[关键词]
[摘要]
目的 研究丹酚酸B抑制碘普罗胺诱导的人肾近端小管上皮细胞HK-2凋亡的作用及机制。方法 将HK-2细胞分为对照组、模型(碘普罗胺)组、丹酚酸B各浓度(1、10、50、100、200 μmol/L)处理组及丹酚酸B(200 μmol/L)对照组。采用CCK-8法检测丹酚酸B对碘普罗胺诱导的HK-2细胞增殖的影响,DAPI染色法观察细胞核形态的变化,DCFH-DA染色荧光显微镜观察结合流式细胞术检测细胞内活性氧(ROS)水平,Western blotting法检测细胞Bax、Bcl-2、cleaved Caspase-3、磷酸化的蛋白激酶B(p-Akt)、磷酸化的细胞外调节蛋白激酶(p-ERK1/2)及Klotho蛋白表达量。结果 与对照组比较,模型组细胞活力显著降低(P<0.01),部分细胞核出现染色质浓缩及核分裂等凋亡特征,ROS水平及Bax、cleaved Caspase-3、p-ERK1/2蛋白表达水平显著增加(P<0.05、0.01),而Bcl-2、p-Akt及Klotho蛋白表达水平显著降低(P<0.05、0.01);丹酚酸B 50、100、200 μmol/L可以部分逆转碘普罗胺的上述效应。但低浓度的丹酚酸B(1、10 μmol/L)对碘普罗胺诱导的HK-2细胞无明显保护作用。结论 丹酚酸B可以抑制碘普罗胺诱导的HK-2细胞凋亡,其机制可能与抗氧化应激,激活Akt信号通路、抑制ERK信号通路及上调Klotho蛋白表达有关。
[Key word]
[Abstract]
Objective To investigate the effect and mechanism of salvianolic acid B on the apoptosis of human renal proximal tubular epithelial cells (HK-2) induced by iopromide. Methods HK-2 cells were divided into eight groups:control group, model group, different concentrations of salvianolic acid B (1, 10, 50, 100, and 200 μmol/L) treatment groups and salvianolic acid B control group (200 μmol/L). The effect of salvianolic acid B on the proliferation of HK-2 cells induced by iopromide was detected by CCK-8 method. The changes of nuclear morphology were observed by DAPI staining. Levels of ROS in different groups were observed by fluorescence microscope and flow cytometry. The expression levels of Bax, Bcl-2, cleaved Caspase-3, p-Akt, p-ERK1/2, and Klotho were detected by Western blotting. Results Compared with control group, the cell viability of HK-2 cells in model group was decreased significantly (P < 0.01), some nucleus appeared apoptotic characteristics such as chromatin condensation and nuclear division, the level of ROS and the expression of Bax, cleaved Caspase-3, p-ERK1/2 were increased significantly (P < 0.05, 0.01); Meanwhile, the expression of Bcl-2, p-Akt, and Klotho were decreased remarkably (P < 0.05, 0.01). However, the above effects of iopromide can be partially reversed by salvianolic acid B at 50, 100, and 200 μmol/L. But low concentration of salvianolic acid B (1 and 10 μmol/L) showed no obvious protective effect on the injury of HK-2 cells induced by iopromide. Conclusion Salvianolic acid B can inhibit the apoptosis of HK-2 cells induced by iopromide, the mechanism may be related to anti-oxidative stress, activation of Akt, inhibition of ERK pathway and up-regulation of Klotho expression.
[中图分类号]
R285.5
[基金项目]
国家自然科学基金资助项目(81560133);贵州省科技合作计划项目(黔科合LH字[2015]7529号);遵义医科大学博士启动基金(F-711)