灯盏花素通过调控TGF-β1介导的Smad和ERK通路干预肾纤维化大鼠的作用机制研究

Mechanism of Breviscapine in Intervening Renal Fibrosis Rats Through Regulating TGF-β1-mediated Smad and ERK Pathway

目的 基于转化生长因子(TGF)-β1介导的Smad和ERK通路探讨灯盏花素对肾纤维化大鼠的干预作用及机制。方法 将60只雄性SD大鼠随机为正常组、模型组、秋水仙碱组(0.45 mg·kg^(-1))及灯盏花素低(5.5 mg·kg^(-1))、中(11 mg·kg^(-1))、高(22 mg·kg^(-1))剂量组,每组10只。采用灌胃腺嘌呤的方式建立肾纤维化模型,连续30 d。第31天开始给药干预,每日1次,连续30 d。采用分光光度法检测血清肌酐(Cr)、尿素氮(BUN)水平及BUN/Cr值;测定肾脏系数;采用HE染色法观察肾脏病理组织形态;采用免疫组化法检测肾脏组织中TGF-β1、Smad2、Smad3、ERK1和ERK2蛋白水平;采用RT-PCR法检测肾脏组织中TGF-β1、Smad2、Smad3、ERK1和ERK2 mRNA水平。结果 与正常组比较,模型组大鼠肾脏系数及血清Cr、BUN水平明显升高(P<0.05),BUN/Cr值明显减小(P<0.05);肾脏出现纤维化病变;肾脏组织中TGF-β1、Smad2、Smad3、ERK1和ERK2蛋白及mRNA水平明显升高(P<0.05)。与模型组比较,灯盏花素低、中、高剂量能明显降低大鼠肾脏系数及血清Cr、BUN水平(P<0.05),增加BUN/Cr值(P<0.05);改善肾脏纤维化病变;降低肾脏组织中TGF-β1、Smad2、Smad3、ERK1和ERK2的蛋白及mRNA水平(P<0.05)。结论 灯盏花素通过抑制TGF-β1介导的Smad和ERK通路来实现对肾纤维化大鼠的保护作用。

Objective To study the intervening effect of breviscapine on renal fibrosis(RF)rats and its mechanism based on TGF-β1-mediated Smad and ERK pathway. Methods Totally 60 healthy rats were randomly divided into normal group,model group,colchicine group(0.45 mg·kg^(-1))and breviscapine low-,medium-and high-dose groups(5.5,11,22 mg·kg,respectively),10 rats in each group. RF rat models were established by gavage administration of adenine for consecutive 30 days. The drug intervention was started on the 31st day,once a day,for consecutive 30 days. Spectrophotometric method was used to detect creatinine(Cr),blood urea nitrogen(BUN),and BUN/Cr. Renal weight was measured and renal index was calculated. Hematoxylin-eosin(HE)staining was used to observe the histopathological characteristics of renal tissue. Immunohistochemistry was used to determine the levels of TGF-β1, Smad2, Smad3, ERK1 and ERK2. RT-PCR was used to determine the mRNA levels of TGF-β1,Smad2,Smad3,ERK1 and ERK2. Results Compared with normal group,the renal index,the serum Cr and BUN levels were significantly increased(P<0.05)in the model group,the ratio of BUN/Cr was significantly reduced(P<0.05). The kidney appeared fibrosis,and the protein and mRNA expressions of TGF-β1,Smad2,Smad3,ERK1and ERK2 were significantly enhanced(P<0.05). Compared with model group,the low-,medium-and high-dose of breviscapine significantly reduced the renal index,the serum Cr and BUN level(P<0.05),increased the ratio of BUN/Cr(P<0.05),and improved the pathological changes of renal tissue. It was also found that the protein and mRNA expressions of TGF-β1,Smad2,Smad3,ERK1 and ERK2 were significantly reduced(P<0.05)in renal tissue. Conclusion Breviscapine has the protective effect on RF rats, and its mechanism may be related to the inhibition of TGF-β1-mediated Smad and ERK pathway.