金匮肾气汤对单侧输尿管梗阻大鼠氧化应激和自噬的影响

The Effect of Jingui Shenqi Decoction on Oxidative Stress and Autophagy in Rats with Unilateral Ureteral Obstruction

目的探讨金匮肾气汤对单侧输尿管梗阻(Unilateral ureteral obstruction,UUO)大鼠肾间质纤维化中氧化应激和自噬的影响。方法将24只雄性SD大鼠随机分为假手术组、模型组、金匮肾气汤组(1.8 g·kg-1)、依那普利组(10 mg·kg-1);左侧输尿管结扎术后第1天开始灌胃给药(10 mL·kg-1),每日1次,第21天处死大鼠。取梗阻肾组织,采用Masson染色法观察肾组织病理改变;化学荧光法检测梗阻肾组织活性氧(ROS)含量;Western Blot法检测NOX4、p22phox、p47phox、LC3II/I、p62、p-mTOR/mTOR及p-AMPK/AMPK等蛋白在梗阻肾组织中的表达。结果与假手术组比较,模型组大鼠梗阻肾组织间质胶原沉积增多,ROS含量显著增高(P<0.01),NOX4、p22phox和p47phox蛋白表达显著上调(P<0.01),LC3II/LC3I蛋白表达比例显著下调、p62蛋白表达显著上调(P<0.01),AMPK磷酸化显著减少、mTOR磷酸化明显增加(P<0.01)。与模型组比较,金匮肾气汤组大鼠梗阻肾组织间质胶原沉积减少,ROS含量显著降低(P<0.05),NOX4、p22phox和p47phox蛋白表达显著下调(P<0.05),LC3II/LC3I蛋白表达比例上调、p62蛋白表达下调(P<0.05),AMPK磷酸化增加、mTOR磷酸化减少(P<0.05)。结论金匮肾气汤可能通过抑制NOX4激活,进而通过AMPK-mTOR途径促进梗阻肾组织自噬,从而改善UUO导致的肾间质纤维化。

Objective To investigate the effect of Jingui Shenqi decoction on oxidative stress and autophagy in unilateral ureteral obstruction(UUO)rats.Methods Twenty-four male SD rats were randomly divided into 4 groups:Sham operation group,model group,Jingui Shenqi decoction treatment group(1.8 g·kg-1),and the enalapril treatment group(10 mg·kg-1)as positive control.One day after left ureteral ligation,rats were intragastric administration with drugs(10 mL·kg-1)for 21 days.On 21 thday,the rats were sacrificed.The obstructive kidney tissues were stained with Masson to observe pathologic changes.Content of reactive oxygen species(ROS)in the obstructed kidney was measured by chemiluminescence.The protein expression of NADPH oxidase 4(NOX4),p22 phox,p47 phox,microtubule-associated proteins 1(light chain 3 II/I(LC3 II/I)),autophagy-related markers p62,phosphorylated mammalian target of rapamycin(p-mTOR)and phosphorylated AMP-activated protein kinase(p-AMPK)in renal tissues were detected using Western Blot.Results Compared with the Sham operation group,collagen deposition increased,ROS content increased(P<0.01),the protein expression of NOX4,p22 phoxand p47 phoxincreased(P<0.01),ratio of the protein expression of LC3 II/LC3 I decreased and p62 increased(P<0.01),AMPK phosphorylation decreased and mTOR phosphorylation increased(P<0.01)significantly in the obstructed kidneys of the model group.Compared with the model group,after treatment with Jingui Shenqi decoction,collagen deposition decreased,ROS content decreased(P<0.05),the protein expression of NOX4,p22 phoxand p47 phox decreased(P<0.05),ratio of the protein expression of LC3 II/LC3 I increased and p62 decreased(P<0.05),AMPK phosphorylation increased and mTOR phosphorylation decreased(P<0.05).Conclusion Jingui Shenqi decoction could inhibit the activation of NOX4 and promote autophagy through AMPK-mTOR pathway,thus improve renal interstitial fibrosis induced by UUO.