阿芬太尼调节SphK1/S1P信号通路保护心肌缺血再灌注损伤大鼠

The protective effect of alfentanil on myocardial ischemia-reperfusion injury in rats by regulating the SphK1/ S1P signaling pathway

[目的]探究阿芬太尼对心肌缺血再灌注损伤(MIRI)大鼠的作用及在该过程中对鞘氨醇激酶1(SphK1)/鞘氨醇-1-磷酸(S1P)信号通路的调节机制。[方法]将SPF级SD雄性大鼠随机分为假手术组、模型组、阳性药物组(复方丹参组)和阿芬太尼低剂量组、阿芬太尼高剂量组、阿芬太尼高剂量+SphK1激动剂组(阿芬太尼+PMA组),每组20只。除假手术组,其余组均利用结扎左前降支冠状动脉后再灌注复制MIRI模型。全自动生物化学分析仪检测血清乳酸脱氢酶(LDH)、肌酸激酶(CK)和谷草转氨酶(AST)的活性;TTC检测大鼠心肌梗死面积;HE染色观察大鼠心肌组织形态学特征;TUNEL染色检测大鼠心肌细胞凋亡;ELISA检测血清肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)及S1P的水平;试剂盒检测心肌组织中丙二醛(MDA)含量和超氧化物歧化酶(SOD)的活性;Western blot检测心肌组织SphK1蛋白表达。[结果]相较于假手术组,模型组大鼠心肌组织病理损伤严重,血清中心肌损伤标志物LDH、CK和AST的活性,心肌梗死面积和心肌细胞凋亡率,TNF-α、IL-6、IL-1β、MDA、S1P水平及SphK1蛋白表达均升高,SOD活性降低(P<0.05);相较于模型组,阳性药物组和阿芬太尼低、高剂量组大鼠心肌组织损伤减轻,血清中心肌损伤标志物LDH、CK和AST的活性,心肌梗死面积和心肌细胞凋亡率,TNF-α、IL-6、IL-1β、MDA、S1P水平及SphK1蛋白表达均降低,SOD活性升高(P<0.05)。SphK1激动剂可逆转高剂量阿芬太尼对上述指标的影响(P<0.05)。[结论]阿芬太尼对MIRI大鼠发挥保护作用,其机制可能与抑制SphK1/S1P信号通路有关。

Aim To investigate the effect of alfentanil on myocardial ischemia-reperfusion injury(MIRI)in rats and its regulatory mechanism on the sphingosine kinase 1(SphK1)/sphingosine-1-phosphate(S1P)signaling pathway dur-ing this process.Methods SPF grade SD male rats were randomly divided into sham surgery group,model group,positive drug group(compound salvia miltiorrhiza group),low dose alfentanil group,high dose alfentanil group,and high alfentanil+SphK1 agonist group(alfentanil+PMA group),with 20 rats in each group.Except the sham operation group,the MIRI model was reproduced by ligating the left anterior descending coronary artery and reperfusion.The activities of serum lactate dehydrogenase(LDH),creatine kinase(CK)and aspartate aminotransferase(AST)were detected by auto-matic biochemical analyzer;TTC was applied to detect the size of myocardial infarction in rats;HE staining was applied to observe the morphological characteristics of rat myocardial tissue;TUNEL staining was applied to detect myocardial cell ap-optosis in rats;ELISA was applied to detect the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),inter-leukin-1β(IL-1β),and S1P;kits were applied to detect content of malondialdehyde(MDA)and activity of superoxide dismutase(SOD)in myocardial tissue;Western blot was applied to detect the expression level of SphK1 protein in myocar-dial tissue.Results Compared with the sham surgery group,the pathological damage to the myocardial tissue of rats was severe in the model group,the activities of serum central muscle injury markers LDH,CK,and AST,myocardial in-farction area,myocardial cell apoptosis rate,the levels of TNF-α,IL-6,IL-1β,MDA,S1P and the expression of SphK1 protein all increased,the activity of SOD decreased(P<0.05).Compared with the model group,the myocardial tissue damage of rats was reduced in the positive drug group and the low and high dose alfentanil groups,the activities of serum central muscle injury markers LDH,CK,and AST,myocardial infarction area,myocardial cell apoptosis rate,the levels of TNF-α,IL-6,IL-1β,MDA,S1P and the expression of SphK1 protein all decreased,the activity of SOD increased(P<0.05).The SphK1 agonist was able to reverse the impact of high-dose alfentanil on the above indicators(P<0.05).Conclusion Alfentanil has protective effect on MIRI rats,and its mechanism may be related to the inhibition of SphK1/S1P signaling pathway.