富马酸二甲酯对糖尿病肾病自噬及足细胞功能的影响

The Effect of Dimethyl Fumarate on Autophagy and Podocyte Function in Diabetic Nephropathy

目的探究富马酸二甲酯(DMF)对糖尿病肾病(DN)自噬及足细胞功能的影响。方法通过对大鼠腹腔注射50 mg/kg的STZ诱导了DN模型,DN组大鼠灌胃2 mL生理盐水,DN+25DMF组、DN+50DMF组和DN+75DMF组分别灌胃25、50和75 mg/(kg·d)的DMF,共治疗12周。检测各组大鼠的血糖、尿蛋白、血清尿素氮和肌酐水平。用Periodic Acid-Schiff(PAS)试剂盒对肾脏进行染色。通过Western blot或免疫荧光染色检测肾脏中nephrin、LC3、p62、p-AMPK、AMPK、p-mTOR、mTOR和p-ULK1的表达。此外,将小鼠足细胞系(MPC5)在高葡萄糖(HG,40 mmol/L)条件下用不同浓度的DMF(10、50和100μmol/L)处理24 h或36 h。使用CCK-8试剂盒测定细胞活力,通过Western blot测定细胞中的LC3和p62蛋白表达水平。结果与DN组比较,DN+25DMF、DN+50DMF和DN+75DMF组大鼠的血糖、尿蛋白、血清尿素氮和肌酐水平显著降低,肾小球系膜基质明显减少,肾组织中nephrin的表达水平显著升高(P<0.05)。与DN组比较,DN+25DMF、DN+50DMF和DN+75DMF组大鼠肾组织中LC3II的表达水平显著升高,而p62显著降低,大鼠足细胞中的自噬体数目显著升高(P<0.05)。与DN组比较,DN+25DMF、DN+50DMF和DN+75DMF组大鼠肾组织中AMPK、mTOR和ULK1磷酸化显著升高(P<0.05)。与HG组比较,HG+10DMF、HG+50DMF和HG+100DMF组MPC5足细胞的活力显著升高,LC3-II蛋白表达水平显著升高,而p62显著降低(P<0.05)。结论DMF通过AMPK/mTOR/ULK1途径增强自噬,从而抑制了DN的进展并提高了足细胞功能。

Objective To investigate the effect of dimethyl fumarate(DMF)on autophagy and podocyte function in diabetic nephropathy(DN).Methods The DN animal model was induced by an intraperitoneal injection of 50 mg/kg of STZ into rats.The rats in the DN group were intragastrically administered with 2 mL of normal saline,while the rats in the DN+25DMF group,DN+50DMF group and DN+75DMF group were intragastrically administered with 25,50 or 75 mg/(kg·d)of DMF,respectively,for a total of 12 weeks of treatment.The blood glucose,urine protein,serum urea nitrogen and creatinine levels of rats in each group were detected.The kidney was stained with Periodic Acid-Schiff(PAS)kit.The expressions of nephrin,LC3,p62,p-AMPK,AMPK,p-mTOR,mTOR and p-ULK1 in kidney were detected by western blot or immunofluorescence staining.In addition,the mouse podocyte cell line(MPC5)was treated with different concentrations of DMF(10,50 or 100μmol/L)under high glucose(HG,40 mmol/L)conditions for 24 or 36 h.The CCK-8 kit was used to determine the cell viability,and the LC3 and p62 protein expression levels in the cells were determined by western blot.Results Compared with the DN group,the blood glucose,urine protein,serum urea nitrogen and creatinine levels of rats in the DN+25DMF,DN+50DMF,and DN+75DMF groups were significantly reduced,also the mesangial matrix of the glomerulus was significantly reduced,while the expression level of nephrin in kidney tissue was significantly increased(P<0.05).Compared with the DN group,the expression level of LC3II in kidney tissue of rats in the DN+25DMF,DN+50DMF and DN+75DMF groups was significantly increased,while p62 was significantly reduced,while the number of autophagosomes in rat podocytes was significantly increased(P<0.05).Compared with the DN group,the phosphorylations of AMPK,mTOR and ULK1 in the kidney tissue of rats in the DN+25DMF,DN+50DMF and DN+75DMF groups were increased significantly(P<0.05).Compared with the HG group,the viability of MPC5 podocytes in the HG+10DMF,HG+50DMF and HG+100DMF groups were significantly increased,also the expression of LC3-II protein was significantly increased,while the p62 was significantly decreased(P<0.05).Conclusion DMF enhances autophagy through AMPK/mTOR/ULK1 pathway,thereby inhibits the progression of DN and improves podocyte function.