大鼠心肌成纤维细胞通过增加基质金属蛋白酶2活性抑制心肌细胞缝隙连接功能  

Rat cardiac fibroblasts inhibit gap junction between cardiomyocytes through increasing matrix metalloproteinase 2 activity

作　　者：柏雪 高鸿 黄祥 严旭 胡廷菊 陈锐 安丽 宋雨婷 BAI Xue;GAO Hong;HUANG Xiang;YAN Xu;HU Tingju;CHEN Rui;AN Li;SONG Yuting(School of Anesthesiology,Guizhou Medical University,Guiyang 550002,China;Guizhou Hospital,Branch of the First Affiliated Hospital of Sun Yat-sen University,Guiyang 550025,China;Department of Anesthesiology,The Affiliated Hospi tal of Guizhou Medical University,Guiyang 550002,China)

机构地区：[1]贵州医科大学麻醉学院,贵州贵阳550002 [2]中山大学附属第一医院贵州医院,贵州贵阳550025 [3]贵州医科大学附属医院麻醉科,贵州贵阳550002

出　　处：《中国病理生理杂志》2024年第3期465-472,共8页Chinese Journal of Pathophysiology

基　　金：贵州省卫生健康委科学技术基金资助项目(No.Gzwkj2021-270)。

摘　　要：目的:探讨缺氧复氧(hypoxia/reoxygenation,H/R)后大鼠心肌成纤维细胞(cardiac fibroblasts,CFs)条件培养液对心肌细胞中缝隙连接蛋白43(connexin 43,Cx43)表达和缝隙连接功能的影响及其分子机制。方法:(1)将H9c2细胞随机分为5组:对照(control)组、常氧(normal)组、基质金属蛋白酶2抑制剂ARP-100(ARP)组、H/R组和H/R+ARP组。荧光划痕技术评价缝隙连接功能,Western blot实验检测Cx43的表达及磷酸化水平,明胶酶谱法测定条件培养液中基质金属蛋白酶的活性。(2)将SD大鼠随机分为control组、ARP组、缺血再灌注(I/R)组和I/R+ARP组,每组8只。微电极阵列技术采集心律失常发生类型和持续时间,免疫组织化学实验检测心肌组织中Cx43蛋白的表达及分布,Western blot实验检测Cx43的表达及磷酸化水平。结果:与control组相比,H/R组Cx43蛋白表达显著下降(P<0.01),磷酸化水平下调(P<0.01),荧光扩散范围变窄(P<0.01),MMP2活性增加(P<0.01);ARP-100可降低大鼠I/R模型中心律失常评分(P<0.01)。结论:H/R处理的大鼠CFs条件培养液可影响大鼠心肌细胞中Cx43的表达、磷酸化水平及缝隙连接功能,其机制可能与H/R诱导的条件培养液中MMP2活性增加有关,抑制MMP2活性可减少大鼠离体全心I/R模型中心律失常评分。AIM:To investigate the effect of conditioned medium from hypoxia/reoxygenation(H/R)-treated rat cardiac fibroblasts(CFs)on gap junction between cardiomyocytes and determine whether its mechanism is related to matrix metalloproteinase 2(MMP2)activity.METHODS:(1)H9c2 cells were randomly divided into five groups:control group,normal group,ARP-100 group,H/R group,and H/R+ARP-100 group.Scrape loading/dye transfer assay was used to assess the gap junction function.Western blot was used to detect the expression and phosphorylation levels of Cx43.Gelatin zymography assay was performed to measure MMP2 activity.(2)SD rats were randomly divided into control group,ARP-100 group,ischemia-reperfusion(I/R)group,and I/R+ARP-100 group,with 8 rats in each group.Microelectrode array technology was used to record the type and duration of arrhythmia.Immunohistochemistry experiment was performed to assess expression levels and distribution of Cx43 in myocardial tissues.RESULTS:Compared with the control group,the H/R group showed decreased protein expression of Cx43(P<0.01),narrowed distance of lucifer yellow diffusion(P<0.01),and increased MMP2 activity(P<0.01).ARP-100 attenuated H/R-induced gap junction dysfunction(P<0.05).The arrhythmia score was also reduced after perfusion with ARP-100(P<0.01).CONCLUSION:H/R-treated rat CFs can inhibit gap junction between cardiomyocytes,and its mechanism may involve increased MMP2 activity.

关 键 词：缺氧复氧 缺血再灌注 心肌细胞 缝隙连接蛋白43 基质金属蛋白酶 

分 类 号：R541[医药卫生—心血管疾病] R363.2[医药卫生—内科学] R329.28[医药卫生—临床医学]