SphK1/S1P信号通路在脂联素恢复七氟烷后处理减轻糖尿病大鼠心肌缺血再灌注损伤中的作用

Role of SphK1/S1P signaling pathway in adiponectin-induced restoration of attenuation of myocardial ischemia-reperfusion injury by sevoflurane postconditioning in diabetic rats

目的:评价SphK1/S1P信号通路在脂联素恢复七氟烷后处理减轻糖尿病大鼠心肌缺血再灌注损伤中的作用。方法:清洁级健康雄性SD大鼠,8~10周龄,体重250~300 g,采用高脂高糖喂养联合腹腔注射1%链脲佐菌素-柠檬酸盐缓冲液40 mg/kg制备大鼠糖尿病模型。取糖尿病模型制备成功的大鼠90只,采用随机数字表法分为6组(n=15):假手术组(Sham组)、缺血再灌注组(I/R组)、七氟烷后处理组(S组)、脂联素预处理组(A组)、脂联素预处理+七氟烷后处理组(AS组)和脂联素预处理+SphK1特异性激活剂(K6PC-5)+七氟烷后处理组(AKS组)。采用结扎左冠状动脉前降支30 min,再灌注120 min的方法制备糖尿病大鼠心肌缺血再灌注损伤模型。于缺血前15 min时A组、AS组和AKS组腹腔注射基因重组脂联素5.0μg/g,AKS组经尾静脉注射SphK1特异性激活剂K6PC-51μg/g,再灌注即刻S组、AS组和AKS组吸入2.5%七氟烷5 min。于再灌注结束时抽取颈内静脉血样,采用ELISA法检测血清cTnI浓度;采用TTC染色法确定心肌梗死体积百分比;采用Western blot法检测心肌SphK1、S1P和磷酸化NF-κB p65(p-NF-κB p65)的表达。结果:与Sham组比较,I/R组血清cTnI浓度和心肌梗死体积百分比升高,心肌SphK1、S1P和p-NF-κB p65表达上调(P<0.05);与I/R组比较,S组各指标差异无统计学意义(P>0.05),A组血清cTnI浓度和心肌梗死体积百分比降低,心肌SphK1、S1P和p-NF-κB p65表达下调(P<0.05);与S组比较,AS组血清cTnI浓度和心肌梗死体积百分比降低,心肌SphK1、S1P和p-NF-κB p65表达下调(P<0.05);与AS组比较,AKS组血清cTnI浓度和心肌梗死体积百分比升高,心肌SphK1、S1P和p-NF-κB p65表达上调(P<0.05)。结论:SphK1/S1P信号通路参与了脂联素恢复七氟烷后处理减轻糖尿病大鼠心肌缺血再灌注损伤的过程。

Objective To evaluate the role of SphK1/S1P signaling pathway in adiponectin-induced restoration of attenuation of myocardial ischemia-reperfusion(I/R)injury by sevoflurane postconditioning in diabetic rats.Methods Healthy clean-grade male Sprague-Dawley rats,aged 8-10 weeks,weighing 250-300 g,in which diabetes mellitus was induced by combination of high-fat and high-sucrose diet and intraperitoneal injection of 1%streptozotoein in citric acid buffer 40 mg/kg,were studied.Ninety rats with diabetes mellitus were divided into 6 groups(n=15 each)using a random number table method:sham operation group(group Sham),group I/R,sevoflurane postconditioning group(group S),adiponectin preconditioning group(group A),adiponectin preconditioning+sevoflurane postconditioning group(group AS)and adiponectin preconditioning+K6PC-5(a specific SphK1 activator)+sevoflurane postconditioning group(group AKS).Myocardial I/R was induced by 30 min occlusion of anterior descending branch of left coronary artery followed by 120 min reperfusion.At 15 min before ischemia,recombinant adiponectin 5.0μg/g was injected intraperitoneally in A,AS and AKS groups,and K6PC-51μg/g was injected via the tail vein in group AKS.In S,AS and AKS groups,2.5%sevoflurane was inhaled for 5 min starting from the onset of reperfusion.Blood samples from the internal jugular vein were collected at the end of reperfusion for determination of serum cardiac troponin I(cTnI)concentration(by enzyme-linked immunosorbent assay),percentage of myocardial infarct volume(by TTC staining)and expression of SphK1,S1P and phosphorylated NF-κB p65(p-NF-κB p65)in myocardial tissues(by Western blot).Results Compared with group Sham,the serum cTnI concentration and percentage of myocardial infarct volume were significantly increased,and expression of SphK1,S1P and p-NF-κB p65 in myocardial tissues was up-regulated in group I/R(P<0.05).Compared with group I/R,no significant change was found in each parameter in group S(P>0.05),and the serum cTnI concentration and percentage of myocardial infarct volume were significantly decreased,and expression of SphK1,S1P and p-NF-κB p65 in myocardial tissues was down-regulated in group A(P<0.05).Compared with group S,the serum cTnI concentration and percentage of myocardial infarct volume were significantly decreased,and expression of SphK1,S1P and p-NF-κB p65 in myocardial tissues was down-regulated in group AS(P<0.05).Compared with group AS,the serum cTnI concentration and percentage of myocardial infarct volume were significantly increased,and expression of SphK1,S1P and p-NF-κB p65 in myocardial tissues was up-regulated in group AKS(P<0.05).Conclusion SphK1/S1P signaling pathway is involved in adiponectin-induced restoration of attenuation of myocardial I/R injury by sevoflurane postconditioning in diabetic rats.