce apoptosis by activating autophagy through the regulation of P38 / AMPK-JNK, PI3K / Akt and Ras / Raf / MEK / ERK pathways.