牡荆素调控Epac1/Rap1通路介导H9c2心肌细胞缺氧复氧损伤的保护作用机制  

Vitexin regulates the Epac1/Rap1 pathway to mediate protective effects against hypoxia-reoxygenation injury in H9c2 cardiomyocytes

作　　者：甘琴 王鑫 杨换花 董六一 GAN Qin;WANG Xin;YANG Huanghua;DONG Liuyi(Department of Basic Medicine,Hefei Technology College,Hefei 230000,Anhui,China;Department of Pharmacology,School of Basic Medicine,Anhui Medical University,Hefei 230032,Anhui,China)

机构地区：[1]合肥职业技术学院基础医学教研室,安徽合肥230000 [2]安徽医科大学基础医学院药理学教研室,安徽合肥230032

出　　处：《中国临床药理学与治疗学》2024年第10期1091-1099,共9页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：国家自然科学基金项目(81470432);安徽省高等学校自然科学研究项目(KJ2021A1387)。

摘　　要：目的:研究Epac/Rap1信号通路在H9c2细胞缺氧复氧损伤中的作用,并探明牡荆素(vitexin,VT)调控Epac/Rap1信号通路保护心肌细胞缺氧复氧损伤的作用机制。方法:采用氧糖剥夺(oxygen glucose deprivation,OGD)构建H9c2心肌细胞缺氧复氧损伤模型。实验随机分为7组:正常对照组、OGD组、OGD+VT组、OGD+Epac1激动剂(8-CPT)+VT组、OGD+Epac1抑制剂(ESI-09)+VT组、OGD+8-CPT+VT+PKA抑制剂(H-89)组、OGD+ESI-09+VT+H-89组。MTT检测细胞活力;LDH检测细胞损伤;Western blot检测H9c2细胞中Epac1及其下游Rap1-GTP、CaMK II和ERK蛋白的表达量;免疫荧光检测H9c2心肌细胞中Epac1和Rap1蛋白的表达;PCR实时荧光定量检测H9c2心肌细胞中Rap1和Epac1的m RNA表达;钙离子荧光探针(Fluo-3 AM)检测细胞内[Ca^(2+)]i含量;Co-IP检测细胞中Epac1与Rap1的相互作用。结果:与正常对照组相比,OGD组H9c2心肌细胞在缺氧5 h复氧1 h后,LDH释放量显著升高,心肌细胞细胞活力显著降低,Epac1蛋白表达显著升高,Rap1活化,Rap1-GTP上调。VT(10μmol/L)可显著抑制OGD后H9c2心肌细胞Epac1的激活,继而抑制其下游Rap1活性形式Rap1-GTP的表达,另外CaMK II蛋白表达下调,但ERK磷酸化增加,同时减轻心肌细胞内钙超载;8-CPT可抵消VT的作用,ESI-09与VT联合处理后具有协同作用;H-89对心肌细胞Epac1及其下游相关蛋白表达无影响。结论:缺氧复氧可介导心肌细胞Epac1/Rap1信号通路激活,VT通过抑制Epac1/Rap1信号通路,下调CaMKⅡ蛋白表达,促进ERK磷酸化,对心肌细胞缺氧复氧损伤起保护作用。AIM:To investigate the role of Epac/Rap1 signaling pathway in hypoxia-reoxygenation injury in H9c2 cells,and to explore the mechanism of vitexin regulating the Epac/Rap1 signaling pathway to protect cardiomyocytes from hypoxia-reoxygenation injury.METHODS:The oxygen glucose deprivation(OGD)model was established using H9c2 cardiomyocytes to simulate hypoxia-reoxygenation injury.The experiment was randomly divided into 7 groups:Normal control group,OGD group,OGD+VT group,OGD+8-CPT+VT group,OGD+ESI-09+VT group,OGD+8-CPT+VT+H-89 group,OGD+ESI-09+VT+H-89 group.Cell viability was measured by MTT.LDH was used to detect cell damage.The expression levels of Epac1 and its downstream Rap1-GTP,CaMK II and ERK proteins in H9c2 cells were detected by Western blot.The expression of Epac1 and Rap1 proteins in H9c2 cardiomyocytes was detected by immunofluorescence.The mRNA expression of Rap1 and Epac1 in H9c2 cardiomyocytes was quantitatively determined by real-time PCR.Calcium ion fluorescence probe(Fluo-3 AM)was used to detect intracellular[Ca^(2+)]i content.The interaction between Epac1 and Rap1 in cells were detected by Co-IP.RESULTS:Compared with the normal control group,after hypoxia for 5 h and reoxygenation for 1 h,the release of LDH,cell viability,Epac1 protein expression,Rap1 activation and RAP1-GTP up-regulation of H9c2 cardiomyocytes in OGD group were significantly increased.VT(10μmol/L)significantly inhibited the activation of Epac1 in H9c2 cardiomyocytes after OGD,and then inhibited the expression of downstream Rap1 active form Rap1-GTP.In addition,the expression of CaMK II protein was down-regulated,but ERK phosphorylation was increased,and intracellular calcium overload was alleviated.Epac1 agonist 8-CPT could counteract the effect of VT,and Epac1 inhibitor(ESI-09)combined with VT had synergistic effect.PKA inhibitor(Hmur89)had no effect on the expression of Epac1 and its downstream related proteins in cardiomyocytes.CONCLUSION:Hypoxiareoxygenation can mediate the activation of Epac1/Rap1 signal pathway in cardio

关 键 词：牡荆素 Epac1 Rap1 缺氧复氧损伤 

分 类 号：R285.5[医药卫生—中药学] R965.2[医药卫生—中医学]