乌司他丁上调P糖蛋白表达对百草枯诱导HK-2细胞损伤的影响

The effects of P-glycoprotein expression induced by ulinastatin on HK-2 cells damage induced by paraquat

目的探讨乌司他丁（ulinastatin，UTI）上调P糖蛋白（P-glycoprotein，P-gp）表达对百草枯（paraquat，PQ）诱导HK-2细胞损伤的保护作用及机制。方法实验分为两部分。第一部分实验分为正常对照组、PQ组、UTI＋PQ组、UTI对照组。第二部分实验分为阴性病毒组（包含对照组、PQ组、PQ＋UTI组、UTI组）和P-gp siRNA组（包含对照组、PQ组、PQ＋UTI组、UTI组）。阴性病毒组：细胞转染空白病毒；P-gp siRNA组：细胞转染携带P-gp siRNA的病毒。常规培养HK-2细胞，在800μmol／LPQ干预后，蛋白免疫印迹法（Westernblot）检测HK-2细胞内P-gp表达量。并以携带P-gp沉默基因的慢病毒技术抑制P-gp表达，应用细胞活性检测试剂盒（CellCountingKit-8，CCK-8）法检测细胞存活率；Westernblot法检测病毒转染后的P-gP的表达量；高效液相色谱法（highperformanceliquidchromatography，HPLC）检测细胞内PQ浓度。结果与正常对照组比较，PQ组细胞内P-gp的表达量无明显变化；与PQ组比较，UTI＋PQ组细胞内P-gP的表达量明显升高，差异有统计学意义（P〈0．05）。与相应阴性病毒组比较，P-gPsiRNA组细胞存活率无明显变化，差异无统计学意义（尸〉0．05）。与阴性病毒＋PQ组比较，P．gPsiRNA＋PQ组细胞内PQ浓度无明显变化，差异无统计学意义（P〉0．05）；与P-gPsiRNA＋PQ组比较，P．gPsiRNA＋PQ＋UTI组细胞内PQ含量明显降低，差异有统计学意义（P〈0．05）。结论UTI在体外能明显减少PQ诱导的HK一2细胞损伤，提高HK．2细胞的存活率，但可能与其上调P-gp的表达关系不大。

Objective To investigate the protective effect of P-glycoprotein up-regulated by ulinastatin （UTI） on HK-2 cells during paraquat （PQ）-induced injury and its underlying mechanisms. Methods The research was divided into two parts. The first part of the research was divided into normal control group, PQ group, UTI＋PQ group, UTI control group. The second part of the research was divided into negative virus group （including control group, PQ group, PQU＋TI group, UTI group） and P-gp siRNA group （including control group, PQ group, PQU＋TI group, UTI group）. Negative virus group： the cells were transfected into the blank virus; siRNA P-gp group： the cells were transfected with P-gp siRNA virus. HK-2 cells were routinely cultured. After 800 p^mol/L PQ treatment, the changes of P-gp protein levels in the HK-2 cells were determined by Western-blot （WB）. Then, transfected lentivirus bringing P-gp silent gene, the cell viability was determined by CCK-8 assay, the expression of P-gp in the cells after transfection was detected by WB and the concentration of PQ in HK-2 cells were measured by high performance liquid chromatography （HPLC）. Results Compared with the normal control group, the P-gp expression of PQ group had no significantly changes （P〉0.05）. Compared with the PQ group, the P-gp expression of UTI＋PQ group significantly increased （P〉0.05）. Compared with the corresponding control siRNA group, the P-gp siRNA group had no significantly changes in cell viability （P〉 0.05）. and significantly decreased in P-gp expression. Compared with the corresponding control siRNA group, the P-gp siRNA group had no significantly changes in PQ concentration in HK-2 cell （P〉0.05）, but compared with P-gp siRNA PQ group, the PQ concentration of P-gp siRNA PQ＋UTI group significantly decrease （P〈0.05）.Conclusion UTI significantly reduced the accumulation of PQ in HK-2 cells and increased the viability of HK- 2 cells in vitro may be not by increased P-gp activity. UTI could significantly reduce HK-2 cell injury induced by PQ in vitro and improve the survival rate of HK-2 ceils. It may not be related to the up regulation of P-gp expression.