肾元颗粒对db/db糖尿病肾病小鼠血管钙化的改善作用及其机制

Improvement effect of Shenyuan Granule on vascular calcification in db/db diabetic nephropathy mice and its mechanism

目的:通过高磷诱导db/db糖尿病肾病(DN)小鼠血管钙化,探讨肾元颗粒对db/db DN小鼠血管钙化的改善作用及其可能机制。方法:将20只SPF级db/db小鼠随机分为模型组和肾元颗粒组(n=10),同系背景野生型(WT)小鼠作为空白组(n=10)。给药12周后,取各组小鼠血清、肾脏和胸主动脉组织。ELISA法检测各组小鼠血清FGF23水平,HE和von Kossa法检测各组小鼠胸主动脉病理形态表现以及钙盐沉积情况,实时荧光定量PCR(Real-time PCR)法检测各组小鼠肾脏组织中Klotho mRNA和胸主动脉组织中Pit-1、Runx2及SM22αmRNA表达水平,Western blotting法检测各组小鼠肾脏组织中Klotho蛋白和胸主动脉组织中Pit-1、Runx2及SM22α蛋白表达水平,免疫组织化学法检测各组小鼠胸主动脉组织中Pit-1蛋白表达水平,免疫荧光法检测各组小鼠胸主动脉组织中SM22α和Runx2蛋白表达水平。结果:与空白组比较,模型组小鼠血清FGF23水平明显升高(P<0.05),肾脏组织中Klotho mRNA和蛋白表达水平明显降低(P<0.01),胸主动脉组织中Pit-1和Runx2 mRNA及蛋白表达水平明显升高(P<0.01),SM22αmRNA和蛋白表达水平明显降低(P<0.01),胸主动脉组织中Pit-1和Runx2蛋白表达水平明显升高(P<0.01),SM22α蛋白表达水平明显降低(P<0.01)。与模型组比较,肾元颗粒组小鼠血清FGF23水平降低(P<0.05),肾脏组织中Klotho mRNA和蛋白表达水平明显升高(P<0.01),胸主动脉组织中Pit-1和Runx2 mRNA及蛋白表达水平明显降低(P<0.01),胸主动脉组织中SM22αmRNA和蛋白表达水平明显升高(P<0.01)。结论:肾元颗粒可通过上调小鼠肾脏组织中Klotho蛋白表达水平,减少Pit-1表达水平,抑制FGF23/Pit-1信号通路,调节血管平滑肌细胞(VSMC)的表型转化,达到血管保护作用。

Objective:To investigate the improvement effect of Shenyuan Granule on the vascular calcification in the db/db diabetic nephropathy(DN)mice induced by high phosphorus and its possible mechanism.Methods:Twenty SPF db/db mice were randomly divided into model group(n=10)and Shenyuan Granule group(n=10),and the wild type(WT)mice were chosen as blank group(n=10).After treated for 12 weeks,the serum,kidney tissue and thoracic aorta tissue of the mice in various groups were taken.The serum FGF23 levels of the mice in various groups were detected by ELISA method;HE and von kossa staining were used to detect the pathomorphology of the thoracic aorta and calcium deposition of the mice in various groups;Real-time PCR method was used to detect the expression levels of Klotho mRNA in kidney tissue and Pit-1,Runx2 and SM22αmRNA in thoracic aorta tissue of the mice in various groups;Western blotting method was used to detect the expression levels of Klotho protein in kidney tissue and Pit-1,Runx2 and SM22αproteins in thoracic aorta tissue of the mice in various groups;immunohistochemisty method was used to detect the expression levels of Pit-1 protein in thoracic aorta tissue of the mice in various groups,and immunofluorescence method was used to detect the expression levels of SM22αand Runx2 proteins in thoracic aorta tissue of the mice in various groups.Results:Compared with blank group,the serum FGF23 level of the mice in model group was significantly increased(P<0.05),the expression levels of Klotho mRNA and protein in kidney tissue were significantly decreased(P<0.01),the expression levels of Pit-1 and Runx2 mRNA and proteins in thoracic aorta tissue were increased(P<0.01),the expression levels of SM22αmRNA and protein in thoracic aorta tissue were decreased(P<0.01),the expression levels of Pit-1 and Runx2 proteins in thoracic aorta tissue were increased(P<0.01),and the expression level of SM22αprotein in thoracic aorta tissue was decreased(P<0.01).Compared with model group,the serum FGF23 level of the mice in Shenyuan Granule group was decreased(P<0.05),the expression levels of Klotho mRNA and protein in kidney tissue were increased(P<0.01),the expression levels of Pit-1 and Runx2 mRNA and protein in thoracic aorta tissue were decreased(P<0.01),and the expression levels of SM22αmRNA and protein in thoracic aorta tissue were increased(P<0.01).Conclusion:Shenyuan Granule can up-regulate the expression level of Klotho protein in kidney tissue of the mice,reduce the expression level of Pit-1,inhibit the FGF23/Pit-1 signaling pathway,and regulate the phenotypic transformation of vasular smooth muscle cell(VSMC)to achieve its protective effect on the blood vessels.