一氧化碳对CYP2E1介导的微粒体酒精氧化应激的保护作用

CARBON MONOXIDE PROTECTS MICROSOMES FROM ETHANOL-DERIVED OXIDATIVE STRESS MEDIATED BY CYP2E1

目的探讨诱导I型血红素氧合酶(heme oxygenase-1,HO-1)催化血红素产生的代谢产物一氧化碳(CO)对大鼠肝微粒体细胞色素P4502E1(cytochrome P4502E1,CYP2E1)酶活性影响及其介导的酒精性氧化应激的保护作用。方法超速离心法制备大鼠肝脏微粒体,加入血红素(hemin)、血红蛋白(Hb)、不同剂量的CO释放剂CORM-2(carbonmonoxide-releasing molecules-2),检测CYP2E1酶活性;在微粒体酒精氧化反应体系中加入血红素、血红蛋白及CORM-2,检测微粒体超氧化物歧化酶(SOD)活力及谷胱甘肽(GSH)、丙二醛(MDA)及氧自由基(ROS)水平。结果 hemin处理后肝微粒体CYP2E1的酶活性下降,联合Hb后CYP2E1酶活性明显升高,外源性CO则显示出类似的CYP2E1酶抑制效应;针对乙醇孵育的肝微粒体,hemin处理后明显抑制了肝微粒体MDA和ROS水平的升高,有效维持了GSH水平和SOD活力,联合Hb处理后明显抑制了上述保护效应,外源性CORM-2也显示出类似的保护效应,而灭活的CORM-2则无明显的保护效应。结论 CO对大鼠肝微粒体酒精性氧化应激具有保护作用,其机制可能与CO对CYP2E1酶活性的直接抑制有关。

Objective To investigate the effect of carbon monoxide derived from hemeoxygenase-1（HO-1） metabolism on CYP2E1（cytochrome p4502E1） activity,and the protective role on ethanol-induced oxidative stress mediated by CYP2E1.Methods Liver microsomes isolated form Sprague-Dawley rat using ultracentrifugation technique,were treated with hemin,Hb or different dose of carbon monoxide-releasing molecule-2（CORM-2） and the catalytic activity of CYP2E1 was detected.In addition,the activity of superoxide dismutase（SOD）,as well as the levels of glutathione（GSH）,malondialdehyde（MDA） and reactive oxygen species（ROS） were assayed when incubated with hemin,Hb or CORM-2.Results Hemin treatment significantly inactivated CYP2E1,which was mimicked by CORM-2 in dose-dependent manner but blocked by Hb.Furthermore,hemin and CORM-2 evidently inhibited the increase of MDA and ROS,and the GSH depletion and SOD inactivation in microsomes induced by ethanol.Likewise,Hb abolished the protective effect of hemin and inactivated CORM-2 failed to exhibit any protective role.Conclusion Carbon monoxide prevented mcrosomes from ethnol-induced oxidative stress,which might be involved in its direct inhibition on CYP2E1.