芪附汤调节ERK1/2信号通路改善UUO阳虚证模型肾间质纤维化的作用和机制

Effects and mechanisms of Qifu decoction ameliorating renal tubulointerstitial fibrosis through inhibiting ERK1/2 signaling pathway in unilateral ureteral obstruction rats with yang deficiency

目的:探讨芪附汤改善阳虚证模型鼠肾间质纤维化(RIF)的作用和机制。方法:将大鼠随机分为假手术组(A组)、模型组(B组)、芪附汤组(C组)、依那普利组(D组)。通过腺嘌呤灌胃联合单侧输尿管接扎术(UUO)的方法而建立RIF模型。造模成功后,C,D组大鼠分别经灌胃给予芪附汤水煎液或依那普利悬浊液;其余2组大鼠经灌胃给予蒸馏水,共干预3周。各在药物和蒸馏水干预前和干预后第1,2,3周末,分别称量大鼠体重,并检测24 h尿蛋白排泄量(Upro)、尿β2-微球蛋白(Uβ2-MG)、尿N-乙酰-β-D-氨基葡萄糖苷(NAG)酶。药物干预第3周末,处死全部大鼠,抽取血液,摘除左肾,称重,并留取相应标本,观察肾脏外观和肾小管/间质形态;检测血清环磷酸腺苷(c AMP)、环磷酸鸟苷(c GMP)、肌酐(Scr)、尿素氮(BUN)和尿酸(UA);检测肾组织中上皮型黏钙蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、转化生长因子-β1(TGF-β1)、结缔组织生长因子(CTGF)、细胞外调节激酶(ERK1/2)和磷酸化ERK1/2(p-ERK1/2)蛋白相对表达量。结果:芪附汤可以改善模型鼠血清中降低的c AMP和c AMP/c GMP;减少模型鼠升高的Uβ2-MG、尿NAG酶,改善肾间质细胞外基质及其胶原沉积,上调肾组织E-cadherin蛋白表达,下调肾组织α-SMA,TGF-β1,CTGF,p-ERK1/2蛋白表达;但是,对Scr,BUN,UA没有影响。芪附汤改善RIF的作用优于依那普利。结论:芪附汤可以改善模型鼠阳虚证指标以及尿β2-MG、尿NAG酶排泄,它可能是通过上调肾组织E-cadherin蛋白表达,下调肾组织α-SMA,TGF-β1,CTGF以及ERK1/2信号通路中关键信号分子——p-ERK1/2蛋白表达,改善肾小管EMT,从而减轻RIF。

Objective ： To demonstrate the effects and mechanisms of Qifu decoction （QFD） on renal interstitial fibrosis （RIF） in model rats with yang-deficiency syndrome. Method： The rats were randomly divided into 3 groups, the Sham group （ Group A）, the Model group（ Group B）, the Qifu decoction group（ Group C）and the Enalapril group（ Group D）. The RIF model was established by adenine administrated and unilateral ureteral obstruction（UUO） of the left ureter. After the model was successfully established, the rats in Group C and D were administrated with QFD or the Enalapril suspension, while the rats in Group A and B were administrated with dis- tilled water. All rats were administrated for 3 weeks. Before administration and at the end of week 1,2 and 3 ,the rats were weighted, and 24 h urinary protein excretion（Upro）, urinary ff2-microglobulin （Ufl2-MG） and urinary N-acetyl-D-glucosaminidase （NAG）were examined,respectively. All rats were killed after administration for 3 weeks. Blood and renal tissues were collected, renal morphology and tubulointerstitial morphology were evaluated,respectively. Serum cyclic adenosine monophosphate（cAMP） ,cyclic guanosine mouophosphate （ cGMP）, blood urea nitrogen （ BUN）, serum creatinine （ Scr ） and uric acid （ UA ） were detected, respectively. The protein expressions of E-cadherin, a-smooth muscle actin（ α-SMA）, transforming growth factor-β1 （TGF-β1）, onnective tissue growth factor（ CT- GF） extracellular signal-regulated protein kinase 1/2 （ ERK1/2 ） and phosphorylated-ERK1/2 （ p-ERK1/2 ） in kidney were evaluated, respectively. Result： QFD ameliorated serum cAMP level and the rate of cAMP/cGMP, attenuated urinary β2-MG level, NAG level and renal, tubulointerstitial fibrosis, increased E-cadherin protein expression, and reduced ct-SMA, TGF-fll, CTGF and p-ERK1/2 protein expressions in the kidney. However, QFD had no influence on renal function in vivo. In addition,these effects were better than those of the model rats treated by Enalapril. Conclusion： QFD could alleviate yang-deficiency parameters, as well as urinary β2-MG level and NAG level in model rats induced by adenine administration and UUO. Moreover, QFD could improve EMT and RIF by up-regulating E-cadherin protein expression, and down-regulating α-SMA, TGF-β1, CTGF and p-ERK1/2 protein expressions, the key molecular in ERK1/2 signaling pathway.