RS47, 642D和Metformin對H9c2細胞缺氧再灌流之保護作用

Abstract

背景：組織缺血後再灌流引起的損傷在臨床上很常見，病人施行心血管手術或器官移植後，或於心肌梗塞、冠狀動脈阻塞及缺血性中風的情況下，都可能引起再灌流損傷，影響預後。希望能研究利用自然界豐富的植物成分及抗糖尿病用藥對心臟的保護作用。本篇主要研究類黃酮成分RS47、642D及降血糖藥物metformin對於H9c2細胞缺氧再灌流造成的損傷，是否具保護作用，並探討其可能的機轉。 方法及結果：H9c2細胞為胚胎大鼠心臟細胞株，利用缺氧艙及無糖無血清的培養液造成6小時的缺氧，之後再灌流12小時。實驗分成不加藥組 (vehicle) 及加藥組 (RS47、642D、metformin) ，加藥組為缺氧前1小時將不同濃度的藥物加入培養液，作用1小時，實驗結束後利用MTT assay觀察細胞存活比率，並利用西方點墨法、PI3K-Akt inhibitor、AMPK inhibitor、ERK inhibitor及LDH assay，更進一步探討RS47、642D及metformin的作用機轉。從MTT assay的結果發現，加藥組顯著的增加了細胞存活率。而西方點墨法觀察藥物相關蛋白質表現量，發現RS47組、642D組及metformin組Akt、AMPK及ERK的磷酸化皆顯著增加；另外，p38、Bax的磷酸化顯著減少，GSK-3β的磷酸化顯著增加。之後利用PI3K-Akt inhibitor LY294002、AMPK inhibitor Compound C及ERK inhibitor PD98059，發現LY294002及Compound C可以抑制三種藥物的細胞存活率增加，PD98059可以抑制metformin的細胞存活率增加。接著利用LDH觀察細胞壞死，其結果與MTT assay符合。 結論：RS47、642D及metformin對於H9c2細胞缺氧再灌流損傷具有保護作用，RS47及642D可能的機轉是透過Akt和AMPK活化，而metformin的機轉可能是經由活化Akt、ERK及AMPK，以減少細胞凋亡及壞死途徑，降低細胞再灌流損傷引起的細胞死亡。

Background：Reperfusion injury during recovery phase of tissue ischemia is important in many clinical situations such as cardiac transplantation, myocardial infarction and stroke, which influence outcome of the patients. We are interested in whether the natural ingredients and anti-diabetic medicines are protective against ischemic heart disease. The aim of the present study was to investigate the protective effects of flavonoids and metformin on H9c2 hypoxia-reoxygenation induced injury. Methods and Results：We used rat heart-derived cell line, H9c2, for the experiments. Cells were placed in hypoxia chamber with no glucose no serum medium to induce hypoxia 6 hr, then changed to normoxic condition for 12 hours. Cells were divided into vehicle and drug group. In drug group, RS47, 642D and metformin were added in the medium 1 hr before hypoxia. MTT assay was used to show cell viability of hypoxia reoxygenation. Western blot、PI3K-Akt inhibitor LY294002、ERK inhibitor PD98059 and AMPK inhibitor Compound C were also used in the present study. In MTT assay, cell viability was significantly increased in the drug group when compared to sham group. Western blot analysis revealed that p-Akt、p-AMPK and p-ERK were increased in the drug group, p-p38 and Bax were decreased, and p-GSK-3β was elevated. The use of Compound C significantly inhibited the protective effect of RS47、642D and metformin. We also measured LDH release, the results matched MTT assay. Conclusion：RS47, 642D and metformin signigicantly increased the cell viability in H9c2 cell hypoxia reoxygenation injury, and this was attributed to a decrease of cell apoptosis and necrosis. The decreased reperfusion injury was related to activation of Akt、AMPK and ERK.