通络益肾方对UUO大鼠肾间质纤维化及piezo1的影响

Effect of Tongluo Yishen Formula on renal interstitial fibrosis and piezo1 in UUO rats

目的探讨通络益肾方对单侧输尿管结扎(unilateral ureteral obstruction,UUO)大鼠肾脏纤维化及压电式机械敏感离子通道组件1(piezo-type mechanosensitive ion channel component 1,piezo1)的影响。方法以UUO大鼠为模型,以通络益肾方为干预手段,以缬沙坦为阳性药,连续给药14天。观察大鼠血肌酐和尿素氮变化。采用苏木素—伊红(hematoxylin-eosin,HE)染色,观察大鼠肾脏病理形态学变化。采用Ⅲ型胶原免疫组化评估大鼠肾间质纤维化程度。采用CD34免疫荧光观察大鼠肾脏肾小管周毛细血管(peritubular capillary,PTC)变化。采用免疫组化、免疫蛋白印记法和转录PCR观察大鼠肾脏piezo1及其下游分子mRNA以及蛋白表达改变。结果与假手术组相比,模型组血清肌酐和尿素氮水平升高(P<0.05),与模型组相比,缬沙坦组和通络益肾方组血清肌酐(P<0.05)和尿素氮(P>0.05)较低。与假手术组相比,模型组镜下观察肾间质纤维化改变明显,PTC明显减少,piezo1表达增加,经通络益肾方和缬沙坦干预后UUO大鼠肾脏间质纤维化程度改善,PTC增多,piezo1表达减少。与假手术组相比,模型组piezo1蛋白表达显著增加(P<0.05),通络益肾方和缬沙坦干预均能降低piezo1蛋白表达(P<0.05);与假手术组相比,模型组大鼠肾组织胎盘生长因子(placental growth factor,PlGF)表达增多(P<0.05);与模型组相比,缬沙坦组和通络益肾方组PlGF表达增多(P<0.05);模型组PlGF的受体血管内皮生长因子受体1(FMS-like tyrosine kinase 1,flt1)表达较假手术组减少(P<0.05),缬沙坦组与通络益肾方组与模型组相比flt1表达水平的升高。结论通络益肾方能明显改善UUO大鼠肾间质纤维化进展,其机制可能是通过下调piezo1蛋白表达,上调ADM、PlGF及其受体flt1表达,减轻PTC损伤,起到拮抗肾间质纤维化的作用。

Objective To explore the effect of Tongluo Yishen Formula on renal fibrosis and piezo1 in rats with unilateral ureteral obstruction(UUO).Methods Using UUO rats as the model,Tongluo Yishen Formula as the intervention method,and valsartan as the positive control substance,the drug was administered continuously for 14 days,to observe the changes in blood creatinine and urea nitrogen in rats.The method of hematoxylin-eosin(HE)staining was used to observe the pathological and morphological changes of kidneys in rats.The method of type III collagen immunohistochemistry were used to evaluate the degree of renal interstitial fibrosis in rats.Immunofluorescence was used to detect CD34 for observing the changes of peritubular capillry(PTC)in kidneys.The methods of immunohistochemistry,Western blot,and RT-PCR were used to observe the changes in mRNA and protein expression of piezo1 and its downstream molecules.Results Compared with the sham group,creatinine and urea nitrogen levels in the model group were increased in serum(P<0.05).Compared with the model group,the serum creatinine(P<0.05)and urea nitrogen(P>0.05)in the valsartan group and the Tongluo Yishen Formula group were decreased.Compared with the sham group,the model group showed significant changes in renal interstitial fibrosis under microscope,with a significant decrease in PTC and an increase in piezo1 expression.After intervention with Tongluo Yishen Formula and valsartan,the degree of renal interstitial fibrosis in UUO rats were improved,with an increase in PTC and a decrease in piezo1 expression.Compared with the sham group,the expression of piezo1 protein in the model group was significantly increased(P<0.05),and both Tongluo Yishen Formula and valsartan intervention could reduce the expression of piezo1 protein(P<0.05).Compared with the sham group,the expression of PlGF in the renal tissue of the model group rats were increased(P<0.05).Compared with the model group,the expression of PlGF in the valsartan group and the Tongluo Yishen Formula group were increased(P<0.05).The receptor flt1 expression of PlGF in the model group were decreased compared with the sham group(P<0.05),while the expression level of flt1 were increased in the valsartan group and the Tongluo Yishen Formula group compared with the model group.Conclusion Tongluo Yishen Formula can significantly improve the progression of renal interstitial fibrosis in UUO rats.Its mechanism may be related to downregulating the expression of piezo1 protein,upregulating the expression of ADM,PlGF and their receptors flt1,alleviating PTC damage,and antagonizing renal interstitial fibrosis.