1,25(OH)_(2)D_(3)对柯萨奇病毒B3诱导小鼠心肌炎症反应的影响及机制  

Effect and mechanism of 1,25(OH)_(2)D_(3) on myocardial inflammation induced by Coxsackie virus B3 in mice

作　　者：刘媛媛[1] 董美娟[1] 卜艳龙 崔兆辉[1] Liu Yuanyuan;Dong Meijuan;Bo Yanlong;Cui Zhaohui(Department of Endocrinology,the Affiliated Huai′an No.1 People′s Hospital of Nanjing Medical University,Huai′an 223300,China)

机构地区：[1]南京医科大学附属淮安第一医院内分泌科,淮安223300

出　　处：《中华医学杂志》2023年第36期2881-2888,共8页National Medical Journal of China

基　　金：国家自然科学基金青年基金(82000475)

摘　　要：目的探讨1,25二羟维生素D_(3)[1,25(OH)_(2)D_(3)]对柯萨奇病毒B3(CVB3)诱导小鼠心肌炎症反应的影响及机制。方法将雄性野生型(WT)小鼠和1α羟化酶基因敲除[1(OH)ase^(-/-)]小鼠分为WT组、WT+CVB3组、1(OH)ase^(-/-)+CVB3组和1(OH)ase^(-/-)+CVB3+VD_(3)组,每组8只。实时荧光定量PCR测定促炎症因子白细胞介素(IL)1β、IL-6、干扰素γ(IFN-γ)和肿瘤坏死因子α(TNF-α)mRNA水平;HE染色观察心肌组织病理学改变;TUNEL染色及流式细胞仪检测心肌细胞凋亡;Fluo-4/AM荧光探针检测心肌细胞内的钙离子含量;Western印迹法检测钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)及内质网应激相关葡萄糖调节蛋白78(GRP78)、CCAAT/增强子结合蛋白同源蛋白(CHOP)的表达水平。结果与WT组相比,WT+CVB3组小鼠心肌中促炎症因子IL-1β(14.88±3.32比1.03±0.02,P=0.009)、IL-6(7.00±1.09比1.81±0.18,P=0.005)、IFN-γ(4.70±1.11比1.34±0.34,P=0.006)、TNF-α(17.20±3.22比1.02±0.12,P<0.001)mRNA水平均升高,炎症细胞浸润增多,心肌细胞凋亡率增加(16.66%±1.09%比7.85%±1.12%,P=0.012),心肌细胞胞质中游离Ca^(2+)增加(2.98±1.05比0.96±0.10,P=0.006),磷酸化CaMKⅡ(pCaMKⅡ)(1.97±0.34比1.00±0,P<0.001)、GRP78(1.78±0.19比1.00±0,P=0.005)及CHOP(1.62±0.09比1.00±0,P=0.002)蛋白表达增加;上述心肌细胞损伤指标在1,25(OH)_(2)D_(3)缺乏的1(OH)ase^(-/-)+CVB3组更显著;而在给予1,25(OH)_(2)D_(3)补充的1(OH)ase^(-/-)+CVB3+VD_(3)组,上述心肌细胞损伤指标均改善(均P<0.05);此外,CaMKⅡ特异性抑制剂可同样降低CVB3感染小鼠的心肌损伤和细胞凋亡率。结论1,25(OH)_(2)D_(3)缺乏可通过CaMKⅡ过度活化加重小鼠心肌炎症反应。Objective To explore the effect and mechanism of 1,25(OH)_(2)D_(3) on myocardial inflammation induced by Coxsackie virus B3(CVB3)in mice.Methods Wild type(WT)and 1α-hydroxylase knockout[1(OH)ase^(-/-)]male mice were divided into four groups:WT group,WT+CVB3 group,1(OH)ase^(-/-)+CVB3 group and 1(OH)ase^(-/-)+CVB3+VD_(3) group,with 8 mice in each group.The indicators for evaluating myocardial cell injury were examined by different methods.The mRNA levels of pro-inflammatory cytokines[interlenkin(IL)-1β,IL-6,interferonγ(IFN-γ)and tumor necrosis factorα(TNF-α)]were determined by quantitative real-time PCR.Hematoxylin-eosin(HE)staining was used to observe the myocardial histopathological changes.The apoptosis of myocardial cells was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)staining and flow cytometry.Fluo-4/AM fluorescence probe was used to detect intracellular calcium ion content.Meanwhile,the expression levels of Ca^(2+)/Calmodulin-dependent protein kinaseⅡ(CaMKⅡ)protein as well as endoplasmic reticulum stress-related proteins like glucose-related protein 78(GRP78)and C/EBP homologous protein(CHOP)in the myocardial tissues were detected by Western blot.Results Compared with WT group,the mRNA levels of pro-inflammatory factors increased in the cardiomyocytes of mice in WT+CVB3 group,including IL-1β(14.88±3.32 vs 1.03±0.02,P=0.009),IL-6(7.00±1.09 vs 1.81±0.18,P=0.005),IFN-γ(4.70±1.11 vs 1.34±0.34,P=0.006)and TNF-α(17.20±3.22 vs 1.02±0.12,P<0.001).Similarly,the infiltration of inflammatory cells,and the apoptosis rate of cardiomyocytes elevated(16.66%±1.09%vs 7.85%±1.12%,P=0.012).The level of calcium ions in myocardial cytoplasm was significantly higher in WT+CVB3 group than that in the WT group(2.98±1.05 vs 0.96±0.10,P=0.006).Likewise,the expression levels of pCaMKⅡ(1.97±0.34 vs 1.00±0,P<0.001),GRP78(1.78±0.19 vs 1.00±0,P=0.005)and CHOP(1.62±0.09 vs 1.00±0,P=0.002)in WT+CVB3 group up-regulated.The above myocardial cell injury markers were more sign

关 键 词：病毒性心肌炎 钙-钙调素依赖性蛋白激酶Ⅱ 炎症 内质网应激 小鼠 

分 类 号：R542.21[医药卫生—心血管疾病]