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J Zhejiang Univ (Med Sci)  2019, Vol. 48 Issue (6): 617-624    DOI: 10.3785/j.issn.1008-9292.2019.12.05
    
Effects of resveratrol on aging of mesenchymal stem cells and its mechanism
ZHANG Dayong1(),LIN Jiuzhou1,WANG Yayan1,XU Shan1,LUO Chengzhuan1,CAI Jiaye1,JIANG Xuefan2,PAN Jianping1,*()
1. School of Medicine, Zhejiang University City College, Hangzhou 310015, China
2. Department of Otolaryngology, Zhejiang Provincial People's Hospital, Hangzhou 310014, China
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Abstract  

Objective: To investigate the effects of resveratrol (Res) on aging of marrow mesenchymal stem cells (MSCs), and to explore its mechanism. Methods: MSCs were isolated from young SD rats and cultured in vitro. The optimal D-gal concentration for induction of MSCs senescence was determined. Then MSCs were randomly divided into four groups, namely the control group, 10μmol/L, 50μmol/L and 100μmol/L Res groups. After the cells were treated with different concentration of Res for 48 h, the senescence-associated changes were examined with senescence-associated-β-galactosidase (SA-β-gal) staining; the expression of p53, p16 and γ-H2AX was evaluated by Western blot. The total active oxygen species (ROS) level was determined by flow cytometry with DCFH-DA staining. In order to assess the effect of Res on the mitochondrial function, MitoSox Red staining was used to detect mitochondrial ROS levels in each group, mitochondrial membrane potential was detected by JC-1 assay, mPTP method was used to detect mitochondrial membrane channel opening level, and Western blot was used to detect the expression level of cytoplasmic cytochrome C (Cyt-C). Results: D-gal 10 and 50 g/L significantly increased the number of SA-β-gal positive cells and the level of mitochondrial ROS (all P < 0.01). Therefore, 10 g/L D-gal was used to induce the senescence of MSCs in subsequent experiment. Compared with the control group, the number of SA-β-gal positive cells in Res groups significantly decreased (all P < 0.01), the expression of p53, p16 and γ-H2AX decreased, and the total and mitochondrial ROS level also decreased (all P < 0.01). Moreover, mitochondrial membrane potential, open level of mitochondrial membrane channels and the levels of cytoplasm Cyt-C in the Res treatment groups decreased compared with the control group (P < 0.05 or P < 0.01). Conclusion: Resveratrol can protect the mitochondrial function of MSCs, and effectively delay the MSC senescence.



Key wordsResveratrol      Bone marrow cells      Mesenchymal stem cells      Mitochondria      Aging      Cells, cultured     
Received: 30 January 2019      Published: 19 January 2020
CLC:  R329.2+4  
Corresponding Authors: PAN Jianping     E-mail: zhangdy@zucc.edu.cn;jppan@zucc.edu.cn
Cite this article:

ZHANG Dayong,LIN Jiuzhou,WANG Yayan,XU Shan,LUO Chengzhuan,CAI Jiaye,JIANG Xuefan,PAN Jianping. Effects of resveratrol on aging of mesenchymal stem cells and its mechanism. J Zhejiang Univ (Med Sci), 2019, 48(6): 617-624.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2019.12.05     OR     http://www.zjujournals.com/med/Y2019/V48/I6/617


白藜芦醇通过保护线粒体功能延缓骨髓间充质干细胞衰老

目的: 探讨白藜芦醇对骨髓间充质干细胞(MSC)衰老的影响及其机制。方法: 分离SD乳鼠(7日龄)MSC,传代培养至第3代后检测0、1、10、50 g/L D-半乳糖对MSC衰老的影响,确定D-半乳糖诱导MSC衰老的最佳浓度。再将细胞随机分为10、50、100 μmol/L白藜芦醇组及对照组。衰老相关β-半乳糖苷酶(SA-β-gal)染色观察各组细胞衰老变化;DCFH-DA染色检测总活性氧水平;MitoSOX Red染色检测线粒体活性氧水平;线粒体膜电位检测试剂盒(JC-1法)检测线粒体膜电位变化;纯化线粒体膜通道孔(mPTP)比色法检测线粒体膜通道开放水平;蛋白质印迹法检测衰老相关蛋白p53、p16、γ-H2AX表达和细胞质内线粒体细胞色素C外泄水平。结果: 10、50 g/L D-半乳糖可明显增加SA-β-gal染色阳性MSC数量和线粒体活性氧水平(均P < 0.01)。与对照组比较,白藜芦醇组SA-β-gal染色阳性细胞数减少(均P < 0.01),p53、p16和γ-H2AX表达减少,细胞内总活性氧和线粒体活性氧水平下降(均P < 0.01),线粒体膜电位下降(P < 0.05或P < 0.01),膜通道开放水平降低(P < 0.05或P < 0.01),细胞质内细胞色素C外泄减少。结论: 白藜芦醇可保护MSC线粒体功能,延缓MSC衰老。


关键词: 白藜芦醇,  骨髓细胞,  间质干细胞,  线粒体,  衰老,  细胞, 培养的 
Fig 1 Effects of different concentrations of D-gal on MSC senescence
Fig 2 Effects of concentrations and treatment time of resveratrol on MSC senescence (senescence-associated-β-galactosidase staining)
Fig 3 Expression of senescence-related protein p53, p16 and γ-H2AX in MSCs treated by resveratrol of different concentrations
Fig 4 Effect of resveratrol on total and mitochondrial reactive oxygen species levels in MSCs
Fig 5 Changes of mitochondrial membrane potential in MSCs treated by resveratrol of different concentrations (n=5)
Fig 6 Changes of the open level of mitochondrial membrane channels in MSCs treated by resveratrol of different concentrations (n=5)
Fig 7 Changes in mitochondrial cyt C excretion level in MSCs treated by resveratrol of different concentrations
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