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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8 mg/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8 mg/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-3 mRNA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-3 mRNA及蛋白的表达较对照组明显增高;治疗组Caspase-3 mRNA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-3 mRNA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。
Objective: To investigate the effects of Shenkang injection (SKI) on renal tubular epithelial cell injury induced by aristolochic acid (AA). Methods: Human renal tubular epithelial cell line (HK-2) was cultured in 90% DMEM medium supplemented with 10% fetal bovine serum. Different concentrations of SKI (4, 6 and 8 mg / mL) and AA 10, 20, 40 μg / mL) for 48 h. MTT assay was used to observe the activity of HK-2 cells. MTT assay was used to determine the cell viability. The best concentration of SKI was 8 mg / mL and the concentration of apoptotic cells was 20 μg / mL. The experiment was divided into three groups: Group: medium without aristolochic acid; AA intervention group: adding AA into the medium (final concentration: 20μg / mL); (3) SKI treatment group: adding AA ) And SKI (final concentration 8 mg / mL). The ultrastructure of the three groups of cells was observed by transmission electron microscope. The percentage of apoptosis was detected by flow cytometry. The mRNA and protein expression of Caspase-3 were detected by real-time quantitative PCR and Western-blot respectively. Results: Compared with the control group, the cell structure of AA intervention group changed, most of the cells in the treatment group were basically normal, and some of the cell structure changed significantly. AA concentration of more than 10μg / mL, HK-2 cell activity was significantly inhibited. After treated with different concentrations of SKI and AA (20μg / mL) for 4, 6 and 8mg / mL, the cell viability increased significantly and the effect became more obvious with the increase of SKI concentration (P <0.05) . Compared with the control group, the proportion of apoptotic cells in AA group was significantly increased (P <0.05), and the proportion of apoptotic cells in SKI group was also increased, but the proportion of apoptotic cells in AA group was significantly decreased. The expression of Caspase-3 mRNA and protein in AA group was significantly higher than that in control group. The expression of Caspase-3 mRNA and protein in AA group was lower than that in AA group. Conclusion: Shenkang injection can inhibit the apoptosis of HK-2 cells by decreasing the expression of Caspase-3 mRNA and protein, and significantly improve the ability of HK-2 cells to resist AA-induced cell injury and protect HK-2 cells effect. This pathway may be one of the mechanisms of Shenkang injection inhibiting HK-2 cell apoptosis.