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
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摘要
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 sphingosine 1-phosphate receptor 1( 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 S1PR1 agonist SEW2871 5 mg/( 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). After30 d 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,0.5 μmol/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 kinase 1/2( 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 48 h. 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 S1PR1 group with Ang Ⅱ stimulation group pretreated with ERK1/2 antagonist U0126( Ang Ⅱ + S1PR1 + U0126). After 48 h of drug treatment,the size of cardiomyocytes was measured by ImageJ software. Results After 30 d 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 SEW2871 group 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 S1PR1 group 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 S1PR1 group [( 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 S1PR1 activation can remarkably improve cardiac function, ameliorate ventricular remodeling and reduce myocardial fibrosis and hypertrophy induced by pressure overload. The expression of S1PR1 in vascular endothelial cells can modify the hypertrophy of cardiac myocytes,which may be accomplished by activating the ERK 1/2 signaling pathway.
Objective To investigate the protective effect and possible mechanism of sphingosine 1-phosphate receptor 1( 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 S1PR1 agonist SEW2871 5 mg/( 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). After30 d 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,0.5 μmol/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 kinase 1/2( 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 48 h. 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 S1PR1 group with Ang Ⅱ stimulation group pretreated with ERK1/2 antagonist U0126( Ang Ⅱ + S1PR1 + U0126). After 48 h of drug treatment,the size of cardiomyocytes was measured by ImageJ software. Results After 30 d 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 SEW2871 group 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 S1PR1 group 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 S1PR1 group [( 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 S1PR1 activation can remarkably improve cardiac function, ameliorate ventricular remodeling and reduce myocardial fibrosis and hypertrophy induced by pressure overload. The expression of S1PR1 in vascular endothelial cells can modify the hypertrophy of cardiac myocytes,which may be accomplished by activating the ERK 1/2 signaling pathway.
引文
[1]陈伟伟,高润霖,刘力生,等.《中国心血管病报告2017》概要[J].中国循环杂志,2018,33(1):1-8.
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[3]SUN J,ZHANG K,XIONG W J,et al.Clinical effects of a standardized Chinese herbal remedy,Qili Qiangxin,as an adjuvant treatment in heart failure:systematic review and meta-analysis[J].BMC Complement Altern Med,2016,16:201.
[4]宝璐尔,张林,范慧敏,等.强心方组分对心肌梗死后心力衰竭小鼠心功能的影响[J].同济大学学报(医学版),2017,38(6):6-11.
[5]ALEWIJNSE A E,PETERS S L,MICHEL M C.Cardiovascular effects of sphingosine-1-phosphate and other sphingomyelin metabolites[J].Br J Pharmacol,2004,143(6):666-684.
[6]CANNAVO A,RENGO G,LICCARDO D,et al.β1-adrenergic receptor and sphingosine-1-phosphate receptor 1(S1PR1)reciprocal downregulation influences cardiac hypertrophic response and progression to heart failure:protective role of S1PR1 cardiac gene therapy[J].Circulation,2013,128(15):1612-1622.
[7]HE P,PHILBRICK M J,AN X,et al.Endothelial differentiation gene-1,a new downstream gene is involved in RTEF-1 induced angiogenesis in endothelial cells[J].PLoS One,2014,9(2):e88143.
[8]JOSIPOVIC I,PFLGER B,FORK C,et al.Long noncoding RNA LISPR1 is required for S1P signaling and endothelial cell function[J].J Mol Cell Cardiol,2018,116:57-68.
[9]JUNG B,OBINATA H,GALVANI S,et al.Flow-regulated endothelial S1P receptor-1 signaling sustains vascular development[J].Dev Cell,2012,23(3):600-610.
[10]KEUL P,VAN BORREN M M,GHANEM A,et al.Sphingosine-1-Phosphate Receptor 1 Regulates Cardiac Function by Modulating Ca2+Sensitivity and Na+/H+Exchange and Mediates Protection by Ischemic Preconditioning[J].J Am Heart Assoc,2016,5(5).pii:ew3393.
[11]OHKURA S I,USUI S,TAKASHIMA S I,et al.Augmented sphingosine 1 phosphate receptor-1 signaling in cardiac fibroblasts induces cardiac hypertrophy and fibrosis through angiotensin II and interleukin-6[J].PLoSOne,2017,12(8):e0182329.
[12]包馨慧,李海霞,陶静,等.一磷酸鞘氨醇/一磷酸鞘氨醇1受体信号通路在大鼠肥大心肌细胞缺血后适应中的作用及其机制[J].中华心血管病杂志,2016,44(5):431-435.
[13]ZHANG F,XIA Y,YAN W,et al.Sphingosine 1-phosphate signaling contributes to cardiac inflammation,dysfunction,and remodeling following myocardial infarction[J].Am J Physiol Heart Circ Physiol,2016,310(2):H250-261.
[14]PIPPIN J W,QU Q,MEIJER L,et al.Direct in vivo inhibition of the nuclear cell cycle cascade in experimental mesangial proliferative glomerulonephritis with Roscovitine,a novel cyclin-dependent kinase antagonist[J].JClin Invest,1997,100(10):2512-2520.
[15]ISHII M,EGEN J G,KLAUSCHEN F,et al.Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates bone homeostasis[J].Nature,2009,458(7237):524-528.
[16]HLA T.Physiological and pathological actions of sphingosine 1-phosphate[J].Semin Cell Dev Biol,2004,15(5):513-520.
[17]HOU J,KANG Y J.Regression of pathological cardiac hypertrophy:signaling pathways and therapeutic targets[J].Pharmacol Ther,2012,135(3):337-354.
[18]PYNE N J,MCNAUGHTON M,BOOMKAMP S,et al.Role of sphingosine 1-phosphate receptors,sphingosine kinases and sphingosine in cancer and inflammation[J].Adv Biol Regul,2016,60:151-159.
[19]CHAVEZ A,SCHMIDT T T,YAZBECK P,et al.S1PR1 Tyr143 phosphorylation downregulates endothelial cell surface S1PR1 expression and responsiveness[J].JCell Sci,2015,128(5):878-887.
[20]HERCIK C,COSMAS L,MOGENI O D,et al.A diagnostic and epidemiologic investigation of acute febrile illness(AFI)in Kilombero,Tanzania[J].PLoS One,2017,12(12):e0189712.
[21]LIU Y,WADA R,YAMASHITA T,et al.Edg-1,the Gprotein-coupled receptor for sphingosine-1-phosphate,is essential for vascular maturation[J].J Clin Invest,2000,106(8):951-961.
[22]KIMURA T,WATANABE T,SATO K,et al.Sphingosine 1-phosphate stimulates proliferation and migration of human endothelial cells possibly through the lipid receptors,Edg-1 and Edg-3[J].Biochem J,2000,348 Pt 1:71-76.
[23]ROHINI A,AGRAWAL N,KOYANI C N,et al.Molecular targets and regulators of cardiac hypertrophy[J].Pharmacol Res,2010,61(4):269-280.
[24]SONG Y,XU J,LI Y,et al.Cardiac ankyrin repeat protein attenuates cardiac hypertrophy by inhibition of ERK1/2 and TGF-βsignaling pathways[J].PLoS One,2012,7(12):e50436.
[25]LI C,CHEN Z,YANG H,et al.Selumetinib,an Oral Anti-Neoplastic Drug,May Attenuate Cardiac Hypertrophy via Targeting the ERK Pathway[J].PLoS One,2016,11(7):e0159079.
[26]IGARASHI J,ERWIN P A,DANTAS A P,et al.VEGF induces S1P1 receptors in endothelial cells:Implications for cross-talk between sphingolipid and growth factor receptors[J].Proc Natl Acad Sci USA,2003,100(19):10664-10669.
[2]SENNI M,PAULUS W J,GAVAZZI A,et al.New strategies for heart failure with preserved ejection fraction:the importance of targeted therapies for heart failure phenotypes[J].Eur Heart J,2014,35(40):2797-2815.
[3]SUN J,ZHANG K,XIONG W J,et al.Clinical effects of a standardized Chinese herbal remedy,Qili Qiangxin,as an adjuvant treatment in heart failure:systematic review and meta-analysis[J].BMC Complement Altern Med,2016,16:201.
[4]宝璐尔,张林,范慧敏,等.强心方组分对心肌梗死后心力衰竭小鼠心功能的影响[J].同济大学学报(医学版),2017,38(6):6-11.
[5]ALEWIJNSE A E,PETERS S L,MICHEL M C.Cardiovascular effects of sphingosine-1-phosphate and other sphingomyelin metabolites[J].Br J Pharmacol,2004,143(6):666-684.
[6]CANNAVO A,RENGO G,LICCARDO D,et al.β1-adrenergic receptor and sphingosine-1-phosphate receptor 1(S1PR1)reciprocal downregulation influences cardiac hypertrophic response and progression to heart failure:protective role of S1PR1 cardiac gene therapy[J].Circulation,2013,128(15):1612-1622.
[7]HE P,PHILBRICK M J,AN X,et al.Endothelial differentiation gene-1,a new downstream gene is involved in RTEF-1 induced angiogenesis in endothelial cells[J].PLoS One,2014,9(2):e88143.
[8]JOSIPOVIC I,PFLGER B,FORK C,et al.Long noncoding RNA LISPR1 is required for S1P signaling and endothelial cell function[J].J Mol Cell Cardiol,2018,116:57-68.
[9]JUNG B,OBINATA H,GALVANI S,et al.Flow-regulated endothelial S1P receptor-1 signaling sustains vascular development[J].Dev Cell,2012,23(3):600-610.
[10]KEUL P,VAN BORREN M M,GHANEM A,et al.Sphingosine-1-Phosphate Receptor 1 Regulates Cardiac Function by Modulating Ca2+Sensitivity and Na+/H+Exchange and Mediates Protection by Ischemic Preconditioning[J].J Am Heart Assoc,2016,5(5).pii:ew3393.
[11]OHKURA S I,USUI S,TAKASHIMA S I,et al.Augmented sphingosine 1 phosphate receptor-1 signaling in cardiac fibroblasts induces cardiac hypertrophy and fibrosis through angiotensin II and interleukin-6[J].PLoSOne,2017,12(8):e0182329.
[12]包馨慧,李海霞,陶静,等.一磷酸鞘氨醇/一磷酸鞘氨醇1受体信号通路在大鼠肥大心肌细胞缺血后适应中的作用及其机制[J].中华心血管病杂志,2016,44(5):431-435.
[13]ZHANG F,XIA Y,YAN W,et al.Sphingosine 1-phosphate signaling contributes to cardiac inflammation,dysfunction,and remodeling following myocardial infarction[J].Am J Physiol Heart Circ Physiol,2016,310(2):H250-261.
[14]PIPPIN J W,QU Q,MEIJER L,et al.Direct in vivo inhibition of the nuclear cell cycle cascade in experimental mesangial proliferative glomerulonephritis with Roscovitine,a novel cyclin-dependent kinase antagonist[J].JClin Invest,1997,100(10):2512-2520.
[15]ISHII M,EGEN J G,KLAUSCHEN F,et al.Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates bone homeostasis[J].Nature,2009,458(7237):524-528.
[16]HLA T.Physiological and pathological actions of sphingosine 1-phosphate[J].Semin Cell Dev Biol,2004,15(5):513-520.
[17]HOU J,KANG Y J.Regression of pathological cardiac hypertrophy:signaling pathways and therapeutic targets[J].Pharmacol Ther,2012,135(3):337-354.
[18]PYNE N J,MCNAUGHTON M,BOOMKAMP S,et al.Role of sphingosine 1-phosphate receptors,sphingosine kinases and sphingosine in cancer and inflammation[J].Adv Biol Regul,2016,60:151-159.
[19]CHAVEZ A,SCHMIDT T T,YAZBECK P,et al.S1PR1 Tyr143 phosphorylation downregulates endothelial cell surface S1PR1 expression and responsiveness[J].JCell Sci,2015,128(5):878-887.
[20]HERCIK C,COSMAS L,MOGENI O D,et al.A diagnostic and epidemiologic investigation of acute febrile illness(AFI)in Kilombero,Tanzania[J].PLoS One,2017,12(12):e0189712.
[21]LIU Y,WADA R,YAMASHITA T,et al.Edg-1,the Gprotein-coupled receptor for sphingosine-1-phosphate,is essential for vascular maturation[J].J Clin Invest,2000,106(8):951-961.
[22]KIMURA T,WATANABE T,SATO K,et al.Sphingosine 1-phosphate stimulates proliferation and migration of human endothelial cells possibly through the lipid receptors,Edg-1 and Edg-3[J].Biochem J,2000,348 Pt 1:71-76.
[23]ROHINI A,AGRAWAL N,KOYANI C N,et al.Molecular targets and regulators of cardiac hypertrophy[J].Pharmacol Res,2010,61(4):269-280.
[24]SONG Y,XU J,LI Y,et al.Cardiac ankyrin repeat protein attenuates cardiac hypertrophy by inhibition of ERK1/2 and TGF-βsignaling pathways[J].PLoS One,2012,7(12):e50436.
[25]LI C,CHEN Z,YANG H,et al.Selumetinib,an Oral Anti-Neoplastic Drug,May Attenuate Cardiac Hypertrophy via Targeting the ERK Pathway[J].PLoS One,2016,11(7):e0159079.
[26]IGARASHI J,ERWIN P A,DANTAS A P,et al.VEGF induces S1P1 receptors in endothelial cells:Implications for cross-talk between sphingolipid and growth factor receptors[J].Proc Natl Acad Sci USA,2003,100(19):10664-10669.