1-磷酸鞘氨醇受体1型信号通路在压力超负荷诱导的心室重构中的作用及机制

Effect of S1PR1 on pressure overload-induced cardiac remodeling in mice

目的研究1-磷酸鞘氨醇受体1型(sphingosine 1-phosphate receptor 1,S1PR1)信号通路在压力超负荷诱导的心室重构中的作用及其机制。方法构建小鼠主动脉弓缩窄(transverse aortic constriction,TAC)模型,模型建立的第2天开始经腹腔注射S1PR1激动剂SEW2871或相应的对照溶剂DMSO,持续给药30 d后观察S1PR1激动剂对TAC术后小鼠心功能的影响。体外实验:首先通过慢病毒感染建立S1PR1基因过表达(S1PR1组)或S1PR1基因沉默(sh RNA组)的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)稳定表达株及相应对照组(NC组),并检测细胞外调节蛋白激酶1/2(ERK1/2)的活性水平。同时,分别收集NC组、S1PR1组和S1PR1组加ERK1/2阻滞剂U0126(S1PR1+U0126组)的HUVEC细胞培养上清液,在0. 5μmol/L的血管紧张素Ⅱ(Ang Ⅱ)的刺激下将各组HUVEC细胞培养上清液分别与H9C2心肌细胞共培养,48 h后测量H9C2心肌细胞大小,分析HUVEC细胞表达的S1PR1通过旁分泌对心肌细胞肥大的影响及可能机制。结果给药30 d后,心超提示TAC术后SEW2871组左室射血分数(59. 65%±6. 12%)较DMSO组(41. 16%±11. 91%)显著提高(P<0. 05)。免疫荧光染色表明TAC术后SEW2871组较DMSO组心肌组织纤维化及心肌肥大程度明显降低(P<0. 05)。蛋白印迹结果提示S1PR1激活ERK1/2信号通路。H9C2心肌细胞的面积测量结果表明S1PR1组[(22. 52±4. 13)μm^2]较NC组[(34. 98±12. 92)μm^2]及S1PR1+U0126组[(80. 60±36. 60)μm^2]的心肌细胞的面积明显降低(P <0. 05)。结论 S1PR1可以显著改善压力超负荷诱导的心室重构,提高心功能,减轻心肌组织纤维化及心肌细胞肥大。内皮细胞表达的S1PR1能改善心肌细胞肥大,可能是通过激活ERK1/2信号通路完成。

Objective To investigate the protective effect and possible mechanism of sphingosine1-phosphate receptor1(S1PR1)on ventricular remodeling induced by stress overload in mice.Methods The transverse aortic constriction(TAC)model was established in female C57BL/6 mice.The TAC mice were given intraperitoneal injection of S1PR1agonist SEW28715mg/(kg·d)(TAC+SEW group,n=5)or Dulbecco's modified eagle medium(DMEM)(TAC+DMSO group,n=5);and the mice in sham operation group were given intraperitoneal injection of DMEM(sham group,n=5).After 30d of administration,the heart function of mice in each group was examined by cardiac ultrasonography.The cardiac H9C2 cells were stimulated with angiotensin II(Ang II,0.5μmol/L)to induce hypertrophy.The S1PR1 gene overexpressing human umbilical vein endothelial cells(HUVECs)(S1PR1 group)and S1PR1 gene silencing HUVECs(shRNA group)were established by corresponding plasmid transfection,and the untransfected HUVESc served as NC group.The protein expression level of extracellular regulated protein kinase1/2(ERK1/2)in HUVEC cells was detected by Western blot.The culture supernatant of HUVEC cells was collected and co-cultured with H9C2 cells under the stimulation of Ang II for 48h.The cells were divided into four groups:the culture supernatant of NC group without Ang II stimulation(CTL),the culture supernatant of NC group with Ang II stimulation(Ang II+NC),the culture supernatant of S1PR1 group with Ang II stimulation(Ang II+S1PR1)and the culture supernatant of S1PR1group with Ang II stimulation group pretreated with ERK1/2 antagonist U0126(Ang II+S1PR1+U0126).After 48h of drug treatment,the size of cardiomyocytes was measured by ImageJ software.Results After 30d of drug treatment,the echocardiography results showed that the left ventricular ejection fraction in SEW2871 group(59.65%±6.12%)was significantly higher than that in DMSO group(41.16%±11.91%)(P<0.05).Immunohistochemistry results showed that the degree of myocardial fibrosis in SEW2871group was lower than that in DMSO group(P<0.05).The results of Western blot showed that the levels of p-ERK1/2 in the S1PR1group was significantly higher than that in NC group and shRNA group(t=3.598,3.200,P<0.05).The area of H9C2 cells in S1PR1group[(22.52±4.13)μm^2]was significantly reduced compared with NC group[(34.98±12.92)μm^2]and S1PR1+U0126 group[(80.60±36.60)μm^2](P<0.05).Conclusion The S1PR1activation can remarkably improve cardiac function,ameliorate ventricular remodeling and reduce myocardial fibrosis and hypertrophy induced by pressure overload.The expression of S1PR1in vascular endothelial cells can modify the hypertrophy of cardiac myocytes,which may be accomplished by activating the ERK1/2signaling pathway.