去甲斑蝥素对梗阻性肾病Wnt/β-catenin表达的影响

Effect of norcantharidin on Wnt/β-catenin expression in obstructive nephropathy

目的:观察去甲斑蝥素(NCTD)对梗阻性肾病(UUO)大鼠模型及人近端肾小管上皮细胞(HK-2)纤维化模型Wnt/β-catenin表达的影响。方法:(1)动物实验:第一批SD大鼠随机分为假手术组(Sham组)、UUO 3d组、UUO 7d组及UUO 14d组,每组各4只。将第二批SD大鼠随机分为假手术组(sham)、模型组(UUO 14d)、NCTD 0.05 mg/kg干预组,NCTD 0.1 mg/kg干预组,每组4只。(2)细胞实验:体外培养HK-2细胞,分对照组、转化生长因子β1(TGF-β1)5 ng/ml刺激组、TGF-β1 5 ng/ml刺激+NCTD(2.5 mg/L、5 mg/L)干预组。免疫组化、Western Blot检测Wnt4、β-catenin蛋白表达情况,Real-time PCR检测Wnt4、β-catenin mRNA表达情况。结果 :随着UUO大鼠梗阻时间延长,β-catenin蛋白表达逐渐升高,UUO14 d表达最强。与UUO组相比,NCTD组Wnt4、β-catenin蛋白及mRNA表达明显下降(P<0.05);与对照组相比,TGF-β1刺激组Wnt4、β-catenin蛋白表达上调;与TGF-β1刺激组相比,NCTD不同浓度干预组Wnt4、β-catenin蛋白表达下调,且呈浓度依赖(P<0.05)。结论:NCTD干预可以下调梗阻性肾病大鼠肾组织及TGF-β1刺激的HK-2细胞中Wnt4和β-catenin的表达。

Objective： To observe the effect of norcantharidin（ NCTD） on the expression of β-catenin and Wnt4 in unilateral ureter obstructive（ UUO） rats model and human proximal renal tubular epithelial cells（ HK-2） fibrosis model.Methodology： Firstly,sixteen male Sprague-Dawley rats were randomly divided into four groups including Sham operation group,UUO 3 days group,UUO 7 days group and UUO 14 days group（ 4 in each group）. Secondly,another sixteen male Sprague-Dawley rats were also randomly divided into four groups： Sham operation group,UUO 14 days group,NCTD（ 0. 05 mg / kg,0. 1 mg / kg） interference group. At last,HK-2 cell were cultured and divided into control group,TGF-β1（ 5 ng /ml） stimulation group,TGF-β1（ 5 ng / ml） stimulation and followed by NCTD（ 2. 5 mg / L,5 mg / L） interference group.Immunohistochemical staining,Western blotting and Real-time PCR were used to detect the protein and mRNA expression of wnt4 and β-catenin. Results： With prolong of ureter obstruction time,the expression of β-catenin protein increased gradually,it was the highest level in UUO 14 days group. So we chose the rat model with UUO 14 days in the following experiments,Compared with the UUO group,protein and mRNA expressions of β-catenin and Wnt4 were decreased in NTCD group,the difference is statistically significant. NTCD can also decrease the protein expression of β-catenin and Wnt4 in the HK-2 cells with TGF-β1 stimulation in the manner of concentration depedence. Conclusion： NTCD can down regulate theβ-catenin and Wnt4 expression in vivo and in vitro.