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目的:研究鞘氨醇激酶1(SPK1)在氧自由基诱导心肌细胞损伤过程中的作用。方法:分离培养Wistar乳大鼠心肌细胞,用不同浓度过氧化氢(H2O2,0,50,100,200,400μmol/L)处理细胞,检测乳酸脱氢酶(LDH)的释放和细胞SPK1酶活性变化;Northern印迹方法分析SPK1催化产物1-磷酸鞘氨醇(S1P)受体的表达。用S1P预处理心肌细胞,然后加入100μmol/L的H2O2继续作用48h,检测细胞培养上清中LDH的活性;用携带人SPK1基因的复制缺陷型重组腺病毒载体(Ad-SPK1)感染Wistar乳大鼠心肌细胞,然后加入100μmol/L的H2O2继续作用48h,检测细胞培养上清中LDH的活性。分析外源性S1P或SPK1高表达对H2O2诱导心肌细胞损伤的保护作用。结果:H2O2作用于心肌细胞后,导致LDH释放增多,SPK1酶活性抑制,其作用呈剂量依赖性;H2O2刺激使S1P受体Edg-1表达水平升高,Edg-3表达水平降低。外源性S1P预处理使H2O2诱导的LDH释放减少,其作用呈剂量依赖性。与对照组相比,Ad-SPK1感染使心肌细胞SPK1酶活性明显升高,细胞培养上清中S1P含量增多,并明显抑制H2O2诱导心肌细胞LDH的释放。结论:SPK1高表达能保护H2O2导致的心肌细胞死亡,这种保护作用主要通过S1P的释放来实现。Edg-1和Edg-3可能参与SPK1对心肌细胞的保护作用。
Objective: To investigate the role of sphingosine kinase 1 (SPK1) in cardiomyocyte injury induced by oxygen free radicals. Methods: Myocardial cells from Wistar rats were isolated and cultured. The cells were treated with different concentrations of H2O2 (0, 50, 100, 200 and 400μmol / L) to detect the release of lactate dehydrogenase (LDH) and the activity of SPK1. Northern blotting The SPK1 catalytic product, sphingosine-1-phosphate (S1P) receptor expression was analyzed. Cardiomyocytes were pretreated with S1P, then treated with 100μmol / L H2O2 for 48h, the activity of LDH in the cell culture supernatant was detected. Wistar milk was infected with replication-defective recombinant adenovirus carrying human SPK1 gene (Ad-SPK1) Rat cardiomyocytes, and then add 100μmol / L H2O2 for 48h, detect the activity of LDH in the cell culture supernatant. The protective effect of exogenous S1P or SPK1 expression on H2O2-induced cardiomyocyte injury was analyzed. Results: After H2O2 treatment, the release of LDH was increased and the activity of SPK1 was inhibited in a dose-dependent manner. H2O2 stimulation increased the expression of Edg-1 on S1P receptor and decreased the expression of Edg-3. Exogenous S1P preconditioning reduced H2O2-induced LDH release in a dose-dependent manner. Compared with the control group, Ad-SPK1 infection significantly increased SPK1 activity in cardiomyocytes, increased S1P content in the cell culture supernatant, and significantly inhibited the release of LDH from H2O2-induced cardiomyocytes. Conclusion: High expression of SPK1 can protect cardiomyocytes from H2O2-induced apoptosis. This protective effect is mainly achieved through the release of S1P. Edg-1 and Edg-3 may participate in the protective effect of SPK1 on cardiomyocytes.