1,25-（OH）2D3通过Ca2+/CaM信号调控内皮细胞自噬抑制动脉粥样硬化钙化  

1,25-（OH）2D3 regulates HUVEC autophagy and inhibits atherosclerotic calcification through Ca2+/CaM signaling

作　　者：杨萍[1] 崔星钢 李波[1] 许尧 王中群[1] 仲威[1] 邵晨[1] 严金川[1] YANG Ping;CUI Xinggang;LI Bo;XU Yao;WANG Zhongqun;ZHONG Wei;Shao Chen;YAN Jinchuan(Department of Cardiology,Affiliated Hospital of Jiangsu University,Zhenjiang,Jiangsu 212001,China)

机构地区：[1]江苏大学附属医院心内科

出　　处：《中国动脉硬化杂志》2018年第11期1091-1098,共8页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金(81670405);江苏省自然科学基金(BK20161355);镇江市社会发展项目(2017030);江苏省研究生科研创新计划(KYCX17_1818)

摘　　要：目的探讨1,25二羟基维生素D3(1,25-(OH)2D3)是否通过信号调控人脐静脉内皮细胞(HUVEC)自噬抑制细胞钙化及小鼠动脉粥样硬化斑块内钙化。方法 (1)将体外培养的HUVEC高脂刺激自噬后予1,25-(OH)2D3干预后,通过荧光显微镜、透射电镜、蛋白免疫印记(Western blot)检测细胞自噬水平;利用慢病毒过表达或者沉默钙调蛋白(CaM),运用Western blot、茜素红染色法、钙检测试剂盒检测细胞钙化指标骨形态发生蛋白2(BMP-2)、碱性磷酸酶(ALP)。(2)取6~8周龄Apo E-/-雄性小鼠高脂饲养形成动脉粥样硬化模型,1,25-(OH)2D3灌胃后尾静脉注射CaM过表达或者沉默慢病毒,Western blot检测组织自噬及钙化水平指标;扫描电镜观察主动脉斑块自噬小体及钙沉积; HE及Von Kossa染色法分别检测小鼠主动脉粥样硬化及钙化程度。结果体外实验显示,1,25-(OH)2D3处理后细胞自噬增强,Ca(2+与CaM上调;与对照组相比,CaM过表达组自噬增强,细胞钙化减轻,ApoE-/-小鼠斑块内钙化明显被抑制; CaM沉默组细胞自噬流不通畅,细胞钙沉积增加; Apo E-/-小鼠斑块内钙化明显增加(P<0.01)。结论 1,25-(OH)2D3通过Ca2+/CaM信号调控HUVEC自噬抑制细胞钙化及小鼠动脉粥样硬化斑块钙化。Aim To explore whether 1,25-(OH)2D3 can regulate the autophagy and calcification of human umbilical vein endothelial cells( HUVEC) and the calcification of mouse atherosclerotic plaque through Ca2+/CaM signal.Methods( 1) HUVEC were incubated in high fat microenvironment to stimulate autophagy. The autophagy level was detected by Western blot,fluorescence microscopy,transmission electron microscopy after HUVEC being stimulated by 1,25-(OH)2D3. Lentivious were used to overexpress or silence calmodulin( CaM),and the calcification was tested by Western blot,calcium ion detection kit,Alizarin red.( 2) Apo E-/-mice fed with high fat diet were randomly divided into 3 groups: control group( 1,25-(OH)2D32.5 μg/( kg·d) dissolved in 0.1 m L soybean oil,Gavage); CaM overexpressed group( control +CaM overexpressed lentivirus,Caudal vein injection) and CaM silenced group( control + CaM silenced lentivirus,Caudal vein injection). Scanning electron microscope was used to observe autophagosomes and calcification of the aorta. HE and Von Kossa staining were used to detect the As degree and calcification of the aorta,respectively.Results The autophagy was promted and the expression of Ca2+ and CaM were up-regulated after HUVEC being stimulated by 1,25-(OH)2D3. Compared with the control group,CaM overexpressed group showed enhanced autophagy and reduced calcification in HUVEC and As plaque in Apo E-/-mice. While the CaM silencing group showed autophagy dysfunction,increased calcium depositions in HUVEC and plaques. Conclusion 1,25-(OH)2D3 regulates HUVEC autophagy to inhibit cell calcification and plaque calcification through Ca2+/CaM signaling.

关 键 词：1 25二羟基维生素D3 人脐静脉内皮细胞 自噬 钙调蛋白 动脉粥样硬化钙化 

分 类 号：R363[医药卫生—病理学]