大黄庶虫虫丸加减对大鼠肾间质纤维化的保护作用及机制

Protective Effect and Mechanism of Modified Dahuang Zhechong Wan on Renal Interstitial Fibrosis in Rats with Obstructive Nephropathy

目的:探讨大黄庶虫虫丸加减对梗阻性肾病大鼠肾间质纤维化的保护作用及机制。方法:采用单侧输尿管结扎(UUO)致大鼠肾间质纤维化模型,将50只SD大鼠随机分为5组,分别为假手术组,模型组,依那普利组(0. 001 g·kg^(-1)),大黄庶虫虫丸加减大、小剂量组(19,9. 5 g·kg^(-1))。术后第15天处死各组大鼠,收集血清,酶法测定血肌酐(SCr),尿素氮(BUN)水平,收集24 h尿液,连苯三酚红钼终点法测定24 h尿蛋白量(24 h-Upro);留取结扎侧肾组织行苏木素-伊红(HE)染色,马松(Masson)染色;采用免疫组化法(IHC)检测转化生长因子-β_1(TGF-β_1),纤连蛋白(FN),α-平滑肌肌动蛋白(α-SMA)表达;蛋白免疫印迹法(Western blot)检测TGF-β_1,p38丝裂原活化蛋白激酶(p38 MAPK),磷酸化p38 MAPK(p-p38 MAPK)蛋白表达。结果:与假手术组比较,模型组大鼠24 h-Upro,SCr,BUN含量明显增高(P <0. 01),光镜下肾组织损伤严重,TGF-β_1,FN,α-SMA含量明显升高(P <0. 05),TGF-β_1,p38 MAPK,p-p38 MAPK蛋白表达明显升高(P <0. 05);与模型组比较,大黄庶虫虫丸加减大、小剂量组24 h-Upro,SCr,BUN明显降低(P <0. 05),肾脏组织损伤减轻,TGF-β_1,FN,α-SMA含量明显降低(P <0. 05),TGF-β_1,p-p38 MAPK蛋白表达明显降低(P <0. 05)。结论:大黄庶虫虫丸加减方可通过降低FN,α-SMA的高表达,下调p38 MAPK信号通路中的p-p38 MAPK,TGF-β_1表达,减少细胞外基质过度沉积和肾脏固有细胞损伤来改善肾间质纤维化。

Objective:To explore the protective effect and mechanism of modified Dahuang Zhechong Wan on renal interstitial fibrosis in rats with obstructive nephropathy.Method:The unilateral ureteral ligation(UUO)-induced renal interstitial fibrosis model was adopted,50 SD rats were randomly divided into 5 groups:sham operation group,model group,enalapril group(0.001g·kg^-1),and high and low-dose modified Dahuang Zhechong Wan group(19,9.5g·kg^-1).Rats in each group were put to death on the 15 thday after operation.The serum levels of serum creatinine(SCr)and urea nitrogen(BUN)were collected by enzyme method.The 24-hour urine was collected for 24-hour urinary protein quantity(24 h-Upro)by pyrogallol red molybdenum end point.The kidney tissue was removed from the ligated side.Hematoxylin-eosin(HE)staining and Masson staining were performed;the expressions of transforming growth factor-β1(TGF-β1),fibronectin(FN)and α-smooth actin(α-SMA)were determined by immunohistochemistry(IHC).Expressions of TGF-β1,p38 mitogen-activated protein kinase(p38 MAPK)and phosphorylated p38 MAPK(p-p38 MAPK)were detected by Western blot.Result:Compared with Sham group,UUO group showed a significant increase in 24 h-Upro,SCr,and BUN(P<0.01),the renal tissue damage was severe under light microscope,and TGF-β1,FN,and α-SMA were increased obviously(P<0.05);TGF-β1,p38 MAPK,and p-p38 MAPK were increased obviously(P<0.05).Compared with the UUO group,24 h-Upro,SCr,BUN decreased obviously(P<0.05);renal tissue damage was alleviated;TGF-β1,FN and α-SMA decreased obviously(P<0.05);TGF-β1 and p-p38 decreased obviously(P<0.05).Conclusion:Modified Dahuang Zhechong Wan may improve renal interstitial fibrosis by reducing the high expressions of FN and α-SMA,down-regulating the expressions of p-p38 MAPK and TGF-β1 in p38 MAPK signaling pathway,and decreasing extracellular matrix over deposition and renal cell damage.