左西孟旦调控LOX-1/p38 MAPK通路减轻冠状动脉微栓塞后心肌损伤

Levosimendan alleviates coronary microembolization-induced myocardial injury through LOX-1/p38 MAPK pathway

目的探讨左西孟旦(levosimendan,Levo)对冠状动脉微栓塞(coronary microembolization,CME)后心肌损伤和LOX-1/p38 MAPK信号通路的影响。方法采用随机数字表法将24只巴马小型猪随机分为假手术组(Sham组)、CME组、CME+左西孟旦预治疗+对照腺相关病毒转染组(CME+Levo+NC组)及CME+左西孟旦预治疗+LOX-1过表达腺相关病毒转染组(CME+Levo+LOX-1组),每组6只。经冠脉介入法于前降支注射栓塞微球构建CME模型;Sham组则注射等量生理盐水代替。CME+Levo+NC组和CME+Levo+LOX-1组于造模前2周分别转染对照病毒和LOX-1过表达病毒,并均于造模前24 h开始持续静滴左西孟旦。心脏超声检测心功能指标;苏木精-伊红(HE)染色和苏木精碱性复红-苦味酸(HBFP)染色分别用于观察心肌组织病理改变和心肌微梗死区域;酶联免疫吸附测定(ELISA)用于检测血清肌钙蛋白Ⅰ(cTnI);脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNLE)检测心肌细胞凋亡率;免疫荧光观察心肌组织LOX-1、Bcl-2、Bax、Caspase-3 p12和p-p38 MAPK蛋白表达情况。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。以P<0.05为差异有统计学意义。结果⑴与Sham组比较,CME组小型猪左心室射血分数(LVEF)[(69.73±4.79)%vs(47.18±2.33)%,P<0.05]、左心室短轴缩短率(LVFS)[(42.47±2.54)%vs(21.43±1.76)%,P<0.05]、心输出量(CO)[(4.42±0.27)L/min vs(2.57±0.17)L/min,P<0.05]均显著下降,而左心室舒张期末径(LVEDd)[(32.37±1.33)mm vs(41.21±1.86)mm,P<0.05]显著增加,心肌微梗死面积占比[(3.73±0.87)%vs(20.72±2.49)%,P<0.05]显著增加,血清cTnI水平[(51.92±16.62)pg/mL vs(236.80±19.56)pg/mL,P<0.05]显著升高,心肌细胞凋亡率[(1.15±0.47)%vs(14.15±3.24)%,P<0.05]显著升高,心肌组织Bax、Caspase-3 p12、LOX-1和p-p38 MAPK蛋白表达明显上调(均P<0.05),Bcl-2蛋白表达明显下调(P<0.05)。⑵与CME组比较,CME+Levo+NC组小型猪LVEF[(47.18±2.33)%vs(55.48±3.92)%,P<0.05]、LVFS[(21.43±1.76)%vs(32.02±1.75)%,P<0.05]、CO[(2.57±0.17)L/min vs(3.45±0.25)L/min,P<0.05]均显著升高,而LVEDd[(41.21±1.86)mm vs(36.78±1.56)mm,P<0.05]显著下降,心肌微梗死面积占比[(20.72±2.49)%vs(11.63±3.12)%,P<0.05]显著减小,血清cTnI水平[(236.80±19.56)(157.40±15.13)pg/mL,P<0.05]显著降低,心肌细胞凋亡率[(14.15±3.24)%vs(8.33±1.28)%,P<0.05]显著下降,心肌组织Bax、Caspase-3 p12、LOX-1和p-p38 MAPK蛋白表达明显下调(均P<0.05),Bcl-2蛋白表达明显上调(P<0.05)。⑶与CME+Levo+NC组比较,CME+Levo+LOX-1组小型猪LVEF[(55.48±3.92)%vs(48.52±1.69)%,P<0.05]、LVFS[(32.02±1.75)%vs(23.80±2.51)%,P<0.05]、CO[(3.45±0.25)L/min vs(4.25±0.23)L/min,P<0.05]均显著降低,而LVEDd[(36.78±1.56)mm vs(41.12±1.57)mm,P<0.05]显著增加,心肌微梗死面积占比[(11.63±3.12)%vs(18.93±1.58)%,P<0.05]显著增加,血清cTnI水平[(157.40±15.13)(244.40±15.97)pg/mL,P<0.05]显著升高,心肌细胞凋亡率[(8.33±1.28)%vs(12.28±1.93)%,P<0.05]显著增加,心肌组织Bax、Caspase-3 p12、LOX-1和p-p38 MAPK蛋白表达明显上调(均P<0.05),Bcl-2蛋白表达明显下调(P<0.05)。结论左西孟旦可通过抑制LOX-1/p38 MAPK信号通路介导的心肌细胞凋亡而减轻CME后心肌损伤。

Objective To study the effect of levosimendan on coronary microembolization(CME)-induced myocardial injury and LOX-1/p38MAPK pathway.Methods Microspheres were injected into coronary anterior descending branch to construct swine CME model,swine was given levosimendan by continuous intravenous drip for 24 h before modeling,and myocardial-specific overexpression of lectin-like oxidized low density lipoprotein receptor 1(LOX-1)was achieved through coronary artery injection of adeno-associated virus(AAVs)at 2 weeks before modeling.Then,echocardiography was used to measure cardiac function;HE staining and HBFP staining were used to observe the pathological changes of myocardium and myocardial microinfarction area,respectively;ELISA was used to detect the serum level of cTnI;TUNLE staining was used to detect cardiomyocyte apoptotic index;the LOX-1,Bax,caspase-3 p12,Bcl-2,and p-p38 MAPK protein in myocardial tissue was observed by immunofluorescence method.Results Compared to the sham group,the LVEF,LVFS,and CO value in the CME group were decreased,while the LVEDd value was increased significantly(all P<0.05);the area of myocardial micro-infarction,serum cTnI level and cardiomyocyte apoptotic rate in the CME group were increased significantly(all P<0.05);the protein levels of Bax,caspase-3 p12,LOX-1,and p-p38 MAPK were increased significantly,while the Bcl-2 level was decreased significantly(P<0.05).Levosimendan pretreatment significantly improved cardiac dysfunction,reduced the area of myocardial micro-infarction and serum cTnI level,alleviated cardiomyocyte apoptosis,and significantly reduced the LOX-1 and p-p38 MAPK protein expression levels following CME(all P<0.05);while pretreatment with levosimendan and LOX-1 overexpression AAVs simultaneously abolished the effects of pretreatment with levosimendan alone(all P<0.05).Conclusion Levosimendan alleviates CME-induced myocardial injury through inhibiting cardiomyocyte apoptosis mediated by LOX-1/p38 MAPK signaling pathway.