自噬在慢性肾病血管钙化中的作用研究

Effects of autophagy on vascular calcification in chronic kidney disease

目的探索自噬与CKD血管钙化之间的关系,以及自噬在CKD血管钙化中的作用。方法首先将30只SD大鼠随机分为对照组(Control组,15只)和腺嘌呤及高磷饮食诱导的模型组(CKD组,15只)。免疫组化检测主动脉中α-SMA、RUNX2、LC3B及Beclin-1蛋白表达,新鲜主动脉做钙含量测定,进一步做它们之间的相关性分析。再构建β-GP诱导的主动脉平滑肌细胞(aorta smooth muscle cell,ASMC)钙化模型。电镜检测细胞内自噬小体,免疫荧光及Western blot检测LC3B蛋白。自噬抑制剂3-MA及激活剂RAP干预自噬后,采用免疫组化、Western blot检测ASMC中α-SMA、RUNX2蛋白表达变化,茜素红染色及钙含量检测ASMC钙化情况。结果模型组成功建立CKD血管钙化模型。与对照组相比,模型组主动脉α-SMA蛋白表达下降,RUNX2、LC3B、Beclin-1蛋白表达及钙含量升高。Beclin-1及LC3B蛋白表达分别与主动脉钙含量及RUNX2蛋白表达呈正相关。β-GP诱导ASMC自噬小体增加,LC3B荧光斑点增多,LC3BII蛋白表达增强(P<0.01)。RAP激活自噬后减少ASMC钙沉积及钙含量(P<0.05),减少RUNX2蛋白表达(P<0.05),促进α-SMA蛋白表达(P<0.05);3-MA抑制自噬后结果与之相反。结论CKD血管钙化同时存在自噬激活,自噬在CKD血管钙化中可能具有内源性保护作用,自噬有望成为CKD血管钙化新的治疗靶目标。

Objective To explore the association and role of autophagy in vascular calcification of chronic kidney disease.Methods Overall,30 SD rats were randomly divided into a control group(Control group,n=15)and adenine and high phosphorus diet-induced model group(CKD group,n=15).Immunohistochemistry was applied to detectα-SMA,RUNX2,LC3B and Beclin-1 in the aorta.Calcium content was measured to further analyze the correlation between them.Aorta smooth muscle cell(ASMC)calcification was induced byβ-GP.Autophagy activation was detected by electron microscopy,immunofluorescence,and Western blot.After 3-MA and RAP regulation of autophagy,immunohistochemistry and Western blot were used to detectα-SMA and RUNX2 protein expression,and alizarin red staining and calcium contents were used to detect calcification of ASMC.Results Compared with the control group,vascular calcification was established in the CKD group.RUNX2,LC3B and Beclin-1 proteins,and calcium content were higher in the CKD group than in the control group,whileα-SMA protein expression had declined.Correlation analysis showed that Beclin-1 and LC3B protein expression were positively correlated with aortic calcium content and RUNX2 protein expression,respectively.β-GP induced ASMC calcium deposition and osteogenic differentiation.β-GP induced an increase of autophagosomes,LC3B fluorescent spots,and LC3BII protein expression(P<0.01).After autophagy activation by RAP,calcium deposition and content(P<0.05)and RUNX2 protein expression(P<0.05)were reduced,whileα-SMA protein expression was promoted(P<0.05).After-autophagy inhibition by 3-MA,calcium deposition and content were increased(P<0.05),RUNX2 protein expression was promoted(P<0.05),andα-SMA protein expression was decreased(P<0.05).Conclusions Autophagy might play a protective role in CKD vascular calcification.Autophagy is expected to become a new therapeutic target for CKD vascular calcification.