右旋硫辛酸对人肝癌HepG2细胞生长增殖及其相关机制研究

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。

Effect of R-(+)-lipoic acid on growth, proliferation and related mechanism in human HepG2 cells

To study the effect of antioxidants R-(+)-lipoic acid(R-LA)on cells growth, proliferation and related mechanisms in human HepG2 cells lines. MTT was used to measure cells growth and proliferation. Reactive oxygen species(ROS)kit was used to analyze ROS level. Cell apoptosis and cell morphological changes were observed by flow cytometry and Hoechst 33258 test. Protein expression levels of apoptosis, autophagy and related pathway were analyzed through Western blot, including Bax, Bcl-2, caspase 3, PARP, ATG5, ATG7, LC3, Beclin1, mTOR, P70S6K, P38, P53, ERK and Akt etc. Results showed that cell growth and proliferation were inhibited in a dose- and time-dependent manner after being treated by lipoic acid. R-LA could increase ROS production, pro-apoptosis proteins Bax levels, activated caspase family and PARP. Meanwhile, R-LA could up-regulate the levels of autophagy-related proteins including ATG5, ATG7, Beclin1 and LC3, and down-regulate phospho-mTOR and P70S6K levels. Signal pathway results showed that R-LA could up-regulate phospho-P38 and phospho-JNK levels, and decrease phospho-Akt and phospho-ERK. When adding 3-methyladenine, autophagy was inhibited. Thus, R-LA might activate autophagy and induce apoptosis by P38/AMPK-JNK, PI3K/AKT and Ras/Raf/MEK/ERK pathways.