肾康注射液对马兜铃酸致肾小管上皮细胞损害的研究

目的：探讨肾康注射液（SKI）对马兜铃酸（AA）导致肾小管上皮细胞损伤的影响。方法：以90％DMEM培养液加10％胎牛血清培养人肾小管上皮细胞系（HK-2），加入不同浓度的SKI（终浓度分别为4、6、8mg／mL）和AA（终浓度为10、20、40μg／mL）培养48h。采用MTT法观察HK-2细胞活性；根据MTT法检测细胞活性的结果，选择SKI最佳干预浓度8mg／mL和AA致细胞明显凋亡浓度20μg／mL，将实验分为3组，①对照组：培养液不加马兜铃酸；②AA干预组：在培养液中加入AA（终浓度为20μg／mL）；（3）SKI治疗组：在培养液中加入AA（终浓度为20μg／mL）和SKI（终浓度为8ing／mL）。用透射电镜观察3组细胞的超微结构；应用流式细胞术检测细胞凋亡百分率；分别采用实时定量荧光PCR及Western—blot技术检测凋亡蛋白Caspase-3mRNA及蛋白的表达情况。结果：与对照组比较，AA干预组细胞结构发生改变，治疗组大部分细胞结构基本正常，部分细胞结构改变明显。AA浓度超过10μg／mL时，HK-2细胞的活性显著抑制。分别以不同浓度4，6，8mg／mL的SKI和AA（20μg／mL）处理HK-2细胞后，则细胞活性明显增加，而且随着SKI浓度的增加其作用愈趋明显（P〈0．05）。与对照组比较，AA组凋亡细胞的比例明显增加（P〈0．05），SKI治疗组凋亡细胞的比例也增加，但比AA组细胞凋亡的比例明显减少。AA组Caspase-3mRNA及蛋白的表达较对照组明显增高；治疗组Caspase-3mRNA及蛋白的表达较AA组降低。结论：肾康注射液通过降低凋亡蛋白Caspase-3mRNA及蛋白的表达抑制HK-2细胞的凋亡，明显提高HK-2细胞抵抗AA致细胞损伤的能力，起到保护HK-2细胞的作用。该途径可能为肾康注射液抑制HK-2细胞凋亡的机制之一。

Effect of Shenkang Injection on Renal Tubular Epithelial Cells Damaged by Aristolochic Acid

Objective ： To investigate the effect of Shenkang Injection （SKI） on renal tubular epithelial cells damaged by aristolochic acid （AA）. Methods ： Human renal tubular epithelial cell line （ HK - 2 ） was cultured in Dulbecco＇ s modi- fied Eagle＇s medium （DMEM） （10% fetal calf serum） with different concentrations of SKI （4,6,8 mg/mL ） and AA （ 10,20,40 μg/mL）. Cell viability was measured by MTF assay. According to the MTT results,the best intervention con- centration （8 mg/mL） of SKI and the best concentration （20 μg/mL） of AA induced cell apoptosis were chosen. Renal tubular epithelial ceils were divided into three groups. The culture fluid was without AA in control group. In AA interven- tion group, HK- 2 cells were cultured with AA （a final concentration of 20 μg/mL） . In SKI group, HK -2 cells were cultured with AA （ a final concentration of 20 μg/mL） and SKI （ a final concentration of 8 mg/mL）. Electron microscopy was performed to observe the ultrastructural changes of HK - 2 cells. The proportion of apoptotic cells was measured by flow cytometry after 48 - hour incubation. Real - time quantitative PCR and Western blot were performed to determine the expressions of Caspase - 3 mRNA and protein in the HK - 2 cells. Results ： Compared with control group, structure of AA group cells changed significantly, structure of most of the SKI group cells were normal and structure of part of the cells changed significantly. AA （final concentration more than 10 μg/mL） could significantly reduce the viability of HK -2 （P 〈 0.05 ）. When cells were cultured with AA （20 mg/mL） and different concentration of SKI （final concentration was 4,6,8 mg/mL,respectively ）, viability of HK - 2 cells was significantly increased （ P 〈 0. 05 ） and dose - dependent. Compared with control group, the proportion of apoptotic cells of AA group was increased significantly （ P 〈 0.05 ）, theproportion of apoptotic cells of SKI group was also increased, but was reduced significantly compared with the AA group. Activity of Caspase - 3 in AA group was significantly higher than that in the control group, cell apoptosis rate was in- creased; activity of Caspase -3 reduced in the SKI treatment group, cell apoptosis rate also significantly reduced contras- ted with AA group. Conclusions ： Treatment with SKI could prevent HK - 2 cells from apoptosis induced by AA through re- ducing the apoptotic protein Caspase -3 mRNA and protein expression. SKI can protect HK -2 cells from injury by in- hibiting the apoptosis cells stimulated by aristolochic acid. The mechanism of inhibition of apoptosis induced by AA with Shenkang Injection is achieved by down - regulating Caspase - 3 mRNA and protein expression of HK - 2 cells.