Vitexin regulates the Epac1/Rap1 pathway to mediate protective ef-fects against hypoxia-reoxygenation injury in H9c2 cardiomyocytes
10.12092/j.issn.1009-2501.2024.10.002
- VernacularTitle: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
- Author:
Qin GAN
1
;
Xin WANG
;
Huanghua YANG
;
Liuyi DONG
Author Information
1. 合肥职业技术学院基础医学教研室,合肥 230000,安徽;安徽医科大学基础医学院药理学教研室,合肥 230032,安徽
- Keywords:
vitexin;
Epac1;
Rap1;
hypoxia-reoxy-genation injury
- From:
Chinese Journal of Clinical Pharmacology and Therapeutics
2024;29(10):1091-1099
- CountryChina
- Language:Chinese
-
Abstract:
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 path-way to protect cardiomyocytes from hypoxia-reoxy-genation injury.METHODS:The oxygen glucose de-privation(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 ex-pression 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 de-tected by immunofluorescence.The mRNA expres-sion 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[Ca2+]i content.The in-teraction between Epac1 and Rap1 in cells were de-tected by Co-IP.RESULTS:Compared with the nor-mal control group,after hypoxia for 5 h and reoxy-genation 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 ac-tive 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 in-hibitor(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:Hypoxia-reoxygenation can mediate the activation of Epac1/Rap1 signal pathway in cardiomyocytes.VT can pro-tect cardiomyocytes from hypoxia-reoxygenation in-jury by inhibiting Epac1/Rap1 signal pathway,down-regulating CaMK Ⅱ protein expression and promoting ERK phosphorylation.
