鞘氨醇-1-磷酸对心脏肥大的作用及对心脏微血管内皮细胞的影响

Effect of sphingosine-1-phosphate on cardiac hypertrophy and cardiac microvascular endothelial cells

目的探讨鞘氨醇-1-磷酸(S1P)对心脏肥大(CH)的作用与对心脏微血管内皮细胞(CMECs)功能及血管新生的调控,及对磷脂酰肌醇3-激酶/蛋白激酶B/血管内皮生长因子(PI3K/AKT/VEGF)通路的影响。方法收集51例CH患者和65例正常志愿者外周血,及其死亡捐赠的左心室心肌组织,采用免疫组织化学法(IHC)检测两组心肌组织S1P含量,酶联免疫吸附测定法(ELISA)检测外周血S1P、人二氢-S1P(DH-S1P)和血管内皮生长因子(VEGF)的含量。采用免疫荧光法(IF)分别对两组心肌组织进行S1P和分化簇31(CD31)、S1P和Alpha-平滑肌肌动蛋白(α-SMA)共定位染色。采用免疫印迹法(WB)检测心肌组织S1P、S1P受体1-3(S1PR1-3)、α-SMA、CD31及PI3K/AKT通路的变化。分离培养原代CMECs,将第3代CMECs接种于12孔板中,建立肥大CMECs模型,分为两组:去氧肾上腺素(PE)组和PE+S1P(OE)组,每组6孔。OE组转染1μg的PDEST42-S1P-V5质粒24 h,IF对细胞进行染色。结果CH患者心肌组织S1P和血清S1P、DH-S1P和VEGF含量显著低于正常志愿者(t=6.994、7.822、5.982、9.811,P<0.05),且血清VEGF和S1P含量呈显著正相关性(r=0.427,P>0.01)。相比于正常志愿者,CH患者心肌组织CD31+S1P+双阳性共定位细胞占CD31阳性细胞比例显著降低(t=18.214,P<0.05);α-SMA+S1P+双阳性共定位细胞占α-SMA阳性细胞比例也显著降低(t=12.451,P<0.05)。与正常志愿者相比,CH患者心肌组织S1P、S1PR3、CD31和α-SMA蛋白含量明显降低(t=4.254、4.492、15.803、9.941,P<0.05),S1PR2含量明显增高(t=6.828,P<0.05),S1PR1含量无明显变化,差异无统计学意义(P>0.05)。CH患者心脏组织p-PI3K、p-AKT和p-eNOS蛋白表达量明显低于正常志愿者(t=12.340、15.597、8.624,P<0.05)。相比于PE组,OE组中S1P和α-SMA双阳性共定位细胞占α-SMA阳性细胞比例显著增加,细胞培养上清中S1P和VEGF蛋白水平显著增加(t=6.894、5.213,P<0.05)。结论低水平的S1P可能通过抑制CMECs血管新生和间充质转换,及下调PI3K/AKT/eNOS通路在CH的发生发展中发挥重要作用。

Objective To investigate the effects of sphingosine-1-phosphate(S1 P)on cardiac hypertrophy(CH),the regulation of cardiac microvascular endothelial cells(CMECs)’s function and angiogenesis,and phosphatidylinositol 3-kinase/protein kinase B/vascular endothelial growth factor(PI3 K/AKT/VEGF)pathway.Methods The peripheral blood(PB)and the left ventricular myocardium of death donors of CH patients(n=51)and normal persons(n=65)were collected.S1 P concentrations of myocardial tissues were detected by immunohistochemistry(IHC).S1 P,Dihydro-S1 P(DH-S1 P)and vascular endothelial growth factor(VEGF)concentrations of PB were detected by enzyme-linked immunosorbent assay(ELISA).S1 P and cluster of differentiation 31(CD31),S1 P and alpha-smooth muscle actin(α-SMA)on myocardial tissues co-localization were assessed by immunofluorescence(IF).Western blot(WB)were used to detect the concentrations of S1 P,S1 PR1-3,α-SMA,CD31 and the changes of PI3 K/AKT/eNOS pathway in myocardial tissues.The primary CMECs were isolated and cultured.The 3 rd generation CMECs were inoculated into a 12-well plates.The CMECs and divided into phenylephrine(PE)group and PE+S1 P(over expression,OE)group,6 pores for each group.1μg of PDEST42-S1 P-V5 plasmid was transfected into the OE group for 24 h,and the cells were stained by IF.Results The concentrations of S1 P in myocardium and S1 P,DH-S1 P and VEGF in serum of CH patients were significantly lower than those in normal volunteers(t=6.994,7.822,5.982,9.811;P<0.05),and VEGF and S1 P levels of serum were significantly positively correlated(r=0.427,P>0.01).Compared with normal volunteers,the proportion of S1 P and CD31 double positive co-localized cells to CD31 positive cells in the CH myocardial tissue was significant decreased(t=18.214,P<0.05).The proportion ofα-SMA and S1 P double positive co-localized cells toα-SMA positive cells in the CH myocardial tissue was also significantly decreased(t=12.451,P<0.05).Compared with normal volunteers,the contents of S1 P,S1 PR3,CD31 and a-SMA protein in CH group were significantly reduced(t=4.254,4.492,15.803,9.941;P<0.05),the content of S1 PR2 was significantly increased(t=6.828,P<0.05),and there was no statistical difference in S1 PR1 content(P>0.05).The expression levels of p-PI3 K,p-AKT and p-e NOS proteins in the heart tissue of CH group were significantly lower than those of normal volunteers(t=12.340,15.597,8.624;P<0.05).Compared with the PE group,the proportion of S1 P and a-SMA double positive co-localized cells to a-SMA positive cells in the OE group increased significantly,and the levels of S1 P and VEGF protein in the cell culture supernatant increased significantly(t=6.894,5.213;P<0.05).Conclusion Low levels of S1 P may play an important role in the development of CH by inhibiting angiogenesis and mesenchymal transition of CMECs,and down-regulating the PI3 K/AKT/e NOS pathway.