虾青素对缺氧复氧心肌细胞的保护作用及机制研究

The Protective Effects and Potential Mechanisms of Astaxanthin on Myocardial Hypoxia/Reoxygenation Injury

目的探讨虾青素(Astaxanthin,AST)对缺氧复氧(H/R)心肌细胞保护作用及对PI3K/AKT/HMGB1通路影响。方法原代心肌细胞随机分为3组:对照组、H/R组与H/R+AST处理组;机制探讨中给予PI3K/AKT抑制剂(LY294002)干预。4 h缺氧联合6 h复氧构建H/R损伤,按分组情况给予AST处理。检测心肌细胞存活率、心肌损伤酶与促炎症介质含量;SOD/MDA联合DHE探针观察氧化应激;流式细胞术检测凋亡;Western blotting检测蛋白表达。结果 AST改善H/R诱导的心肌细胞损伤,表现为细胞活性上调、LDH/CK-MB酶含量降低。此外,AST可减轻心肌细胞凋亡、抑制IL-6/TNF-α释放、降低ROS产生、增加SOD活性并下调MDA含量。在机制研究中,AST激活PI3K/AKT并抑制HMGB1表达,而阻断PI3K/AKT后AST心肌保护功能被逆转。结论 AST通过PI3K/AKT/HMGB1依赖性途径在H/R心肌细胞中发挥保护功能。

Objective To investigate roles of astaxanthin（AST） on hypoxia/reoxygenation（H/R）-exposed cardiomyocytes and the possible mechanisms implicated with PI3 K/AKT/HMGB1 pathway. Methods Cultured neonatal rat cardiomyocytes（NRCMs） are allocated into three groups（n = 3）： control group（Con）, H/R group and H/R plus AST administration group（H/R＋AST group）. In addition, the PI3 K/AKT inhibitor（LY294002） is added in mechanistic experiments. Four hours of hypoxia followed with 6 h of reoxygenation are carried out to induce H/R injury in isolated NRCMs, paralleling AST administration（10 μm） in indicated group. Cell viability and necrotic enzymes（LDH/CK-MB） activities are measured by CCK-8 and ELISA assay, respectively. The responses of inflammation, apoptosis, ROS and molecular alternations are systematically estimated. Results AST treatment contributed to attenuated NRCMs＇ damage caused by H/R, as exhibited by elevated cellular viability and reduced activities of LDH/CK-MB（H/R＋AST group vs. H/R group, P〈0.05）. Moreover, the upregulated inflammation, apoptosis and ROS are significantly reverted by AST due to diminished apoptotic rate, limited releases of IL-6/TNF-α and repressed ROS generation in concert with MDA/SOD alternation. Mechanistic evaluations demonstrated that AST markedly promoted the levels of pro-survival PI3 K/AKT axis but repressed HMGB1 expression, and blocking PI3 K/AKT via pharmacological inhibitor blunted H/R-inhibitory effects rendered by AST under H/R. Conclusion AST ameliorates myocardial H/R injury in part via a PI3 K/AKT/HMGB1-dependent avenue.