脂联素通过下调NOX2表达减轻急性酒精性心肌损伤

Adiponectin attenuates acute alcoholic myocardial injury by down regulating NOX2 expression

目的探讨脂联素(APN)对小鼠急性酒精性心肌损伤的影响及相关机制,为急性酒精性心肌损伤及酒精性心肌病的预防和治疗提供实验依据及新思路。方法将8周龄SPF级C57BL/6J雄性小鼠随机分为正常对照组(n=10)和正常模型组(n=15),将8周龄雄性纯合子脂联素基因敲除鼠(APN^(-/-))随机分为APN^(-/-)对照组(n=10)和APN^(-/-)模型组(n=15),各模型组均给予腹腔注射乙醇3 g/(kg·d),各对照组均给予等量生理盐水腹腔注射。4组小鼠均正常摄食、饮水,连续3天后进行各项指标监测:观察小鼠一般情况;检测血清乳酸脱氢酶(LDH)、血浆N末端B型脑钠肽原(NT-pro BNP);用动物心脏超声机测定心脏结构及功能指标;测定Caspase 3活性及TUNEL法检测心肌细胞凋亡;制备心肌组织HE染色切片及Masson染色切片;制备心肌组织匀浆测定活性氧簇(ROS)、丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性;Western blot检测心肌组织NADPH氧化酶2(NOX2)蛋白表达情况。结果与正常对照组相比,正常模型组射血分数明显下降(P<0.01);血清LDH、血浆NT-pro BNP浓度分别升高1.98倍、5.13倍(P<0.01);HE染色及Masson染色显示心肌结构及肌纤维改变、胶原纤维增多,胶原容积分数(CVF)值升高2.63倍(P<0.01);心肌组织内Caspase 3活性升高2.58倍(P<0.01),TUNEL法显示阳性凋亡心肌细胞增加12.67倍(P<0.01);心肌组织内ROS、MDA含量分别升高1.68倍、2.87倍(P<0.01),SOD活性升高2.92倍(P<0.01);NOX2蛋白表达量升高1.87倍(P<0.01)。与APN^(-/-)对照组相比,APN^(-/-)模型组上述指标均明显升高(均P<0.01)。与正常模型组相比,APN^(-/-)模型组小鼠射血分数下降更为明显(P<0.01),血清LDH、血浆NTpro BNP浓度分别升高1.30倍、1.25倍(P<0.01);HE及Masson染色显示心肌结构及肌纤维改变、胶原纤维增多,CVF值升高1.55倍;心肌组织内Caspase 3活性升高1.66倍(P<0.01),TUNEL法显示阳性凋亡心肌细胞比例升高1.64倍(P<0.01);心肌组织内ROS、MDA含量分别升高1.42倍、1.39倍(P<0.01),SOD活性降低28%(P<0.01);NOX2蛋白表达量升高1.44倍(P<0.01)。结论脂联素可以减轻小鼠急性酒精性心肌损伤,其机制与脂联素抑制NOX2蛋白表达发挥抗氧化应激作用有关。

Aim To explore the effect of adiponectin （APN） on acute alcohol induced myocardial injury in mice and related mechanism, and to provide experimental basis and new ideas for the prevention and treatment of acute alcoholic myocardial injury and alcoholic cardiomyopathy. Methods 8 week old C57BL/6J male mice were randomly divided into normal control group （n=10） and normal model group （n=15）. 8 week old male SPF homozygous adiponectin knockout （APN^-/-） mice were randomly divided into APN^-/- control group （n=10） and APN^-/- model group （n=15）. Model groups were given intraperitoneal injection of ethanol 3 g/（kg·d）, control groups received normal saline intraperitoneal injection. 4 groups of mice were normal feeding, drinking water. After 3 days, the indicators were monitored：the general condition of mice was observed, serum lactate dehydrogenase （LDH） and N-terminal B type natriuretic peptide （NT-proBNP） were detected, cardiac structure and function index were detected by mouse heart ultrasonic machine, myocardial cell apoptosis was detected by TUNEL and the activity of Caspase 3 was determined, myocardial tissue HE staining and Masson staining were prepared, myocardial tissue homogenate reactive oxygen species （ROS） content, malondialdehyde （MDA） content and superoxide dismutase （SOD） activity were detected, Western blot was used to detect NADPH oxidase 2 （NOX2） protein expression in myocardial tissue. Results Compared with normal control group, ejection fraction of normal model group decreased significantly （P〈0.01）; serum LDH and plasma NT-proBNP concentration of normal model group were increased by 1.98 times, 5.13 times （P〈0.01）; HE staining and Masson staining showed myocardial structure and muscle fiber changes, collagen fibers increased, collagen volume fraction （CVF） value increased 2.63 times in normal model group （P〈0.01）; the activity of Caspase 3 in myocardial tissue increased 2.58 times （P〈0.01）, positive myocardial cell apoptosis increased 12.67 times by TUNEL in normal model group （P〈0.01）; ROS content and MDA content in myocardium were increased by 1.68 times, 2.87 times （P〈0.01）, SOD activity increased 2.92 times in normal model group （P〈0.01）; the expression of NOX2 protein increased 1.87 times in normal model group （P〈0.01）. Compared with control group, the above indicators in APN^-/- model group were significantly increased （P〈0.01）. Compared with normal group, serum LDH and plasma NT-proBNP concentration were increased by 1.30 times, 1.25 times in APN^-/- model group （P〈0.01）; HE and Masson staining showed the myocardial structure and muscle fiber changes, collagen fibers increased, CVF value increased by 1.55 times in APN^-/- model group; the activity of Caspase 3 in myocardial tissue increased 1.66 times （P〈0.01）, the myocardial cell apoptosis ratio was 1.64 times higher in APN^-/- model group （P〈0.01）; ROS content and MDA content in myocardium were increased by 1.42 times, 1.39 times （P〈0.01）, SOD activity decreased 28% in APN^-/- model group （P〈0.01）, the expression of NOX2 protein increased 1.44 times in APN^-/- model group （P〈0.01）. Conclusion Adiponectin can reduce acute alcoholic myocardial injury in mice, and its mechanism may be related to the inhibition of NOX2 protein expression and the effect of anti-oxidative stress.