摘要
目的探讨血必净（xuebijing，XBJ）对百草枯（paraquat, PQ）诱导人近端肾小管上皮细胞系（human kidney cell line-2，HK-2）细胞凋亡的保护作用及机制。方法常规培养HK-2细胞。（1）PQ及XBJ干预后细胞生长抑制实验：PQ组分为0、200、400、800、1 600、3 200 μmol/L PQ组，干预24、48、72 h后检测细胞存活率；XBJ组分为0、5、10、20、40 mg/ml XBJ组，干预24、48、72 h后检测细胞存活率，以确定进行干预时合理的XBJ和PQ的浓度和作用时间。（2）XBJ拮抗PQ诱导HK-2细胞生长抑制实验：分为正常对照组、PQ组（800 μmol/L）、不同浓度XBJ＋PQ组（分别以5、10、20 mg/ml的XBJ预培养1 h后以800 μmol/L的PQ继续培养），各组分别培养24、48、72 h后检测细胞存活率。（3）将HK-2细胞分为正常对照组、PQ组（800 μmol/L的PQ作用24 h）、PQ＋XBJ组（10 mg/ml的XBJ预处理1 h后以800 μmol/L的PQ作用24 h）、XBJ对照组（10 mg/ml的XBJ作用24 h），用流式细胞仪检测细胞凋亡情况，蛋白免疫印迹法检测各组细胞内Bcl-2和BAX的蛋白表达情况，caspase-3及caspase-9活力试剂盒检测细胞内caspase-3及caspase-9活力。结果（1）PQ能明显降低HK-2细胞的存活率，并且呈现出时间和浓度依赖性，其中800 μmol/L的PQ作用24 h后HK-2细胞的存活率约为55%；20 mg/ml以下的XBJ在72 h内对HK-2细胞的存活率无明显影响。（2）与PQ组比较，PQ＋XBJ组HK-2细胞的存活率明显升高，差异有统计学意义（P〈0.05）。（3）与正常对照组比较，PQ组细胞凋亡率明显增加，差异有统计学意义（P〈0.05）。与PQ组比较，PQ＋XBJ组细胞凋亡率明显降低，差异有统计学意义（P〈0.05）。（4）与正常对照组比较，PQ组细胞内Bcl-2蛋白表达明显较低，BAX蛋白表达明显升高，差异有统计学意义（P〈0.05）；与PQ组比较，PQ＋XBJ组细胞内Bcl-2蛋白表达明显升高，BAX蛋白表达明显降低，差异有统计学意义（P〈0.05）。（5）与正常对照组比较，PQ组细胞内caspase-3及caspase-9活力明显升高，差异有统计学意义（P〈0.05）；与PQ组比较，PQ＋XBJ组细胞内caspase-3及caspase-9活力明显降低，差异有统计学意义（P〈0.05）。结论XBJ（10 mg/ml）对PQ（800 μmol/L）诱导的HK-2细胞损伤有明显的保护作用，其可以通过减轻PQ诱导HK-2细胞凋亡，来提高细胞的存活率。
ObjectiveTo investigate the protective effect and mechanism of Xuebijing （XBJ） on paraquat （PQ） -induced apoptosis in Human kidney cell line-2 （HK-2） cells.MethodsRoutinely cultured HK-2 cells, （1） Cell growth inhibition experiment after PQ and XBJ intervention： PQ was divided into 0、200、400、800、1600 and 3200 μmol/L PQ groups, and the cell survival rate was detected after intervening 24、48 and 72 h. XBJ was divided into 0、5、10、20、40 mg/ml XBJ groups, and the cell survival rate was detected after intervening 24、48 and 72 h.To determine the rational drug concentration and the duration of action of XBJ and PQ. （2） PQ-induced HK-2 cell growth inhibition experiment antagonized by XBJ： The cells were divided into normal control group, PQ group （800 μmol/L） and PQ＋XBJ group （The cells were pretreated with 5、10 and 20 mg/ml XBJ for 1 h, then cultured with PQ of 800 μmol/L） , After cultured 24 h、48 h and 72 h separately, the cell survival rate was detected. （3） HK-2 cells were divided into normal control group、PQ group （800 μmol/L PQ cultured for 24 h） 、PQ＋XBJ group （pretreated with 10 mg/ml XBJ for 1 h, and then 800 μmol/L PQ cultured for 24 h） and XBJ group （10 mg/ml XBJ cultured 24 h）. The apoptosis of cells was detected by flow cytometry. The protein expression of Bcl-2 and BAX in each group was detected by Western blotting. The expressions of caspase-3 and caspase-9 were detected by caspase-3 and caspase-9 activity kit active.Results（1） PQ could significantly reduced the survival rate of HK-2 cells and showed time and concentration dependence. The survival rate of HK-2 cells was about 55% after 800 μmol/L PQ contacted 24 h, XBJ under 20 mg/ml was no significant effect on the survival rate of HK-2 cells after cultured 72 h. （2） Compared with the PQ group, the survival rate of HK-2 cells of PQ＋XBJ group was significantly increased （P〈0.05）. （3） Compared with the normal control group, the cell apoptosis rate of PQ group was significantly increased （P〈0.05）. Compared with the PQ group, the cell apoptosis rate of PQ＋XBJ group was significantly decreased （P〈0.05）. （4） Compared with the normal control group, Bcl-2 protein expression in PQ group was significantly decreased and BAX protein expression in PQ group was significantly increased （P〈0.05） ; compared with PQ group, Bcl-2 protein expression in PQ＋XBJ group was significantly increased, BAX protein expression in PQ＋XBJ group was significantly decreased （P〈0.05）. （5） Compared with the normal control group, the activities of caspase-3 and caspase-9 in PQ group were significantly increased （P〈0.05）. Compared with PQ group, the activities of caspase-3 and caspase-9 in PQ＋XBJ group were decreased significantly （P〈0.05） .ConclusionXBJ （10 mg/ml） has obvious protective effect on HK-2 cell injuried by PQ （800 μmol/L） , It can improve the survival rate of cells through reducing the apoptosis of HK-2 cells which induced by PQ.
作者
田昕
张万里
胡丽丽
余兴蓉
洪广亮
陈黎明
曹凯强
卢中秋
Tian Xin;Zhang Wanli;Hu Lili;She Xingrong;Hong Guangliang;Chen Liming;Cao Kaiqiang;Lu Zhongqiu(The Fifth Affiliated Hospital of Wenzhou Medical University, Lishui 323000, Chin)
出处
《中华劳动卫生职业病杂志》
CAS
CSCD
北大核心
2018年第1期1-6,共6页
Chinese Journal of Industrial Hygiene and Occupational Diseases
基金
温州市科技计划项目（Y20160310）
浙江省中医药重点学科计划（2012-XK-A28）
浙江省“十二五”重点学科建设项目（2012-207）
浙江省医学创新学科建设项目（11-CX26）
作者简介
通信作者：卢中秋，E-mail：lzq640815@163．com
