肾衰方及其拆方对肾间质纤维化VEGF、VEGFR2、a-SMA的影响

Effects of Shenshuai Formula and Its Decomposed Formulae on VEGF,VEGFR2 and a-SMA in Renal Interstitial Fibrosis

目的探讨肾衰方及其拆方对单侧输尿管梗阻(UUO)大鼠肾间质纤维化(RIF)及VEGF、VEGFR2、a-SMA的影响。方法采用单侧输尿管梗阻建立RIF大鼠模型。将108只无特定病原体(SPF)级健康成年SD雌性大鼠随机分为假手术组、模型组、肾衰方组、祛邪方组、补虚方组、贝那普利组,每组18只。UUO术后1 d起连续灌胃给药3周,肾衰方组、补虚方组、祛邪方组大鼠分别按19.5、9.5、11.0 g/(kg·d)进行灌胃,贝那普利组给予1.05 mg/(kg·d),假手术组和模型组每日给予等量生理盐水灌胃。造模后各组分别在第7、14、21天各处死6只大鼠,检测其血清肌酐(Scr)、尿素氮(BUN),同时计算左肾肾脏脏器指数;采用苏木精-伊红染色和马松染色进行肾脏组织病理检测,采用Western Blot检测VEGF、VEGFR2、a-SMA的表达,实时荧光定量RT-PCR检测VEGF、VEGFR2、a-SMAmRNA的表达。结果(1)与假手术组比较,同时期模型组、肾衰方组、祛邪方组、补虚方组、贝那普利组血清BUN、Scr、左肾肾脏脏器指数均升高(P<0.05)。与模型组相比,同时期肾衰方组、祛邪方组、补虚方组、贝那普利组血BUN、Scr、肾脏脏器指数均降低(P<0.05),与其他治疗组相比,肾衰方组在降低BUN、Scr、肾脏脏器指数更明显(P<0.05)。(2)肾脏病理改变:与假手术组比较,同时期模型组、肾衰方组、祛邪方组、补虚方组、贝那普利组肾小管间质损伤指数和胶原阳性面积均增加(P<0.05)。与UUO模型组比较,同时期肾衰方组、祛邪方组、补虚方组、贝那普利组肾小管间质损伤指数和胶原阳性面积均降低(P<0.05);与其他治疗组相比,肾衰方组在减轻肾小管间质损伤指数和胶原阳性面积更明显(P<0.05)。(3)与假手术组比较,同时期模型组、肾衰方组、祛邪方组、补虚方组、贝那普利组VEGF、VEGFR2、a-SMA蛋白和VEGF、VEGFR2、a-SMAmRNA表达量均增加(P<0.05);与模型组相比,同时期肾衰方组、祛邪方组、补虚方组、贝那普利组VEGF、VEGFR2、a-SMA和VEGF、VEGFR2、a-SMAmRNA表达均有不同程度降低(P<0.05),与其他治疗组相比,肾衰方组降低VEGF、VEGFR2、a-SMA蛋白和VEGF、VEGFR2、a-SMAmRNA表达量更明显。结论肾衰方改善肾功能,减轻肾脏病理损害可能是通过抑制VEGF、VEGFR2、a-SAM蛋白和mRNA表达。

Objective To investigate the effects of Shenshuai Formula and its decomposed formulae on renal interstitial fibrosis(RIF),expressions of VEGF, VEGFR2 and a-SMA in UUO rats. Methods RIF rat model was established by unilateral ureteral obstruction(UUO). A total of 108 healthy adult SD female rats without specific pathogen(SPF) were randomly divided into sham operation group, model group, Shenshuai Formula group, Quxie Formula group, Buxu Formula group and benazepril group, 18 in each group. UUO was administered by intragastric administration for 3 weeks from the 1 st day after operation. The rats in the Shenshuai Formula group, Buxu Formula group and Quxie Formula group were given the corresponding formula with the dose of 19.5 g/(kg·d), 9.5 g/(kg·d) and 11.0 g/(kg·d) respectively. The benazepril group was given benazepril with a dose of 1.05 mg/(kg·d). The sham operation group and the model group were given the same amount of normal saline per day. After model establishment, 6 rats died on the 7 th, 14 th and 21 st day, and their serum levels of creatinine(Scr) and urea nitrogen(BUN) were detected. The left kidney organ index was also calculated. The kidney tissue was detected by HE staining and Masson staining. The expressions of VEGF, VEGFR2 and a-SMA were detected by Western blot. The expressions of VEGF, VEGFR2 and a-SMAmRNA were detected by real-time quantitative RT-PCR. Results(1)Compared with those of the sham operation group, the serum levels of BUN, Scr, and left kidney organ index were increased in the model group, Shenshuai Formula group, Quxie Formula group, Buxu Formula group and benazepril group(P<0.05). Compared with those of the model group, the levels of BUN and Scr and kidney organ index of the Shenshuai Formula group,Quxie Formula group, Buxu Formula group and benazepril group were decreased(P<0.05). Compared with those of the other treatment groups,the Shenshuai Formula group’s levels of BUN, Scr and kidney organ index were significantly lower(P<0.05).(2)Renal pathological changes: Compared with those of the sham operation group, the interstitial injury index and collagen-positive area of the model group, Shenshuai Formula group, Quxie Formula group, Buxu Formula group and benazepril group increased(P<0.05). Compared with those of the model group, the kidney tubulointerstitial injury index and collagen positive area of the Shenshuai Formula, Quxie Formula group, Buxu Formula group and benazepril group were decreased(P<0.05). Compared with those of the treatment groups, the Shenshuai Formula group was more effective on reducing the tubulointerstitial injury index and collagen positive area(P<0.05).(3)Compared with those of the sham operation group, the expression levels of VEGF, VEGFR2, a-SMA protein and VEGF, VEGFR2 and a-SMA mRNA in the model group,Shenshuai Formula, Quxie Formula group, Buxu Formula group and benazepril group increased(P<0.05). Compared with those of the model group, the expressions of VEGF, VEGFR2, a-SMA and VEGF, VEGFR2 and a-SMA mRNA in the Shenshuai Formula group, Quxie Formula group, Buxu Formula group and benazepril group were decreased to different degrees(P<0.05). Compared with those of the other treatment groups, Shenshuai Formula group reduced the expressions of VEGF, VEGFR2, a-SMA protein and VEGF, VEGFR2, a-SMA mRNA. Conclusion Shenshuai Formula can improve renal function and alleviate renal pathological damage by inhibiting the protein and mRNA expressions of VEGF, VEGFR2 and a-SAM.