丹参酮Ⅱ_A对万古霉素损伤HK-2细胞凋亡的影响及其机制的研究

Effect of Tanshinone Ⅱ_A Sulfonate Injection against HK-2 apoptosis induced by Vancomycin and the probable mechanism

目的观察丹参酮Ⅱ_A(TSⅡ_A)对万古霉素(VAN)损伤的人肾小管上皮细胞(HK-2)细胞凋亡的影响,探讨其可能机制。方法体外传代培养HK-2细胞,常规培养至细胞数约为1×106个/m L,分为空白组、VAN组、TSⅡ_AL组、TSⅡ_AM组、TSⅡ_AH组,除空白组加入PBS溶液外,其他各组加入终浓度为100μg/L VAN建立VAN损伤HK-2细胞模型,培养24 h后,空白组、模型组加入等体积的PBS溶液,TSⅡ-A各剂量组分别加入不同浓度的TSⅡ_A,继续培养24 h。MTT法测定细胞存活率,比色法测定细胞悬液中乳酸脱氢酶(LDH)、过氧化氢酶(CAT)活性;荧光素DCFH-DA探针法测定细胞内ROS含量,Annexin V-FITC/PI双染法检测细胞凋亡率。结果与空白组比较,VAN组细胞存活率、CAT活性显著降低,LDH活性和细胞内ROS水平显著升高,细胞凋亡率明显增加(P<0.05);与VAN组比较,TSⅡ_AL组、TSⅡ_AM组、TSⅡ_AH组的细胞存活率、CAT活性显著升高,LDH活性和细胞内ROS水平显著降低,细胞凋亡率明显减少(P<0.05),且作用均有浓度依赖性。结论 TSⅡ_A磺酸钠注射液能显著提高VAN损伤HK-2细胞的存活率,减少VAN损伤的HK-2细胞的凋亡,其机制可能与降低VAN损伤HK-2细胞LDH水平和细胞内的ROS水平及升高CAT等氧化应激反应有关。

Objective To explore the effect of TanshinoneⅡA Sulfonate Injection against HK-2 cell damage induced by Vancomycin and the probable mechanism. Methods HK-2 cells were cultured in vitro till the number was up to 1×10^6/mL,randomly divided into five groups： control group, VAN group, low dose group of TSⅡA Sulfonate Injection, middle dose group of TSⅡA Sulfonate Injection, hight dose group of TSⅡA Sulfonate Injection. Except for the control group adding PBS, other groups were added 100μg/L VAN for 24 hours. Then, the control group and VAN group were respectively added to the same volume of PBS, and the high dose group, middle dose group and low dose group of TSⅡA respectively added to different concentrations of TSⅡA . The cell viability was detected with MTT method, the activity of LDH were measured and the cell viability was detected with the content of LDH. DCFH-DA was used as the fluorescence probe to detect the level of ROS by a fluorescence microplate reader. Annexin V-FITC/PI double dyeing method was used to detect apoptosis rate. Results Compared with the control group, the survival rate of the cells and the activity of CAT were decreased, meanwhile the level of ROS, the activity of LDH and apoptosis rate were increased in the VAN group（P〈0.05）. Compared with the VAN group, the survival rate of the cells and the activity of CAT were increased, meanwhile the level of ROS, the activity of LDH and apoptosis rate were decreased in the low, middle and hight dose group of TSⅡA Sulfonate Injection（P〈0.05）, and there was a concentration dependence. Conclusion TSⅡA Sulfonate Injection can promote the proliferation of HK-2 cells in vitro,and improve the biochemical parameters and enzyme levels. The results suggest that TSⅡA Sulfonate Injection has a protective effect on HK-2 cell, and the protective mechanisms may be related with its antioxidant effect.