迷迭香酸抑制自噬诱导HepG2细胞凋亡的作用研究

Effect of Rosmarinic acid on HepG2 cell apoptosis and autophagy inhibition

目的:观察迷迭香酸(RA)对HepG2细胞凋亡和自噬的作用,探讨自噬与凋亡之间的相互作用。方法:用不同浓度迷迭香酸作用于HepG2细胞24h、48h、72h。MTT法检测细胞存活率,观察迷迭香酸对HepG2细胞核形态的影响,流式细胞术检测细胞凋亡变化,台盼蓝染色法观察细胞自噬泡情况,透射电镜观察HepG2细胞的自噬变化,蛋白质印迹法检测凋亡相关蛋白Cleaved Caspase-3、Bcl-2和自噬相关蛋白(LC3)、p62的表达水平。采用迷迭香酸+3-甲基腺嘌呤(3-MA,自噬抑制剂)或迷迭香酸+雷帕霉素(RAP,自噬诱导剂)分别培养处理HepG2细胞,观察细胞增殖、细胞凋亡情况及相关蛋白表达变化。结果:与对照组相比,迷迭香酸(6.25、12.5、25、50、100、200μmol/L)均能降低HepG2细胞的存活率,诱导HepG2细胞发生凋亡,具有量效和时效关系,减少细胞内自噬泡和自噬小体,降低LC3Ⅱ/LC3Ⅰ的比值,减少Bcl-2的表达,增加Cleaved Caspase-3、p62蛋白的表达。与迷迭香酸25μmol/L处理组相比,迷迭香酸25μmol/L+3-MA 1.0μM/mL应用可诱导HepG2细胞凋亡增加,可进一步减少细胞内自噬泡和自噬小体数目,降低LC3Ⅱ/LC3Ⅰ比值,进一步抑制HepG2细胞的自噬水平,减少Bcl-2的表达,增加Cleaved Caspase-3和p62表达;迷迭香酸25μmol/L+RAP 1.0μM/mL处理HepG2细胞,增加细胞内自噬泡和自噬小体数目,升高LC3Ⅱ/LC3Ⅰ比值,可以抑制和逆转迷迭香酸诱导的细胞自噬水平下降,增加Bcl-2的表达,减少Cleaved Caspase-3和p62表达,细胞凋亡水平下降,这说明抑制或增强自噬时,迷迭香酸能改变对HepG2细胞的凋亡影响作用。结论:迷迭香酸可通过抑制自噬促进HepG2细胞的凋亡。

Objective: To investigate the effect of Rosmarinic acid( RA) on autophagy and apoptosis of HepG2 cells and the interaction between autophagy and apoptosis. Methods: HepG2 cells were cultured in vitro and stimulated with different concentrations of RA for 24 h,48 h and 72 h. Cell proliferation was detected by MTT assay. The effect of RA on cell apoptosis was detected by flow cytometry. Trypan blue staining was used to observe the cell autophagy and changes of autophagy in HepG2 cells were observed by transmission electron microscopy. Then,the expressions of apoptosis-related protein cleaved caspase-3、Bcl-2 and autophagy-related proteins,including microtubule-associated protein1 light chain 3( LC3) and p62,were detected by Western blotting. HepG2 cells were stimulated with RA combined with autophagy inhibitor3-methyladenine( 3-MA) or autophagy inducer Raypamycin( RAP),and then the changes of cell proliferation,cell apoptosis and expressions of the above proteins were analyzed. Results: Compared with the control group,the cell proliferation in the RA groups( 6. 25,12. 5,25,50,100,200μmol/L) were significantly decreased and HepG2 cells were induced to apoptosis. The results showed that RA inhibited HepG2 cells in the concentration dependent manner and the time-dependent manner. Intracellular autophagy vesicles and autophagosomes were reduced and the expression of Bcl-2 and the ratio of LC3 Ⅱ/LC3 Ⅰin HepG2 cells were significantly decreased. The expressions of cleaved caspase-3 and p62 were significantly increased. Compared with RA( 25μmol/L) group,the cell apoptosis in RA( 25μmol/L) combined with3-MA( 1. 0μM/mL) group was significantly increased. Intracellular autophagy vesicles and autophagosomes were decreased and the autophagy was reduced. The expressions of Bcl-2 and the ratio of LC3Ⅱ/LC3Ⅰin HepG2 cells were significantly decreased. The expressions of cleaved caspase-3 and p62 were also significantly increased. On the contrary,the cell apoptosis in RA( 25μmol/L) combined with RAP( 1. 0μM/mL) group were significantly decreased. Intracellular autophagy vesicles and autophagosomes were significantly increased and reduction of autophagy was reversed. The expression of Bcl-2 and the ratio of LC3Ⅱ/LC3Ⅰin HepG2 cells were also clearly increased. The expressions of cleaved caspase-3 and p62 were also significantly decreased. This indicated that when autophagy was inhibited or enhanced,rosmarinic acid had a significant effect on HepG2 cell apoptosis. Conclusion: RA can induce the apoptosis of HepG2 cells by inhibiting autophagy.