黄芪甲苷联合糖皮质激素治疗嘌呤霉素氨基核苷肾病大鼠的疗效及其机制

Effect of astragaloside IV combined with glucocorticoids on puromycin aminonucleoside-induced rat nephropathy model and the associated mechanism

目的:观察黄芪甲苷(astragaloside IV,AS-IV)联合糖皮质激素干预治疗嘌呤霉素氨基核苷(puromycin aminonucleoside,PAN)肾病大鼠的疗效,并探讨其相关机制。方法:40只150~180 g无特定病原体级健康雄性Wistar大鼠被随机分为对照组、模型组(PAN组)、黄芪甲苷干预组(PAN+AS-IV组)、甲泼尼龙(MP)干预组(PAN+MP组)和AS-IV+MP干预组(PAN+AS-IV+MP组)。采用单次尾静脉注射50 mg/kg PAN的方法建立PAN肾病模型。药物干预组于造模同时分别给予40 mg·kg^(-1)·d^(-1)AS-IV灌胃和15 mg·kg^(-1)·d^(-1)MP腹腔注射,连续治疗10 d,实验第11天收集各组大鼠血液、24 h尿液和肾组织标本。用全自动生化仪检测尿蛋白量、尿肌酐、血清白蛋白(Alb)水平;Western印迹法检测足细胞标志蛋白nephrin、Rho家族蛋白RhoA、Rac/Cdc42的相对表达量;免疫荧光法检测各组大鼠肾组织nephrin、synaptopodin蛋白的表达。结果:与对照组比较,PAN组大鼠尿蛋白/肌酐比值(uPCR)显著升高,Alb水平显著降低,足细胞标志蛋白nephrin相对表达量明显下调,RhoA、Rac/Cdc42蛋白相对表达量显著上调(均P<0.01)。与PAN组比较,PAN+AS-IV组和PAN+AS-IV+MP组血清Alb水平显著升高(均P<0.01),PAN+MP组(P<0.05)和PAN+AS-IV+MP组(P<0.01)uPCR显著降低。Western印迹结果显示,与PAN组比较,各药物干预组(PAN+AS-IV组、PAN+MP组和PAN+AS-IV+MP组)肾组织nephrin相对表达量显著升高,RhoA、Rac/Cdc42相对表达量显著降低(均P<0.01)。免疫荧光结果显示,PAN组足细胞标志蛋白nephrin、synaptopodin表达较对照组明显减少,各药物干预组肾组织nephrin、synaptopodin表达较PAN组均升高,其中PAN+AS-IV+MP组改善最显著。结论:AS-IV及糖皮质激素均可改善PAN诱导的肾足细胞损伤,且两者联合使用有协同增强的保护作用,可能与抑制Rho家族信号的激活有关。

Objective To investigate the effect and mechanism of astragaloside IV(AS-IV)combined with glucocorticoids in the treatment of puromycin aminonucleoside(PAN)rat nephropathy model.Methods Forty specific pathogen-free healthy male Wistar rats(150-180 g)were randomly divided into 5 groups:control group,PAN group,AS-IV treatment group(PAN+AS-IV group),methylprednisone(MP)treatment group(PAN+MP group),and AS-IV+MP treatment group(PAN+AS-IV+MP group).The model was established by a single tail vein injection of PAN(50 mg/kg body weight).The treatment groups were given 40 mg·kg^(-1)·d^(-1) AS-IV by intragastric administration and 15 mg·kg^(-1)·d^(-1) MP by intraperitoneal injection for 10 consecutive days at the same time of modeling.Urine sample was collected on the 11th day of the experiment.The urine protein,urine creatinine and blood albumin were detected by biochemical analyzer.The changes of nephrin and synaptopodin in renal tissues were detected by immunofluorescence assay,and the expressions of nephrin,RhoA and Rac/Cdc42 proteins were detected by Western blotting.Results Compared with the control group,urine protein creatinine ratio(uPCR)was significantly increased,serum albumin(Alb)was significantly decreased in the PAN group,nephrin expression was significantly down-regulated,and the expressions of RhoA and Rac/Cdc42 were significantly up-regulated in the renal tissue of the PAN group(all P<0.01).Compared with PAN group,serum Alb levels in PAN+AS-IV group and PAN+AS-IV+MP group were significantly increased(both P<0.01),and the uPCR levels in PAN+MP group(P<0.05)and PAN+AS-IV+MP group(P<0.01)were significantly decreased(all P<0.05).Compared with the PAN group,the relative expressions of nephrin in renal tissue of all drug intervention group(PAN+AS-IV group,PAN+MP group and PAN+AS-IV+MP group)were significantly increased,while the relative expressions of RhoA and Rac/Cdc42 were significantly decreased(all P<0.01).The immunofluorescence results suggested that the expressions of nephrin and synaptopodin in renal tissue of PAN group were significantly down-regulated compared with the control group,which were reversed in all treatment groups,and the reversion was most pronounced in the PAN+AS-IV+MP group.Conclusion Both AS-IV and glucocorticoid can improve PAN-induced podocyte injury,and the combination of the two has synergistic action,which may be related to inhibiting the activation of Rho family signaling pathway.