摘要
目的:白藜芦醇对高糖作用下肾小管上皮细胞(HK-2)氧化应激介导的凋亡影响及作用机制。方法:体外培养肾小管上皮细胞,高糖作用于肾小管上皮细胞诱导其凋亡,设置空白对照组、模型组(30mmol·L^-1高糖处理)、不同浓度(5、10μmol·L^-1)白藜芦醇组,LY294002(PI3K抑制剂)组(10μmol·L^-1)。使用MTT法检测各组细胞活力;流式细胞术检测各组细胞凋亡率;JC-1检测线粒体膜电位(MMP);DHE荧光探针检测ROS的变化;特定试剂盒检测乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH-PX)含量;Western-blot法检测各组细胞色素C(CYT-C)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达水平以及Akt的磷酸化情况。结果:与空白对照组相比高糖损伤组能够显著降低肾小管上皮细胞的活力,增加细胞中ROS的含量和凋亡率,LDH(细胞外)、MDA含量显著增加,SOD、GSH-PX活性显著下降,同时降低线粒体膜电位,增加了CYT-C、Caspase-3的表达,而p-Akt/Akt表达降低(P<0.01)。不同浓度白藜芦醇(起效剂量5μmol·L^-1,高剂量10μmol·L^-1)和高糖同时作用肾小管上皮细胞时,细胞存活率逐渐上升,ROS含量及凋亡率逐渐下降,LDH、MDA含量逐渐下降,SOD、GSH-PX活性随之上升,线粒体膜电位提升,CYT-C、Caspase-3表达显著下降,p-Akt/Akt表达增加(P<0.01)。LY294002(PI3K抑制剂)组上述指标的检测结果与白藜芦醇组相反。结论:白藜芦醇可能部分通过激活PI3K/Akt通路抑制氧化应激及其诱导的凋亡,从而对肾小管上皮细胞发挥保护作用。
Objective: To investigate the effect of resveratrol on oxidative stress-mediated apoptosis in renal tubular epithelial cells (HK-2 )under high glucose action and its mechanism of action. Methods:HK-2 was cultured in vitro.High glucose acted on HK-2 to induce apoptosis mediated by oxidative stress.The cell viability of each group was detected by MTT assay .The apoptosis rate of cells in each group was detected by flow cytometry.The MMP of each group was detected by JC-1 .The ROS of each group was detected by DHE.The LDH,MDA,SOD and GSH-PX of each group was detected by specific kit.Western blot was used to detect the expression of protein (CYT-C,Caspase-3 and p-Akt/Akt )in each group. Results:Resveratrol significantly enhanced HK-2 viability and increased the activities of antioxidant enzymes (superoxide dismutase,catalase ),scavenged reactive oxygen species (ROS ),raised MMP.Resveratrol also reduced the rate of apoptosis of cells.Resveratrol downregulated oxidative stress (CYT-C )and its mediated apoptosis pathway related protein (Caspase-3 )expression.Resveratrol also activated phosphorylation of Akt,whereas the addition of LY294002,a pharmacological inhibitor of PI3K,blocked resveratrol-induced downregulation of protein (CYT-C,Caspase-3 )expression and cytoprotection. Conclusion:Resveratrol can prevent apoptosis of HK-2 through inhibiting apoptosis mediated by oxidative stress,which may protect vascular endothelial cells and cardiovascular system.The main mechanism responsible for the inhibition of oxidative stress is the activation of the PI3K-Akt pathway.
作者
杨莎莎
周利
YANG Sha-sha;ZHOU Li(Department of Nephrology,Taihe Hospital Affiliated to Hubei Medical College,Shiyan 442000,China)
出处
《中国现代中药》
CAS
2019年第7期913-919,共7页
Modern Chinese Medicine
基金
湖北省教育厅科学基金(B2013117)
