乙醛脱氢酶2改善亚急性酒精中毒对急性心肌梗死损害的作用及机制研究

Protective effects of aldehyde dehydrogenase-2 mediated by PI3K/Akt signal pathway on acute myocardial infarction in mice with sub-acute alcoholism

目的观察亚急性酒精中毒对急性心肌梗死（acute myocardial infarction, AMI）小鼠心脏结构和功能的影响，探讨乙醛脱氢酶2（aldehyde dehydrogenases-2, ALDH2）在其中的作用及机制。方法将20只ALDH2基因野生型小鼠（wild type，WT）和32只敲除型小鼠（knockout type，KO）随机（随机数字法）分为四组，即WT组（n＝10）、野生型＋乙醇组（WT＋E）（n＝10）、KO组（n＝16）、基因敲除＋乙醇组（KO＋E）（n＝16），其中WT＋E组及KO＋E组经口灌胃大剂量乙醇[2 g/（kg·d），连续8 d]，WT组及KO组在相同时间经口灌胃等量生理盐水。乙醇干预结束后所有小鼠均建立AMI模型，于建模第7天检测血清乙醇浓度、肝功能、心功能、心肌梗死面积、ALDH2活性、caspase-3 mRNA及PI3K、p-Akt蛋白的表达水平。采用SPSS 17.0统计软件进行分析，计量资料采用单因素方差分析，计数资料采用χ2检验。结果（1）AMI建模后第7天上述四组小鼠病死率依次为20.0 %、30.0 %、31.3 %、37.5 %，差异无统计学意义（P〉0.05）。（2）WT＋E组小鼠血清乙醇浓度（34.99±4.38）mg/dL和KO＋E组（38.70±6.35）mg/dL显著高于WT组（26.72±4.22）mg/dL和KO组（27.19±5.06）mg/dL，差异有统计学意义（均P〈0.05）；同时，WT＋E组和KO＋E组总胆红素、谷丙转氨酶、谷草转氨酶显著高于WT组和KO组，差异有统计学意义（均P〈0.05）。（3）WT组小鼠FS值（57.37±10.21）%、WT＋E组（54.63±13.18）%均高于KO组（39.15±11.86）%、KO＋E组（36.78±15.33）%，差异有统计学意义（均P〈0.05）；WT组小鼠LVEF值（68.58±11.68）%、WT＋E组（65.16±13.34）%均高于KO组（42.44±14.33）%、KO＋E组（40.12±15.72）%，差异有统计学意义（均P〈0.05）；四组小鼠LVEDD差异无统计学意义（P〉0.05）。（4）四组小鼠心肌梗死面积依次为（20.32±10.03）%、（34.16±11.46）%、（51.60±13.52）%、（66.78±12.10）%，差异均有统计学意义（均P〈0.05）。（5）WT组小鼠心肌ALDH2活性（5.92±1.14）mmol NADH/（min·mg）高于WT＋E组（3.53±1.07）mmol NADH/（min·mg）、KO组（3.15±0.96）mmol NADH/（min·mg）、KO＋E组（1.07±0.89）mmol NADH/（min·mg）（均P〈0.05）；KO＋E组小鼠心肌ALDH2活性低于KO组小鼠（P〈0.05）。（6）四组小鼠心肌组织PI3K表达水平差异无统计学意义（P〉0.05）; p-Akt相对表达量依次为（0.88±0.10）、（0.63±0.14）、（0.50±0.21）、（0.22±0.09），差异有统计学意义（均P〈0.05）；caspase-3 mRNA相对表达量依次为（0.23±0.07）、（0.46±0.11）、（0.70±0.14）、（0.91±0.20），差异有统计学意义（均P〈0.05）。结论亚急性酒精中毒会加剧AMI后心脏结构损害，保护ALDH2功能可有效拮抗乙醇对AMI的损害作用，其机制与PI3K/Akt信号通路介导的心肌细胞凋亡减少有关。

Objective To evaluate the effects of sub-acute alcoholism on cardiac structure and function, and investigate the mechanisms of aldehyde dehydrogenases-2 （ALDH2） alleviating the damage of heart caused by acute myocardial infarction. Methods The wild mice with ALDH2 （ ＋/＋ ） （WT group） and mice with knockout type ALDH2 （-/-） genotypes （ KO group） were raised and then divided into four groups according to the presence or absence of sub-acute alcoholism： WT group （n = 10）, KO group （n = 16）, WT ＋alcoholism group （WT ＋ E, n = 10） and KO ＋ alcoholism group （KO ＋ E, n = 10） . The mice of WT ＋ E group and KO ＋ E group were fed with high-dose of ethanol （2 g/kg per day for 8 days）, while the mice of WT group and KO group were treated with equal amount of saline instead. Acute myocardial infarction models were established in all mice after ethanol administration, and blood ethanol concentration, cardiac function, myocardial infarct size, the activity of ALDH2, and the key molecules of PI3K/Akt signal pathway and caspase-3 mRNA were detected one week after modeling. Statistical analysis was performed using SPSS 17.0. Differences in levels of detected biomarkers between groups were assessed using Chi-squared or One way ANOVA, and P 〈 0. 05 was considered to be statistically significant. Results （1） The mortality rates of WT group, KO group, WT ＋ E group and KO ＋ E group were 20.0%, 30.0%, 31.3% and 37.5%, respectively. （2） Compared with WT group and KO group, the blood ethanol concentration was higher and the damage of liver was more severe in WT ＋ E group and KO ＋ E group （P 〈 0. 05 ）. （3） The fraction shortening of short axis of left ventricle （FS） and left ventricular ejection fraction were higher in WT group and WT ＋ E group compared with KO group and KO ＋ E group （P 〈 0.05 ）. （4） The area of myocardial infarction was largest in KO ＋ E group, followed by KO group, WT ＋ E group, and WT group （all P 〈 0. 05 ） . （5） The activity of ALDH2 in WT group was higher than that in other groups, and the ALDH2 activity in KO ＋ E group was lower than that in KO group （P 〈0. 05）. （6） There was no significant difference in expressions of PI3K among four groups. The level of p-Akt was highest in WT group, followed by WT ＋ E group, KO group, and KO ＋ E group （ all P 〈 0. 05 ）. The levels of caspase-3 mRNA was highest in KO ＋ E group, followed by KO group, WT ＋ E group, and WT group （all P 〈 0.05）. Conclusions Myocardial damage caused by acute myocardial infarction can be aggravated by sub-acute alcoholism, while ALDH2 protection can effectively alleviate the damage effects of sub-acute alcoholism on myocardial infarction. The mechanism of protective effects of ALDH2 on acute myocardial infarction may be related to attenuation of cardiocytes apoptosis mediated by PDK/Akt signal pathway.