六味地黄丸含药血清减低高糖诱导的肾小管上皮细胞的氧化损伤和凋亡的研究  

Serum containing Liuwei dihuang wan attenuates the proliferation and inflammatory cytokines expression induced by high glucose in cultured rat mesangial cells

作　　者：谭颖颖[1] 屈直[1] 张琪[1] 

机构地区：[1]陕西中医学院,陕西咸阳712046

出　　处：《时珍国医国药》2015年第7期1566-1569,共4页Lishizhen Medicine and Materia Medica Research

基　　金：国家自然科学基金(No.31171101;No.81100175);教育部科学技术研究重点项目(No.212173);陕西省教育厅科学研究计划项目(No.2013JK0819;No.2013JK0763)

摘　　要：目的观察六味地黄丸含药血清对高糖环境下培养的肾小管上皮细胞的保护作用,以及对高糖诱导的线粒体氧化损伤和凋亡的影响。方法制备不同剂量大鼠六味地黄丸含药血清,并培养高糖诱导的肾小管上皮细胞(HK-2),采用四甲基偶氮唑盐法和乳酸脱氢酶释放实验确定药物最适作用浓度。六味地黄丸含药血清处理高糖诱导的HK-2细胞24和48h后,2,7-二氯氢化荧光素二酯染色检测细胞内ROS含量,流式细胞仪测定线粒体膜电位变化,real-time PCR方法检测线粒体DNA的拷贝数,Hoechst 33258染色检测细胞凋亡,以及使用分光光度法检测细裂解液中Caspase-3和Caspase-9酶的活性。结果与正常对照组比较,高糖组在培养的24、48 h内HK-2细胞的ROS含量、线粒体膜电位、mt DNA拷贝数、细胞凋亡率、Caspase-3和Caspase-9酶活性显著上调(P<0.01)。与高糖组比较,10%六味地黄丸含药血清均能逆转上述变化(P<0.05),且抑制作用呈一定的时间依赖关系。结论六味地黄丸可能通过调控高糖环境下肾小管上皮细胞的线粒体ROS生成、改善线粒体膜电位和mt DNA拷贝数、抑制Caspase蛋白的级联激活反应,降低肾小管上皮细胞的氧化损伤和细胞凋亡,从而有利于延缓糖尿病肾病的间质纤维化的进展。Objective To examine the protective effects of serum containing Liuwei dihuang wan on renal tubular epithelial cells in a high glucose environment culture,and the effect of mitochondrial oxidative damage and apoptosis induced by high glucose.Methods Serum containing Liuwei dihuang wan was prepared from rats,and renal tubular epithelial cells（ HK2） were cultured in high glucose medium.The optimal concentration of serum containing Liuwei dihuang wan were determined by MTT assay and lactate dehydrogenase release experiment.After treatment of high glucose induced HK2 cells with serum containing Liuwei dihuang wan for 24 or 48 h,ROS in cell were detected by DCFH- DA staining.The changes of mitochondrial membrane potential were measured by flow cytometry,and mitochondrial DNA was detected by real- time PCR method.Hoechst 33258 staining was used to detect cell apoptosis,and caspase- 3 and caspase- 9 enzyme activity was examined by spectrophotometry.Results Compared with control group,high glucose culture in HK2 cell for 24 or 48 h significantly up- regulated the ROS content,mitochondrial membrane potential,copy number of mt DNA,apoptosis rate,and the activity of caspase- 3 and Caspase- 9（ P〈0.01）.Compared with high glucose group,10% of serum containing Liuwei dihuang wan could reverse these changes（ P〈0.05）,and the inhibition was time dependent.Conclusion Liuwei dihuang wan could reduce the oxidative damage and apoptosis of renal tubular epithelial cells induced by high glucose through regulating the mitochondrial ROS generation,improving the mitochondrial membrane potential and mt DNA copy number,and inhibiting the activation of caspase cascade reaction,which contributes to delaying the progression of diabetic nephropathy fibrosis.

关 键 词：糖尿病肾病 六味地黄丸 氧应激 线粒体 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]