人参皂苷Rg1通过自噬抑制Raw 264.7巨噬细胞凋亡

目的探讨人参皂苷Rg1在血清剥夺诱导的Raw 264.7巨噬细胞自噬和凋亡中的作用及其机制。方法体外培养小鼠Raw 264.7巨噬细胞随机分为空白对照组、不同时间(12、24、36、48和60h)血清剥夺处理组、人参皂苷Rg1(50μmol/L)+不同时间(24、36和48h)血清剥夺处理组;依据最佳血清剥夺时间进一步分为空白对照组、血清剥夺(36h)处理组、人参皂苷Rg1(50μmol/L)+血清剥夺(36h)处理组、人参皂苷Rg1(50μmol/L)+血清剥夺(36h)+3-甲基腺嘌呤(3-MA)(5mmol/L,1h)处理组。采用Western blotting检测LC3、Atg 5、Beclin 1、cleaved Caspase-3、Bcl-2及Bax蛋白水平的表达变化;采用免疫荧光双标记检测细胞内LC3蛋白水平表达的变化;采用Hoechst 33342/PI荧光双染检测细胞凋亡的变化。结果不同时间(12、24、36、48和60h)血清剥夺诱导Raw264.7巨噬细胞凋亡;与血清剥夺处理组相比,人参皂苷Rg1处理组LC3、Atg 5及Beclin 1蛋白表达水平显著上调;加入3-MA抑制剂后,凋亡细胞较人参皂苷Rg1处理组明显增多,且Bcl-2蛋白表达水平明显下调的同时,cleaved Caspase-3和Bax蛋白表达水平则显著上调。结论人参皂苷Rg1通过促进血清剥夺诱导的Raw 264.7巨噬细胞自噬,发挥抗凋亡的保护作用。

Ginsenoside Rg1 inhibits apoptosis by inducing autophagy in Raw 264.7 macrophages

Objective To investigate the regulation role and mechanism of ginsenoside Rg1 on autophagy and apoptosis in Raw 264.7 macrophages stimulated with serum free. Methods Raw 264.7 macrophages were cultured and treated differently in vitroand randomly divided into the control group, the serum free group (12, 24, 36, 48 and 60 hours) in which ginsenoside Rg1 (50μmol/L) was added with serum free treatment group (24, 36 and 48 hours), pretreatment with autophagy inhibitor 3-methyladennine (3-MA) (5mmol/L) for 1 hour then added ginsenoside Rg1 (50μmol/L) with serum free treatment group and corresponding control group. The protein expressions of microtubule-associated protein 1 light chain 3 (LC3), Atg5, Beclin 1, cleaved Caspase-3, Bcl-2 and Bax were detected by Western blotting respectively. The variation of protein expression level of LC3 was measured by double immunofluorescence labeling. The morphology of cell nucleus was measured by Hochest 33342/PI double fluorescent double staining. Results Different time (12, 24, 36, 48 and 60 hours) of the serum free groups induced autophagy. Compared with different time of the serum free groups, the ginsenoside Rg1 (50μmol/L) was added with serum free group up-regulated the protein expression of LC3, Atg 5 and Beclin 1. Compared with the ginsenoside Rg1 (50μmol/L) group, pretreatment with 3-MA (5mmol/L) inhibited the protein expression of Bcl-2 and up-regulated the protein expression of cleaved Caspase-3 and Bax and the quantity of apoptosis in Raw 264.7 macrophages. Conclusion Ginsenoside Rg1 effectively attenuates serum free-induced apoptosis by inducing the autophagy in Raw 264.7 macrophages.