摘要
目的 探讨麦冬皂苷D调节鞘氨醇激酶1(SphK1)/1-磷酸鞘氨醇(S1P)/鞘氨醇1磷酸酯受体1(S1PR)通路对心肌缺血再灌注损伤(MIRI)大鼠心肌炎症的影响.方法 将大鼠随机分为MIRI组、麦冬皂苷D组、SphK1激活剂(K6PC-5)组、麦冬皂苷D+K6PC-5组、假手术组,每组12只.除假手术组外,其它组大鼠均通过结扎冠状动脉左前降支法构建MIRI模型.建模成功后,立即给药处理,每日1次,持续2周.超声成像系统评估大鼠左心室射血分数(LVEF)、左心室分数缩短(LVFS)变化;HE染色检测心肌组织的病理;ELISA法检测心肌组织中肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)、白细胞介素(IL)-1β、肿瘤坏死因子α(TNF-α)水平;TUNEL染色检测心肌组织的心肌细胞凋亡;免疫组化染色心肌组织中Bim、天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)阳性细胞数占比;Western Blot法检测心肌组织中S1P、SphK1、S1PR1蛋白水平.结果 与假手术组比较,MIRI组大鼠心肌结构损伤、纤维排列紊乱、心肌细胞减少、细胞核固缩且有大量炎症细胞浸润;LVFS、LVEF降低(P<0.05);心肌组织中CK-MB、LDH、IL-1β、TNF-α水平,心肌细胞凋亡率,Bim、Caspase-3阳性细胞数占比及S1P、SphK1、S1PR1蛋白水平升高(P<0.05).与MIRI组比较,麦冬皂苷D组大鼠心肌结构损伤、纤维排列紊乱、心肌细胞减少、细胞核固缩及有大量炎症细胞浸润现象有所缓解;LVFS、LVEF升高(P<0.05);心肌组织中CK-MB、LDH、IL-1β、TNF-α 水平,心肌细胞凋亡率,Bim、Caspase-3阳性细胞数占比及S1P、SphK1、S1PR1蛋白水平降低(P<0.05).K6PC-5逆转了麦冬皂苷D对MIRI大鼠心功能障碍、心肌炎症及心肌细胞凋亡的影响(P<0.05).结论 麦冬皂苷D抑制MIRI大鼠心肌炎症及心肌细胞凋亡的机制可能与抑制SphK1/S1P/S1PR1通路有关.
Abstract
Objective To investigate the effect of ophiopogonin D on myocardial in rats with ischemia-reperfusion injury (MIRI) by regulating the sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P)/sphingosine 1-phosphate receptor 1 (S1PR1) pathway. Methods Rats were randomly separated into MIRI group,ophiopogonin D group,SphK1 activator (K6PC-5)group,ophiopogonin D+K6PC-5 group,and sham group,with 12 rats in each group. Except for the sham group,MIRI models were constructed by ligating the left anterior descending coronary artery of rats in all other groups. After successful modeling,medication treatment was carried out immediately,once a day for 2 weeks. The ultrasound imaging system was applied to evaluate changes in left ventricular ejection fraction (LVEF) and left ventricular fractional shortening(LVFS)in rats. HE staining was applied to detect the pathology of myocardial tissue. ELISA was applied to detect levels of creatine kinase isoenzyme (CK-MB),lactate dehydrogenase (LDH),interleukin-1β(IL-1β),and tumor necrosis factor-α(TNF-α)in myocardial tissue. TUNEL staining was applied to detect myocardial cell apoptosis in myocardial tissue. Immunohistochemical staining was applied to detect the proportions of Bim and Caspase-3 positive cells in myocardial tissue. Western Blot was applied to detect S1P,SphK1,and S1PR1 proteins in myocardial tissue. Results Compared with the sham group,rats in the MIRI group showed myocardial structural damage,disordered fiber arrangement,decreased myocardial cells,nuclear pyknosis,and a large number of inflammatory cell infiltration. The LVFS and LVEF decreased,the levels of CK-MB,LDH,IL-1β,TNF-α,myocardial cell apoptosis rate,proportions of Bim and Caspase-3 positive cells,and S1P,SphK1,and S1PR1 proteins in myocardial tissue were elevated (P<0.05). Compared with the MIRI group,the rats in the ophiopogonin D group showed relief of above-mentioned phenomenon,including myocardial structural damage,disordered fiber arrangement,decreased myocardial cells,nuclear pyknosis,and infiltration of a large number of inflammatory cells. The LVFS and LVEF increased,the levels of CK-MB,LDH,IL-1β,TNF-α,myocardial cell apoptosis rate,proportions of Bim and Caspase-3 positive cells,and S1P,SphK1,and S1PR1 proteins in myocardial tissue decreased (P<0.05). K6PC-5 reversed the effects of ophiopogonin D on cardiac dysfunction,myocarditis,and myocardial cell apoptosis in MIRI rats. Conclusion The mechanism by which ophiopogonin D inhibits myocarditis and myocardial cell apoptosis in MIRI rats may be related to the inhibition of the SphK1/S1P/S1PR1 pathway.
维普
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