解剖学报

解剖学报 ›› 2016, Vol. 47 ›› Issue (5): 599-606.doi: 10.16098/j.issn.0529-1356.2016.05.004

• 细胞和分子生物学 • 上一篇    下一篇

人参皂苷 Rg1 通过自噬抑制 Raw 264.7 巨噬细胞凋亡

凌露1 杨萍2 盖盛坤3 刘燃1 陈媛丽2 陆地4* 孙林1*   

  1. 1.昆明医科大学第二附属医院心血管内科，昆明 650101; 2.昆明医科大学基础医学院人体解剖学与组织学胚胎学系，昆明 650500;  3.临汾市人民医院心内科，山西  临汾 041000; 4.昆明医科大学生物医学工程研究中心，昆明 650500
  • 收稿日期:2016-03-22 修回日期:2016-05-30 出版日期:2016-10-06 发布日期:2016-10-06
  • 通讯作者: 陆地,孙林 E-mail:ludi20040609@126.com
  • 基金资助:

    人参皂苷 Rg1在 动脉粥样硬化治疗中的作用及其信号转导;天麻素在心肌缺血再灌注损伤治疗中的 信号调控机制;天麻素在心肌缺血再灌注损伤治疗中的作用及其信号机制研究;人参皂苷Rg1在 心肌缺血再灌注损伤中心肌保护作用机制研究;天麻素抑制小胶质细胞激活抗阿尔茨海默氏病的信号网络研究;人参皂苷 Rg1抑制小胶质细胞激活的信号转导机制研究

Ginsenoside Rg1 inhibits apoptosis by inducing autophagy in Raw 264.7 macrophages

LING Lu1 YANG Ping2 GAI Sheng-kun3 LIU Ran1 CHEN Yuan-li2 LU Di 4* SUN Lin 1*   

  1. 1. Department of Cardiology，the Second Affiliated Hospital of Kunming Medical University，Kunming 650101，China; 2. Department of Anatomy and Histology，Kunming Medical University College of Basic Medicine, Kunming 650500, China;3. Department of Cardiology，Linfen City People’s Hospital， Shanxi Linfen 041000, China; 4. Kunming Medical University Biomedical Engineering Reseach Center, Kunming  650500, China

  • Received:2016-03-22 Revised:2016-05-30 Online:2016-10-06 Published:2016-10-06
  • Contact: LU Di,SUN Lin E-mail:ludi20040609@126.com

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摘要：

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 Raw 264.7 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。

关键词: 人参皂苷 Rg1, 自噬；Raw 264.7 巨噬细胞, 免疫印迹法, 免疫荧光双标记法, Hochest 33342/PI荧光双染, 小鼠

Abstract:

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.

Key words: Ginsenoside Rg1, Autophagy, Raw 264.7 macrophage, Western blotting, Double immunofluorescent labeling, Hochest 33342/PI fluorescent double staining, Mouse