线粒体乙醛脱氢酶2对脓毒症心肌的保护作用及其机制研究

Myocardial depression is a major contributor to mortality and morbidity in patients with sepsis. One of the major molecular mechanisms that may be involved in myocardial depression during sepsis is mitochondrial dysfunction. Aldehyde dehydrogenase 2 (ALDH2), which is located in mitochondria, has been reported to be a critical modulator of cardiac myocyte survival and death. Our primarily study showed that increase in ALDH2 activity improved the mitochondrial function, which led to alleviate sepsis-induced myocardial dysfunction. However the exact mechanism is still unknown. The aims of present study are to identify which of the critical phosphorylation sites are contributed to the ALDH2 activity and caridoprotection by using site directed mutagenesis method; to investigate whether the cardiopretection of ALDH2 is due to a reduction in opening of mitochondrial permeability transition pore; to explore whether mitochondrial ATP sensitive potassium channel and mitochondrial connexin 43 are the key mitochondrial effectors/targets of ALDH2 by using pharmacological method and co-immunoprecipitation assay. This study may help to elucidate the molecular mechanism of ALDH2 against sepsis-induced myocardical injury, provide new ideas and theoretical basis for the treatment of septic cardiomyopathy.

心脏是脓毒症的易损靶器官之一，线粒体功能障碍是脓毒症心肌损害的主要原因。线粒体乙醛脱氢酶2（aldehyde dehydrogenase 2，ALDH2）在心肌保护中起着非常关键的作用。我们的预实验表明，增加ALDH2活性可改善心肌细胞线粒体功能，减轻脓毒症心肌损伤。但具体分子机制尚不明了。本课题将采用DNA定点突变的方法，分析影响ALDH2活性的关键磷酸化位点，确定哪些磷酸化位点是决定ALDH2心肌保护作用的关键；并进一步利用药理学、免疫共沉淀等实验技术，寻求与ALDH2相互作用的相关线粒体蛋白，阐明ALDH2的心肌保护作用是否与参与线粒体渗透性转换孔调节的关键蛋白（线粒体ATP敏感性钾离子通道、线粒体缝隙连接蛋白43）有关。通过对本课题的研究将阐明ALDH2对抗脓毒症心肌损伤的分子作用机制，从而为临床上心肌保护药物的研究提供新的思路和理论依据。

心脏是脓毒症的易损靶器官之一，线粒体功能障碍是脓毒症心肌损害的主要原因。乙醛脱氢酶2（aldehyde dehydrogenase 2，ALDH2）是线粒体内一种醛类氧化酶，在心肌保护中起着非常关键的作用。本课题主要研究增加ALDH2活性是否可改善心肌细胞线粒体功能，减轻脓毒症心肌损伤；并阐明其可能机制。.本课题采用腹腔注射脂多糖（lipopolysaccharide，LPS）建立雄性SD大鼠脓毒症模型，测定心功能指标。体外培养H9c2细胞，构建ALDH2 S279A真核表达质粒，观察心肌细胞生存率和凋亡发生率。比色法测定ALDH2和iNOS活性、心肌NO水平、以及MPT孔开放程度。WB法测定ALDH2、CX43、bid等蛋白表达情况。研究发现：（a）大鼠腹腔注射LPS（0.01-1 μg/mL）后每搏输出量和心输出量逐渐下降；ALDH2蛋白表达水平无明显改变，但ALDH2活性明显降低，且呈现浓度依赖性特征。（b）ALDH2特异性小分子激动剂Alda-1可增加脓毒症大鼠心肌组织ALDH2活性，同时抑制脓毒症大鼠心脏MPT孔开放和心功能的下降，降低大鼠死亡率；此作用可被MPT开放剂苍术苷所阻断。iNOS选择性抑制剂可部分阻断LPS诱导的心肌ALDH2活性下降，并可改善LPS诱导的心功能障碍。（c）Alda-1（20 μM）孵育H9c2细胞后，LPS引起的心肌线粒体功能和细胞生存率下降、凋亡增加等情况得到了明显抑制。将H9C2细胞转染ALDH2 S279A后，细胞ALDH2活性明显下降；同时可部分取消Alda-1对抗LPS诱导的心肌细胞损伤作用。（d）Alda-1孵育H9c2细胞后，mitoKATP通道开放程度和CX43线粒体转位明显增加。Alda-1可明显抑制LPS诱导的凋亡蛋白bid裂解，MPT孔的开放；而此作用可被mitoKATP通道抑制剂5-HD和Cx43 siRNA所部分阻断。.以上结果提示，S279磷酸化位点是影响ALDH2活性改变的关键。增加ALDH2活性可保护脓毒症大鼠心功能，其机制可能与促进下游CX43磷酸化水平增加，导致CX43线粒体转位、以及增加mitoKATP通道开放有关。本课题的研究结果将为临床上心肌保护药物的研究提供新的思路和理论依据。