黄芪甲苷对高糖腹透液诱导的腹膜间皮细胞线粒体损伤的干预机制研究

李正红; 史俊; 赵君谊; 张露; 高坤; 盛梅笑

黄芪甲苷对高糖腹透液诱导的腹膜间皮细胞线粒体损伤的干预机制研究

1.
南京中医药大学附属医院，江苏南京 210029
2.
南京中医药大学第一临床医学院，江苏南京 210023

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出版历程
- 刊出日期: 2019-07-10

Intervention Mechanism of Astragaloside Ⅳ on Mitochondrial Damage Induced by High Glucose Dialysate in Peritoneal Mesothelial Cells

1.
Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, 210029, China
2.
The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, 210023, China

摘要

摘要: 目的研究黄芪甲苷对高糖腹透液诱导的人腹膜间皮细胞（HPMCs）线粒体损伤的影响，并探讨其干预机制。方法 ①人腹膜间皮细胞株（HMrSV5）在RPMI 1640（含1%FCS）培养液中培养24 h后分为6组：空白组：正常培养液2 mL；高糖腹透液组：含4.25% PDS 1 mL+正常培养液1 mL；低剂量黄芪甲苷组：黄芪甲苷剂量终浓度10 μmol/mL；中剂量黄芪甲苷组：黄芪甲苷剂量终浓度20 μmol/mL；高剂量黄芪甲苷组：黄芪甲苷剂量终浓度40 μmol/mL；阳性药对照组：吡菲尼酮50 μmol/mL。②分别采用CCK-8法测定HMrSV5的增殖活性；流式细胞仪检测细胞活性氧族（ROS）水平变化；罗丹明123荧光染色检测HMrSV5膜电位水平变化；JC-1法测定HMrSV5线粒体膜电位的去极化；Western blot检测HPMCs线粒体形态和功能相关蛋白的表达情况。结果 ①CCK-8检测结果显示，与模型组相比，黄芪甲苷处理组OD值明显升高，细胞存活率也相应增加，呈浓度依赖性，黄芪甲苷对HPMCs有保护作用；②流式检测提示高糖腹透液刺激后线粒体ROS产生增加，而黄芪甲苷可有效降低ROS水平；③JC-1和罗丹明123染色后流式检测发现随着黄芪甲苷浓度的增加，线粒体膜电位的去极化程度随之减少，同时有效增加线粒体膜电位，线粒体膜通透性降低，黄芪甲苷能部分修复高糖腹透液对线粒体的损伤；④Western blot检测结果显示，黄芪甲苷处理的细胞线粒体分裂蛋白DRP1、FIS1的表达水平明显降低，线粒体外膜结合蛋白OPA1、TOM70的表达水平明显升高。结论 ①高糖腹透液能诱导HPMCs线粒体损伤模型；②黄芪甲苷能够通过促进HPMCs线粒体外膜OPA1、TOM70蛋白的表达，降低DRP1、FIS1蛋白表达水平，减少线粒体膜电位去极化，同时有效增加膜电位及降低线粒体膜通透性，进而改善高糖腹透液诱导的HPMCs线粒体氧化应激损伤，保护其结构功能的稳定。
- 黄芪甲苷 /
- 人腹膜间皮细胞 /
- 活性氧族 /
- 线粒体损伤
Abstract: OBJECTIVE To study the effect of Astragaloside Ⅳ on mitochondrial damage of human peritoneal mesothelial cells (HPMCs) induced by high-glucose peritoneal dialysis and explore its intervention mechanism. METHODS ①HPMCs HMrSV5 were cultured in RPMI 1640 (containing 1% FCS) medium for 24 hours and divided into six groups: blank group: normal culture medium 2 mL; High-glucose peritoneal dialysis group: 4.25% PDS 1 mL + normal culture medium 1 mL; Low-dose Astragaloside Ⅳ group: the final concentration of Astragaloside Ⅳ was 10 μmol/mL; Middle-dose Astragaloside Ⅳ group: The final concentration of Astragaloside Ⅳ was 20 μmol/mL；High-dose Astragaloside Ⅳ group: The final concentration of Astragaloside Ⅳ was 40 μmol/mL； Positive drug control group: pifenidone 50 μmol/mL. ②The proliferative activity of HMrSV5 was measured by CCK-8 method， the level of reactive oxygen species (ROS) was measured by flow cytometry， the level of membrane potential of HMrSV5 was measured by Rhodamine 123 fluorescence staining， the depolarization of mitochondrial membrane potential of HMrSV5 was measured by JC-1 method， and the expression of mitochondrial morphology and function-related proteins of HPMCs was detected by Western blot. RESULTS ①The results of CCK-8 showed that compared with the model group， the OD value and survival rate of HPMCs in Astragaloside Ⅳ treatment group increased significantly， which was concentration-dependent. Astragaloside Ⅳ had protective effect on HPMCs. ②Flow cytometry showed that the mitochondrial ROS production of HPMCs increased after high glucose peritoneal dialysis stimulation， while Astragaloside Ⅳ could effectively reduce the ROS level. ③After JC-1 and Rhodamine 123 staining， flow cytometry showed that with the increase of Astragaloside Ⅳ concentration， the depolarization degree of mitochondrial membrane potential of HPMCs decreased. At the same time， the mitochondrial membrane potential of HPMCs increased effectively， and the permeability of HPMCs mitochondrial membrane decreased. Astragaloside Ⅳ could partly repair the damage of HPMCs mitochondria caused by high glucose peritoneal dialysis. ④Western blot results showed that the expression levels of mitochondrial mitotic proteins DRP1 and FIS1 were significantly decreased， while the expression levels of mitochondrial extracorporeal membrane binding proteins OPA1 and TOM70 were significantly increased. CONCLUSION ①High glucose peritoneal dialysis can induce mitochondrial damage in HPMCs. ②Astragaloside Ⅳ can improve the oxidative stress injury of HPMCs mitochondria induced by high glucose peritoneal dialysate and protect the stability of their structure and function by promoting the expression of OPA1 and TOM70 proteins， reducing the expression levels of DRP1 and FIS1 proteins， reducing the depolarization of mitochondrial membrane potential， effectively increasing the membrane potential and reducing the permeability of mitochondrial membrane.
- Astragaloside Ⅳ /
- human peritoneal mesothelial cells /
- reactive oxygen species /
- mitochondrial damage

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