益气化湿通络方对实验性肾功能衰竭大鼠肾脏氧化应激损伤及纤维化的改善作用

目的:观察益气化湿通络方对5/6肾切除肾衰竭模型大鼠残留肾脏氧化应激损伤及纤维化的改善作用。方法:采用Platt法建立5/6肾切除慢性肾衰竭大鼠模型。术后2周抽检大鼠确认造模成功后,将大鼠随机分为:模型组(Model)、益气化湿通络方组(YHT)、贝那普利组(BH)、假手术组(Sham),每组8只。每日灌胃治疗1次(YHT组免煎颗粒水溶液0.276 g/100 g;BH组盐酸贝那普利片剂水溶液0.09 mg/100 g灌胃;Sham及Model 1 ml/100 g生理盐水灌胃),连续治疗12周。12周末用代谢笼收集24 h尿液,检测尿蛋白含量。之后麻醉大鼠腹主动脉取血、摘取肾脏,检测血清血肌酐(Scr)、血尿素氮(BUN)含量;HE、Masson染色观察左肾病理改变;检测肾组织匀浆超氧化物歧化酶(SOD)的活性和丙二醛(MDA)的含量,检测肾组织中核因子NF-E2相关因子(Nrf2)、Kelch样环氧氯丙烷相关蛋白-1(Keap1)、NADPH氧化酶4(Nox4)、转化生长因子-β1(TGF-β1)、I型胶原蛋白(Collagen1)的表达以及Nrf2在肾组织细胞核内的表达。结果:与Sham组比较,Model组大鼠肾小球损伤较重,纤维化明显;Scr、BUN、MDA水平和24 h尿蛋白的排出量,Keap1、Nox4、TGF-β1、Collagen1的蛋白表达均明显升高(P＜0.01),SOD活性、Nrf2表达明显降低(P＜0.01);与Model组比较,经YHT或BH干预后肾小球病变程度减轻,纤维化较少,Scr、BUN、MDA水平和24 h尿蛋白的排出量,Keap1、Nox4、TGF-β1、Collagen1的蛋白表达均明显减少(P＜0.01),SOD活性、Nrf2表达明显升高(P＜0.01)。结论:益气化湿通络方通过影响Nrf2/Keap1信号通路、下调TGF-β1蛋白表达,从而改善肾衰竭模型大鼠残留肾脏的氧化应激损伤及纤维化程度。

The improvement effects of Yiqi Huashi Tongluo Formula on oxidative stress and renal fibrosis of experimental CRF rats

Objective:Effects of Yiqi Huashi Tongluo Formula on oxidative stress and renal fibrosis of residual kidney were investigated in five/sixth nephrectomy rats.Methods:The rat model of chronic renal failure after nephrectomy was established by Platt method.Two weeks after the operation,the rats were randomly divided into model group,Yiqi Huashi Tongluo Formula(YHT)group,benazepril online(BH)group and sham group,with 8 rats in each group.Treatment was initiated once a day for 12 weeks after successful modeling.Animals were treated once a day with intragastric administration for 12 weeks.(Aqueous solution of free decoction granules in YHT group was 0.276 g/100 g·d.BH group benazepril hydrochloride tablet aqueous solution 0.09 mg/100 g·d gavage;sham group and model group were gavage with 1 ml/100 g normal saline).Urine was collected with a metabolic cage at the end of the 12th week,and urine protein content was detected for 24 hours.The rats were then anesthetized to extract blood from the abdominal aorta and the kidneys.The pathological changes of left kidney were observed by HE staining and Masson staining.The activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA)in kidney homogenate were determined by colorimetry.Western blot assay was used to detect the expressions of nuclear factor-erythroid 2-related factor 2(Nrf2),kelch-like ech-associated protein-1(Keap1),NADPH oxidase 4(Nox4),transforming growth factor-binding 1(TGF-β1),type I collagen(Collagen1)and Nrf2 in the nucleus in renal tissue.Results:Compared with sham group,model group rats had severe glomerular injury and obvious fibrosis.Levels of Scr,BUN,MDA and 24-hour urine protein excretion,protein expressions of Keap1,Nox4,TGF-β1 and Collagen1 were significantly increased(P<0.01),while SOD activity and Nrf2 expression were significantly decreased(P<0.01).Compared with the model group,the degree of glomerular lesion was reduced and fibrosis was less after YHT or BH intervention,and the levels of Scr,BUN,MDA,24-hour urine protein excretion,protein expressions of Keap1,Nox4,TGF-β1 and Collagen1 were significantly decreased(P<0.01),while SOD activity and Nrf2 expression were significantly increased(P<0.01).Conclusion:Through affecting the Nrf2/Keap1 signaling pathway and down-regulating the expression of TGF-β1 protein,Yiqi Huashi Tongluo Formula improved the oxidative stress damage and fibrosis degree of residual kidney in the model rats with renal failure.