葡萄籽提取物对缺血再灌注导致衰老大鼠肾小管上皮细胞凋亡的保护作用

GSPE protects the apoptosis of aging rats′ renal tubular epithelial cell induced by ischemia-reperfusion

目的探讨葡萄籽提取物(Grape seed extract proanthocyanidin,GSPE)对缺血再灌注(I/R)导致衰老大鼠肾小管上皮细胞凋亡的保护作用。方法分离培养24月龄Wistar雄性大鼠的原代肾小管上皮细胞,第3代后,将细胞分为对照组、10μmol/L Antimycin A处理组、GSPE干预组以及Antimysin A+GSPE组。Anexin V/PI染色,流式细胞仪检测肾小管上皮细胞凋亡情况,定量PCR和Western印迹法分别检测Caspase-3mRNA和蛋白表达水平,化学发光法检测细胞Capsase-3活性及过氧化物丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。激光共聚焦检测细胞内活性氧(ROS)的浓度。结果(1)衰老大鼠肾小管细胞经Antimycin A处理后,凋亡率增高,Caspase-3,MDA,ROS明显增加,SOD下降;(2)经GSPE干预后,其凋亡情况明显好转,caspase-3、MDA、ROS明显降低。SOD含量部分恢复(P<0.05)。结论老年大鼠肾小管细胞在I/R损伤时,ROS堆积,线粒体损伤导致肾小管上皮细胞凋亡。GSPE可清除ROS从而达到抑制肾小管上皮细胞凋亡、减轻I/R损伤的作用。

Objective To explore the protective effects of Grape seed extract proanthocyanidin（GSPE）to the renal tubular epithelial cells of aging rats induced by ischemia and reperfusion. Methods The 24 month Wistar rats renal tubular epithelial cells were Isolated and cultured. After the third passage, the cells were divided into control, and 10 μmol/L Antimysin A, GSPE, Antimysin A ＋ GSPE treatment groups. The apoptosis of the renal tubular epithelial cells was determined by flow cytometry, which was dyed by Anexin V/PI. PCR and Western blot were used to detect the expressions of mRNA and protein of caspase-3 respectively. Chemoluminescence technique was used to detect the activity of caspase-3, the content of MDA and the activity of SOD. Laser confocal microscopy was used to test the concentration of the ROS in the cells. Results （1）The rates of apoptosis and the levels of caspase-3,MDA,ROS were increased obviously,and the level of SOD was decreased in aging rats renal tubular epithelial cells by Antimysin A; （2）After the treatment of GSPE, the apoptosis was improved, the levels of caspase-3, MDA and ROS were decreased significantly. The content of SOD was partial recovered（P〈0.05）. Conclusions At the I/R injury of aging rats kidney tubular cells, the accumulation of ROS and the damage of mitochondria lead to the apoptosis of renal tubular epithelial cell. GSPE can remove ROS and prevent the apoptosis of kidney tubular epithelial cells, lessen the impaired effects of I/R.