马蒂达,吴洋,王显,苏玉强,顾勇
MA Dida,WU Yang,WANG Xian,SU Yuqiang,GU Yong

• 1:陕西中医药大学第一临床医学院
• 2:北京中医药大学第一临床医学院
• 3:北京中医药大学东直门医院心内科
• 4:西安医学院临床医学院

摘要(Abstract)：

目的观察桃叶珊瑚苷对心肌梗死(MI)大鼠的心功能的影响,并探讨AMP活化激酶(AMPK)/NOD样受体蛋白3(NLRP3)通路在其中的作用机制。方法 60只SD大鼠均分为假手术(Sham)组、MI模型组、MI+桃叶珊瑚苷组及MI+桃叶珊瑚苷+抑制剂组组,Sham组大鼠做手术但不结扎冠脉血管、后3组大鼠采用冠状动脉左前降支结扎法建立大鼠MI模型,前2组大鼠手术后灌胃生理盐水、后2组大鼠手术后分别灌胃100 mg/kg桃叶珊瑚苷、100 mg/kg桃叶珊瑚苷+0.2 mg/kg AMPK抑制剂Dorsomorphin(CC)、均14 d;记录各组大鼠左室射血分数(LVEF)、左室收缩压(LVSP)、左室收缩末期容积(LVESV)、左室短轴缩短率(LVFS)、左室舒张压(LVEDP)、左室舒张末期容积(LVEDV),红四氮唑(TTC)-伊文思蓝(EB)及免疫荧光法检测心肌梗死体积和心肌细胞焦亡比例,苏木素伊红(HE)染色观察心肌细胞形态,ELISA法检测心肌匀浆中白细胞介素-1β(IL-1β)、IL-18及乳酸脱氢酶(LDH)水平,Werstern blot法检测心肌组织AMPK、凋亡半胱氨酸酶前体(procaspase1)、p-AMPK、cleaved-caspase1、NLRP3、凋亡斑点样蛋白(ASC)及胃泌素D-N (GSDMD-N)蛋白表达。结果 Sham组大鼠心肌无梗死、细胞形态及排列正常,MI组出现心肌梗死、心肌细胞增大且排列混乱、有大量炎症细胞;与Sham组相比,MI组、MI+桃叶珊瑚苷+抑制剂组大鼠LVEF、LVSP、LVFS、p-AMPK/AMPK蛋白水平降低(P <0.05),LVESV、LVEDP、LVEDV、心肌梗死体积、心肌细胞焦亡比例、IL-1β、IL-18、LDH及ASC、NLRP3、cleaved/pro-caspase1、GSDMD-N蛋白水平升高(P <0.05);与MI组相比,MI+桃叶珊瑚苷组大鼠LVEF、LVSP、LVFS、p-AMPK/AMPK蛋白水平升高(P <0.05),LVESV、LVEDP、LVEDV、心肌梗死体积、心肌细胞焦亡比例、IL-1β、IL-18、LDH及ASC、NLRP3、cleaved/pro-caspase1、GSDMD-N蛋白水平降低(P <0.05);与MI+桃叶珊瑚苷组相比,MI+桃叶珊瑚苷+抑制剂组大鼠LVEF、LVSP、LVFS、p-AMPK/AMPK蛋白水平降低(P <0.05),LVESV、LVEDP、LVEDV、心肌梗死体积、心肌细胞焦亡比例、IL-1β、IL-18、LDH水平及ASC、NLRP3、cleaved/pro-caspase1、GSDMD-N蛋白水平升高(P <0.05)。结论桃叶珊瑚苷可改善MI大鼠心功能,其机制可能与活化AMPK、抑制NLRP3小体形成、减轻心肌细胞炎症和焦亡有关。
Objective To investigate the effect of aucubin on myocardial infarction(MI) in rats and the role of AMP-activated protein kinase(AMPK)/NOD-like receptor protein 3(NLRP3) pathway.Methods Sixty rats were divided into MI group,MI + aucubin group and MI + aucubin + inhibitor group and sham operation(Sham) group. Rats of Sham group received operation but coronary vessels were not ligated,the other three groups received coronary artery left anterior descending artery ligation to establish MI models; two groups received intragastric normal saline after operation,two groups received 100 mg/kg aucubin,100 mg/kg aucubin + 0. 2 mg/kg AMPK inhibitor Dorsomorphin(CC)respectively for 14 consecutive days. Left ventricular ejection fraction(LVEF),left ventricular systolic pressure(LVSP),left ventricular end-systolic volume(LVESV),LVFS,LVEDP,and LVEDV of all groups were recorded. TTC-EB and immunofluorescent assay were adopted to detect infarction size and ratio of cardiomyocyte pyroptosis. Morphology of cardiomyocytes was observed by hematoxylin eosin(HE) staining; the levels of IL-1β,IL-18,and LDH in myocardial homogenate were detected by ELISA kit. The protein expression of AMPK,pro-caspase 1,p-AMPK,cleaved-caspase 1,NLRP3,apoptosis-associated speck-like protein(ASC),and GSDMD-N was detected by Western blot.Results There was no myocardial infarction in Sham group,and the cell morphology and arrangement were normal. There was myocardial infarction,cardiomyocytes were enlarged and disarrayed with a lot of inflammatory cells. Compared with those in Sham group,the LVEF,LVSP,LVFS,and protein level of p-AMPK/AMPK in MI group and MI + aucubin + inhibitor group were lowered(P < 0. 05);LVESV,LVEDP,LVEDV,infarction size,proportion of cardiomyocyte pyroptosis,levels of IL-1β,IL-18,LDH,and protein levels of ASC,NLRP3,cleaved/pro-caspase1,GSDMD-N were increased(P <0. 05). Compared with those in MI group,the LVEF,LVSP,LVFSm,and protein level of p-AMPK/AMPK in MI + aucubin group were increased(P < 0. 05); LVESV,LVEDP,LVEDV,infarction size,ratio of cardiomyocyte pyroptosis,levels of IL-1β,IL-18,LDH,and protein levels of ASC,NLRP3,cleaved/pro-caspase1,GSDMD-N were decreased(P < 0. 05); compared with those in MI + aucubin group,the LVEF,LVSP,LVFS,and protein level of p-AMPK/AMPK in MI + aucubin+ inhibitor group were decreased(P < 0. 05); LVESV,LVEDP,LVEDV,infarction size,ratio of cardiomyocyte pyroptosis,levels of IL-1β,IL-18,LDH,and protein levels of ASC,NLRP3,cleaved/pro-caspase1,GSDMD-N were increased(P < 0. 05). Conclusion Aucubin can inhibit the formation of NLRP3 body,reduce myocardial inflammation,and pyroptosis,and improve cardiac function,which may be achieved by activating AMPK.

关键词(KeyWords)： 大鼠;桃叶珊瑚苷;心肌梗死;AMP活化的激酶/NOD样受体蛋白3通路;细胞焦亡;心功能
rats;aucubin;myocardial infarction(MI);AMP-activated protein kinase(AMPK)/NOD-like receptor protein 3(NLRP3) pathway;apoptosis;cardiac function

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81601891);; 陕西省社会发展科技攻关项目(2016SF-166)

作者(Author): 马蒂达,吴洋,王显,苏玉强,顾勇
MA Dida,WU Yang,WANG Xian,SU Yuqiang,GU Yong

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