冬虫夏草通过Sirt1发挥对HK2细胞缺血-再灌注损伤的保护作用  

Cordyceps sinensis protects HK2 cells from ischemia-reperfusion injury through Sirt1 pathway

作　　者：张影莉 敖翔[1] 李慧 邓松筠 肖舟[1] 彭卫生[1] 项金华 周巧玲[1] 

机构地区：[1]中南大学湘雅医院肾内科,长沙410008 [2]长沙市第三医院肾内科,长沙410015 [3]长沙市第一医院呼吸内科,长沙410005 [4]中南大学湘雅医院重症监护室,长沙410008

出　　处：《中南大学学报（医学版）》2017年第11期1263-1269,共7页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(81070552)~~

摘　　要：目的:研究冬虫夏草(Cordyceps sinensis,CS)对缺血-再灌注损伤(ischemia reperfusion injury,IRI)的HK2细胞凋亡水平和Sirt1表达的影响,探讨CS对HK2细胞IRI的保护机制。方法:不同浓度CS(10,20,40,80,160,320 mg/L)与HK2细胞共培养24 h,测定细胞增殖率,确定其最佳干预浓度;体外培养的HK2细胞用0.01μmol/L抗霉素A处理模拟缺血过程,2 h后恢复含血清培养基模拟再灌注过程。将HK2细胞分为对照组,I/R组,I/R+CS组(160 mg/L),I/R+CS(160 mg/L)+sirtinol(25μmol/L)组,24 h后提取各组细胞总RNA和蛋白。四甲基偶氮唑盐(MTT)比色法检测细胞增殖;qRT-PCR及Western印迹检测Sirt1和凋亡相关基因(cleaved caspase-3)mRNA及蛋白表达的变化,Annexin V-FITC/PI双染法及流式细胞仪检测细胞凋亡率。结果:CS 10~160 mg/L与HK2细胞作用24 h,对细胞增殖影响不明显(P>0.05);当浓度增大到320 mg/L时,出现明显抑制HK2细胞增殖的现象(P<0.01)。与对照组相比,I/R组Sirt1,cleaved caspase-3 mRNA和蛋白表达增加(P<0.01),细胞凋亡率明显增加(P<0.01);相对于I/R组,I/R+CS组Sirt1mRNA和蛋白水平增加(P<0.01),而cleaved caspase-3 mRNA和蛋白表达明显下降(P<0.01),细胞凋亡率降低(P<0.05)。给予Sirt1抑制剂sirtinol后,I/R+CS+sirtinol(25μmol/L)组Sirt1 mRNA和蛋白明显降低(P<0.05),cleaved caspase-3 mRNA和蛋白表达明显升高(P<0.05),细胞凋亡率较I/R+CS组增加(P<0.05)。结论:CS对HK2细胞IRI具有保护作用,其机制可能与CS促进Sirt1表达有关。Objective： To investigate the effects of Cordyceps sinensis （CS） on cellular apoptosis and Sirt1 expression in HK2 cells followed by ischemia-reperfusion （I/R）. Methods： HK2 cells were incubated with different concentrations of CS （10, 20, 40, 80, 160, 320 mg/L） for 24 hours, and the optimal concentration of CS was selected by measuring cell proliferation. The confluent HK2 cells were incubated with 0.01 μmol/L antimycin A for 2 hours to induce ischemia in vitro, and then the reperfusion was achieved by incubating cells with glucose-replete complete growth medium for 24 hours. HK2 cells were divided into 4 groups： a control group, an I/R group, an I/R＋CS （160 mg/L） group, and an I/R＋CS （160 mg/L）＋Sirtinol （25 μmol/L） group. Twenty-four hours later, total RNA and protein were collected. The cell proliferation was evaluated by MTT assay; the mRNA and protein expression of Sirt1 and the cleaved caspase-3 were measured by qRT-PCR and Western blot, respectively. The cellular apoptosis rate was determined by Annexin V-FITC/PI double staining and flow cytometry. Results： Certain concentrations （10–160 mg/L） of CS did not show effect on the proliferation of HK2 cells （P〉0.05）, while 320 mg/L of CS inhibited cell proliferation significantly （P〈0.01）; compared with the control group, the mRNA and protein expressions of Sirt1 and the cleaved caspase-3 in the I/R group were up-regulated （P〈0.01） and the apoptosis rate was extremely high; compared with the I/R group, CS significantly up-regulated Sirt1 mRNA and protein expression （P〈0.01） while down-regulated cleaved caspase-3 mRNA and protein levels （P〈0.01）, and reduced apoptosis rate （P〈0.05）. The effects of CS were blocked in the presence of sirtinol, an inhibitor of CS. Conclusion： CS protects HK2 cells from I/R injury through activation of Sirt1 pathway.

关 键 词：冬虫夏草 肾缺血-再灌注损伤 SIRT1 

分 类 号：R285.5[医药卫生—中药学]