Toll样受体4激动剂LPS腹腔注射对大鼠心肌缺血再灌注损伤的预防作用及其作用机制

Preventive effect of Toll-like receptor 4 agonist LPS on myocardial ischemia-reperfusion injury in rats and its mechanism

目的 探讨Toll样受体4(TLR4)激动剂LPS腹腔注射对大鼠心肌缺血再灌注损伤(MIRI)的预防作用及其作用机制。方法 将60只大鼠根据随机数字表法分为LPS干预组(0.1 mg/kg、0.5 mg/kg、1 mg/kg,均为小剂量)、维拉帕米干预组、模型组、假手术组,每组15只。构建MIRI模型前7 d,LPS干预组给予0.1 mg/kg、0.5 mg/kg、1 mg/kg LPS腹腔注射,维拉帕米干预组给予2.5%维拉帕米(2 mg/kg)腹腔注射,假手术组与模型组给予等容量生理盐水腹腔注射;均每日1次。然后,除假手术组外,其余组大鼠进行缺血再灌注处理。处理后72 h,采用超声检测各组大鼠心功能,苏木精—伊红(HE)染色观察各组大鼠心肌组织病理学变化,2,3,5-氯化三苯基四氮唑(TTC)染色测算各组大鼠心肌梗死面积;采用Western blotting法检测大鼠心肌组织FoxO3a、pFoxO3aS253、Beclin-1及LC蛋白。结果 与假手术组比较,模型组大鼠左心室射血分数(LVEF)、短轴缩短率(LVFS)减小(P均<0.05);心肌损伤严重,心肌梗死面积百分比升高(P均<0.05);心肌组织中pFoxO3aS253蛋白相对表达量降低(P<0.05),FoxO3a、Beclin-1、LC蛋白相对表达量升高(P均<0.05)。与模型组比较,维拉帕米干预组及LPS干预组大鼠LVEF、LVFS增加(P均<0.05);0.1 mg/kg及0.5 mg/kg LPS干预组大鼠心肌病变改善不明显,维拉帕米干预组和1 mg/kg LPS干预组大鼠心肌纤维水肿、断裂、坏死及炎细胞浸润程度均减轻;维拉帕米干预组及LPS干预组大鼠心肌梗死面积百分比降低(P均<0.05),pFoxO3aS253蛋白相对表达量升高(P均<0.05),FoxO3a、Beclin-1、LC蛋白相对表达量下降(P均<0.05)。结论 小剂量TLR4激动剂LPS可预防大鼠MIRI,其作用机制可能与促使FoxO3a磷酸化及抑制Beclin-1、LC激活有关。

Objective To investigate the preventive effect of Toll-like receptor 4(TLR4) agonist Lipopolysaccharides(LPS) on myocardial ischemia-reperfusion injury(MIRI) in rats and its mechanism.Methods Sixty rats were divided into the LPS intervention group(0.1 mg/kg,0.5 mg/kg,1 mg/kg,all were small doses),verapamil intervention group,model group and sham operation group,with 15 rats in each group.At 7 days before the establishment of MIRI models,rats in the LPS intervention group were given intraperitoneal injection of 0.1 mg/kg,0.5 mg/kg and 1 mg/kg LPS,rats in the verapamil intervention group were given intraperitoneal injection of 2.5% verapamil(2 mg/kg),rats in the sham operation group and model group were given intraperitoneal injection of equal volume normal saline.All were performed once a day.Then,except for the sham operation group,rats in the other groups underwent ischemia-reperfusion treatment.At 72 h after treatment,the cardiac function of rats in each group was detected by ultrasound,the myocardial histopathological changes were observed by hematoxylin-eosin(HE) staining,and the myocardial infarction size was observed by 2,3,5-triphenyltetrazolium chloride(TTC) staining.The proteins of forkhead box proteinO3a(FoxO3a),phospho-forkhead box protein O3a(pFoxO3aS253),Beclin-1 and LC in rat myocardial tissues were detected by Western blotting.Results Compared with the sham operation group,left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS) decreased(both P<0.05),myocardial injury was more serious and the percentage of myocardial infarction area increased(both P<0.05),the relative expression of pFoxO3aS253 protein in the myocardial tissues decreased(P<0.05),and the relative expression levels of FoxO3a,Beclin-1,and LC proteins increased in the model group(all P<0.05).Compared with the model group,LVEF and LVFS increased in the verapamil intervention group and LPS intervention group(both P<0.05);there was no obvious improvement in cardiomyopathy in the 0.1 mg/kg and 0.5 mg/kg LPS intervention groups,and the degree of myocardial fibrosis edema,fracture,necrosis and inflammatory cell infiltration were reduced in the verapamil intervention group and 1 mg/kg LPS intervention group;the percentage of myocardial infarction area decreased(all P<0.05),the relative expression level of pFoxO3aS253 protein increased(P<0.05),and the relative expression levels of FoxO3a,Beclin-1 and LC proteins decreased in the verapamil intervention group and LPS intervention group(all P<0.05).Conclusion Low-dose TLR4 agonist LPS can prevent MIRI in rats,and its mechanism may be related to promoting phosphorylation of FoxO3a and inhibiting activation of Beclin-1 and LC.