螺内酯对高糖环境下鼠肾小管上皮细胞衰老机制的干预研究

Effects of spironolactone to senescence of renal tubular epithelial cell in high glucose environment

目的 探讨螺内酯对高糖环境下鼠肾小管上皮细胞衰老机制的干预作用.方法 将DMEM高糖培养基培养的鼠肾小管上皮细胞NRK-52E分为5组：高糖组（25.6 mmol/L,a组）,醛固酮组（10^-5mmol/L,b组）,醛固酮加不同剂量螺内酯干预组：低剂量螺内酯（10^-7 mmol/L,c组）,中剂量螺内酯（10^-6mmol/L,d组）,高剂量螺内酯（10^-5 mmol/L,e组）;去血清同步化24 h,分别给予上述干预后继续培养24、48、72 h,进行细胞衰老β-半乳糖酐酶检测.培养72 h的细胞给予提取RNA及蛋白质,分别进行Klotho、p53、p21、β-肌动蛋白（β-actin）荧光定量PCR的检测及相应的蛋白检测.多组间比较采用方差分析,两两比较采用q检验.结果 （1）72h各组阳性衰老细胞构成比：a组（47.9±4.7）％,b组（49.0±5.2）％,c组（38.2±4.1）％,d组（39.7±3.8）％,e组（42.1±3.9）％;x2=45.850,均P＜0.001,24、48 h不同剂量的螺内酯组（c、d、e组）衰老细胞的构成比均低于a及b组.（2） 72 h各组细胞Klotho mRNA表达结果：与a组相比,b组Klotho mRNA表达明显下降（P＜0.05）,a组1.13±0.09,b组0.85±0.02,c组1.42±0.11,d组1.37±0.08,e组1.14±0.06; F=8.134,P＜0.01）.（3） p53 mRNA表达结果：与a组相比,p53mRNA表达明显升高（a组0.62±0.13,b组0.93±0.15,c组0.45±0.06,d组0.51 ±0.09,e组0.61 ±0.09;F=9.629,P＜0.05）.（4） p21 mRNA表达结果：与a组相比,p21 mRNA表达明显升高（a组0.74 ±0.06,b组1.05±0.05,c组0.42±0.02,d组0.61 ±0.07,e组0.72 ±0.03;F=5.450,P＜0.05）.与b组相比,c、d、e组Klotho mRNA与蛋白表达呈螺内酯剂量依赖性上升（P＜0.05）,p53与p21则呈剂量依赖性下降（P＜0.05）.Klotho与p53、p21蛋白表达之间呈负相关（r=-0.744,r=-0.627,P＜0.05）.结论 醛固酮增多可以加速高糖环境下肾小管上皮细胞衰老,而螺内酯可以干预衰老的发生,干预机制可能通过MR、Klotho、p53、p21信号通路.

Objective To explore the effect of spironolactone to NRK-52E＇s senescence in high glucose culture.Methods NRK-52E ceils was divided into five groups,which were a,b,c,d and e group.Group a：cells were in only high glucose culture （25.6 mmol/L glucose）.Group b was interfered with aldosterone（10^-5 mmol/L）,while the other groups were treated by different dose of spironolactone（group c：low dose with 10^-7 mmol/L spironolactone,group d：medium dose with 10^-6 mmol/L spironolactone,and group e：high dose with 10^-5 mmol/L spironolactone） before aldosterone was added into high glucose culture.After these interventions,cell aging would be tested by β-galactosidase while mRNA and protein expression of Klotho,p53,p21 and β-actin would be detected by qPCR and Western Blot.Results （1） After different exposure as above for 72 hours,the senescent cells proportion of group a,b,c,d and e were （47.9±4.7）%,（49.0±5.2）%,（38.2±4.1）%,（39.7 ± 3.8）%,（42.1 ± 3.9）%,respectively.Comparing the aging cells positive rate of each group in 72 hour,the chi-square was 45.850.The aging cells ratio increased despending the time.Compared with group a and b,the ratio of group c,d and e decreased followed by different doses of spironolactone（P〈0.01）.（2） Compared with group a,the Klotho mRNA expression of group b decreased while the p53 mRNA and p21 mRNA increased obviously （P〈0.05）.The Klotho mRNA levels of each group were 1.13 ± 0.09,0.85 ± 0.02,1.42 ± 0.1 1,1.37 ± 0.08 and 1.14 ± 0.06,respectively （F=8.134,P〈0.01）.The expression of p53 mRNA of each group were 0.62±0.13,0.93±0.15,0.45±0.06,0.51 ±0.09 and 0.61±0.09,respectively （F=9.629,P〈0.05）.As p21 mRNA,they were 0.74±0.06,1.05±0.05,0.42±0.02,0.61 ±0.07,0.72±0.03,respectively （F=5.450,P〈0.05）.（3） Comparison with group b,Klotho of group c,d and e,Whatever mRNA or protein expression,increased at different level,while p53,p21 had the opposite results.Klotho protein expression had the negative correlation with p53 and p21 （r=-0.744,r=-0.627,P〈0.05）.Conclusion Aldosterone may accelerate NRK-52E aging,while spironolaetone can prevent it through MR/Klotho/p53/p21 signal way.