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目的:观察螺内酯对高糖诱导的肾小球系膜细胞氧化应激的影响,并且探讨该作用是否与MAPKs家族信号通路相关。方法:采用高糖和(或)螺内酯处理人肾小球系膜细胞株(HMC)后,DHE荧光染色检测细胞内活性氧(ROS)水平,NBT试剂盒法检测细胞内超氧化物歧化酶(SOD)活性,Western blot法检测MAPKs家族ERK、JNK、p38MAPK的磷酸化及总蛋白水平。结果:DHE染色结果显示,高糖可明显增加HMC内ROS含量,而给予螺内酯干预处理后,细胞内ROS水平明显降低,与高糖刺激组相比,螺内酯干预组细胞内SOD活性明显增加(P<0.05);Western blot结果表明,高糖可明显增加HMC细胞内ERK及p38MAPK的磷酸化水平,给予螺内酯处理后可逆转高糖的上述作用(P<0.05),而各组之间磷酸化JNK及总ERK、p38MAPK、JNK蛋白水平无明显变化。结论:螺内酯可能通过下调MAPK家族信号蛋白的磷酸化水平,降低细胞内ROS含量,增加SOD活性,拮抗由高糖所引起的氧化应激,从而起到保护肾小球系膜细胞,延缓肾小球硬化的作用。
AIM: To investigate the effect of spironolactone on oxidative stress induced by high glucose in mesangial cells and to explore whether this effect is related to the MAPKs signaling pathway. Methods: Human glomerular mesangial cell line (HMC) was treated with high glucose and / or spironolactone, and the level of reactive oxygen species (ROS) was measured by DHE staining. The activity of superoxide dismutase SOD) activity, phosphorylation and total protein levels of MAPKs family ERK, JNK, p38MAPK were detected by Western blot. Results: The results of DHE staining showed that high glucose could significantly increase the content of ROS in HMC. However, the intracellular ROS level was significantly decreased after treatment with spironolactone. Compared with high glucose group, the activity of SOD in the intervention group was significantly increased (P <0.05). Western blot results showed that high glucose could significantly increase the phosphorylation of ERK and p38MAPK in HMC cells, which could reverse the effect of high glucose (P <0.05), and the phosphorylation of JNK And total ERK, p38MAPK, JNK protein levels did not change significantly. Conclusion: Spironolactone may protect glomerular mesangial cells and delay renal damage by down-regulating phosphorylation of MAPK family signaling proteins, decreasing intracellular ROS content, increasing SOD activity and antagonizing oxidative stress caused by high glucose The role of ball hardening.