NRK-52E缺血再灌注损伤时Kim-1、NO变化及虫草对其的干预作用  

Variation of Kim-1 and NO in ischemia-reperfusion injuried NRK-52E cells and effects of cordyceps sinensis on them

作　　者：洪学敏[1] 周巧玲[1] 唐荣[1] 何楠[1] 刘抗寒[1] 杨敬华[1] 

机构地区：[1]中南大学湘雅医院肾内科,湖南长沙410008

出　　处：《中国现代医学杂志》2011年第31期3866-3871,共6页China Journal of Modern Medicine

摘　　要：目的通过抗霉素A(Antimycin A,AA)刺激大鼠肾小管上皮细胞(NRK-52E)模拟体外缺血再灌注损伤过程,观察缺血再灌注损伤时NRK-52E中肾损伤分子-1(kidney injury molecule-1,Kim-1)及NO表达的变化及冬虫夏草对其的干预作用。方法将体外培养的NRK-52E分为对照组、模型组和虫草组。虫草组予含40 mg/L虫草原液的培基预处理。培养72 h后NRK-52E去除培基,予10-10 mol/L抗霉素A(AA)刺激1 h,模拟体外缺血过程,1 h后再加入培基,模拟体外再灌注损伤过程。分别在AA刺激前,AA刺激后1 h,加入培基再灌注10 min,30 min,60 min,120 min共6个时间点终止实验。用流式细胞仪以Annexin V/PI双染法检测细胞凋亡;分别用硝酸还原酶法和ELISA检测细胞上清液Kim-1与NO浓度;用RT-PCR法观察Kim-1 mRNA表达。结果 NRK-52E细胞经AA刺激后,细胞凋亡率显著增高,同时Kim-1表达上调;缺血60 min较缺血前Kim-1蛋白水平增加了3.1倍,再灌注10 min增加了3.6倍(P<0.05)。Kim-1 mRNA也呈现类似结果。NO浓度随灌注时间延长发生类似变化。与模型组比较,虫草组对缺血再灌注损伤显示出较明显的耐受性,主要表现为在细胞凋亡率降低的同时Kim-1表达及NO浓度下降(P<0.05)。结论抗霉素A能诱导NRK-52E细胞出现以凋亡为主的缺血再灌注损伤状态,伴随Kim-1 mRNA的早期高表达及NO浓度升高;冬虫夏草可通过下调Kim-1的表达及降低NO浓度,抑制细胞凋亡,发挥肾保护作用。【Objective】 To investigate the expression of kidney injury molecule-1（Kim-1） and nitric oxide（NO） in rat renal tubular epithelial cells（NRK-52E） after ischemia-reperfusion injury（IRI） induced by Antimycin A and observe the effects of Cordyceps Sinensis（CS） on it.【Methods】 NRK-52E cells cultured in vitro,were randomly divided into three groups： control,IRI group（model group）,CS group.CS group was pretreated for 72 hours with medium containing 40 mg/L stock solution of cordyceps sinensis,other two groups were cultured by blank medium for the same time.After 72 hours,according to documented ways,NRK-52E cells were removed from the media and incubated with Antimycin A for 1 hour,and then the media were recovered to simulate the IRI in vitro.At six time points in the course（the beginning,ischemia for 60 min,ischemia 60 min /reperfusion 10 min,ischemia 60 min /reperfusion 30 min,ischemia 60 min /reperfusion 60 min,ischemia 60 min /reperfusion 120 min.）,we detected the apoptosis ratio of cells by Annexin V-FITC/PI double staining and flow cytometry,and evaluated Kim-1 expression by ELISA and RT-PCR and NO by nitrate reductase method.【Results】 Using the method of Antimycin A for 1 hour to simulate the ischemia-reperfusion injury of NRK-52E cells,we observed the increasing expression of Kim-1（P 0.05）.The apoptosis ratio of NRK-52E cells significantly elevated in cells by a time-dependent manner compared with the control group（P 0.05）.The NO expression varied like the expression of Kim-1 as the reperfusion time prolonged.Compared to model group,the NRK-52E cells preincubated by CS showed apparent tolerance to ischemia-reperfusion injury,which was manifestated by the decreased apoptosis ratio,decreased expression of Kim-1 and NO.【Conclusions】 Antimycin A can induce NRK-52E cells to ischemia-reperfusion injury characteristed by apoptosis,accompanied by early high expression of Kim-1 and high concentration of NO in this process.Cordyceps sinensis can protect the NRK-52E ce

关 键 词：肾小管上皮细胞 缺血再灌注损伤 肾损伤分子-1 NO 凋亡 冬虫夏草 

分 类 号：R-332[医药卫生]