目的 分析枸杞多糖对过氧化氢所致人肾小管上皮细胞(human kidney,HK-2)的损伤情况、细胞内自噬蛋白表达以及细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)通路的激活情况,探讨枸杞多糖改善其氧化损伤的作用机制.方法 体外培养人源性HK-2细胞,根据处理方式不同分为对照组、枸杞多糖组、氧化应激组及干预组.对照组使用细胞培养液;枸杞多糖组使用含有枸杞多糖的细胞培养液;氧化应激组使用含有过氧化氢的细胞培养液;干预组先用枸杞多糖细胞培养液孵育24 h,再滴加过氧化氢孵育30 min,每组处理时间相同.观察每组细胞的生长状态;使用细胞活力检测盒分析各组细胞的存活率;比色法分析胞质所含丙二醛(mal-ondialdehyde,MDA)的量及超氧化物歧化酶(superoxide dismutase,SOD)的活性;蛋白印迹评估胞质内自噬蛋白微管相关蛋白轻链 3(microtubule-associated protein light chain 3,LC3)及 ERK/mTOR 通路相关蛋白 ERK、磷酸化-ERK(phosphoryla-tion ERK,p-ERK)、mTOR和磷酸化-mTOR(phosphorylation mTOR,p-mTOR)的含量.结果 与对照组比较,枸杞多糖组细胞生长状态、存活率、胞质所含MDA含量和SOD活性、自噬蛋白LC3及ERK/mTOR通路相关蛋白ERK、p-ERK、mTOR和p-mTOR的含量比较,差异均无统计学意义(均P＞0.05);氧化应激组细胞皱缩、悬浮较多,存活率[(51.16±4.85)％]下降、MDA[(4.78±0.81)mmol/L]升高、SOD[(10.11±1.17)U/L]降低,胞质内 LC3Ⅱ/LC3Ⅰ[(0.391±0.058)]降低、p-ERK/ERK[(1.526±0.185)]、p-mTOR/mTOR[(1.423±0.174)]升高(均P＜0.05).干预组与氧化应激组相比,细胞状态好转,悬浮少,存活率[(86.29±4.55)％]上升、MDA[(2.22±0.46)mmol/L]下降、SOD[(18.25±2.33)U/L]升高,胞质内 LC3Ⅱ/LC3 Ⅰ[(0.846±0.091)]升高、p-ERK/ERK[(0.906±0.127)]、p-mTOR/m[TOR(0.820±0.092)]降低(均P＜0.05).结论 枸杞多糖能抑制过氧化氢刺激的HK-2内的ERK/mTOR信号通路,增强细胞的自噬作用,提高细胞存活率,改善其氧化损伤.
Effects of lycium barbarum polysaccharides on hydroperoxide induced autophagy and activation of ERK/mTOR pathway in HK-2 cells
Objective To analyze the lycium barbarum polysaccharides on hydroperoxide-induced autophagy of renal tubular epithelial cells(human kidney,HK-2)and the activation of extracellular regulated protein kinases(ERK)/mammalian target of rapamycin(mTOR)pathway in cells,and to explore the mechanism of lycium barbarum polysaccharides in improving oxidative damage.Methods The HK-2 cells were cultured in vitro and arranged into control group,lycium barbarum polysaccharide group,oxidative stress group and treatment group according to different treatment methods.The control group was given cell culture medium.The lycium barbarum polysaccharide group was given cell culture medium containing lycium barbarum polysaccharide.The oxidative stress group was given cell culture medium containing hydrogen peroxide.The treatment group was firstly incubated with lycium barbarum polysaccharide cell culture solution for 24 hours,and then added hydrogen peroxide for 30 minutes.The treatment time of each group was the same.The growth state of cells in each group was observed.The cell viability test box was used to analyze the survival rate of cells in each group.The content of malondialdehyde(MDA)in the cytoplasm and the activity of superoxide dismutase(SOD)were analyzed by colorimetry.The contents of cytoplasmic autophagy protein microtubule-associated protein light chain 3(LC3)and ERK/mTOR pathway related proteins ERK,p hosphorylation ERK(p-ERK),mTOR and phosphorylation mTOR(p-mTOR)were evaluated by Western blot.Results In contrast to control group,the lycium barbarum polysaccharide group had no significant difference in cell growth status,survival rate,MDA content and SOD activity in the cytoplasm,the content of autophagy protein LC3 and ERK/mTOR pathway related proteins ERK,p-ERK,mTOR and p-mTOR(all P＞0.05).In the oxidative stress group,the number of cells shrinkage and suspension was more,the survival rate[(51.16±4.85)％]decreased,MDA[(4.78±0.81)mmol/L]increased,SOD[(10.11±1.17)U/L]decreased,the intracellular LC3Ⅱ/LC3 Ⅰ(0.391±0.058)decreased,p-ERK/ERK(1.526±0.185),and the p-mTOR/mTOR(1.423±0.174)increased(all P＜0.05).In contrast to oxidative stress group,treatment group had better cell status,less suspension,higher survival rate[(86.29±4.55)％],lower MDA[(2.22±0.46)mmol/L],higher SOD[(18.25±2.33)U/L],higher intracellular LC3Ⅱ/LC3 Ⅰ(0.846±0.09±),lower p-ERK/ERK(0.906±0.127),and lower p-mTOR/mTOR(0.820±0.092)(all P＜0.05).Conclusion The lycium barbarum polysaccharide can inhibit the ERK/mTOR signal pathway in HK-2 stimulated by hydrogen peroxide,enhance the autophagy of cells,raise the survival rate of cells and improve their oxidative damage.
Lycium barbarum polysaccharideAutophagyRenal tubular epithelial cellsExtracellular regulated protein kinases ERK/Mammalian target of rapamycin signal pathway
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