低分子肝素改善子痫前期中的内皮损伤的机制研究

Mechanism of low molecular heparin in improving endothelial injury in preeclampsia

目的探讨低分子肝素对子痫前期(preeclampsia,PE)患者胎盘组织中血管内皮细胞的Notch信号通路配体Delta样配体4(Delta-like ligand 4,DLL4)表达的影响,及其对内皮细胞损伤的作用及相关机制.方法选取2018年3月至2019年4月在海南医学院第二附属医院妇产科住院分娩的早发型重度PE患者60例,按随机数字表法将其分为PE组(单用硫酸镁)与低分子肝素组(联合使用硫酸镁与低分子肝素),各30例.选取同期正常妊娠剖宫产妇女30例为对照组.免疫组织化学法观察对比3组产妇胎盘组织中DLL4的表达水平;ELISA检测3组孕产妇外周血中内皮损伤相关细胞因子内皮素-1(endothelian-1,ET-1)、可溶性血管细胞黏附分子-1(soluble vascular cell adhesion molecule-1,sVCAM-1)的表达;体外培养脐静脉内皮细胞,预先应用低分子肝素进行干预,再应用PE患者血清进行刺激,TUNEL染色,内皮细胞通透性检测观察低分子肝素对PE血清诱导的内皮细胞凋亡,内皮细胞完整性的影响;Western blot检测低分子肝素对PE血清诱导的内皮细胞中凋亡蛋白Bcl-2、Bax、Cleaved caspase-3表达的影响,并使用Notch信号通路阻滞剂MK-0752进行干预,观察低分子肝素是否通过Notch/DLL4信号通路抑制PE血清诱导的内皮细胞凋亡.结果DLL4主要表达于胎盘组织的血管内皮细胞中,且与对照组(63.5%)相比,PE组及低分子肝素组的胎盘组织中DLL4的表达阳性率(分别为14.7%、36.9%)均明显更低,而低分子肝素组较PE组中DLL4的表达明显更高,差异均有统计学意义(P<0.05);与对照组相比,PE组及低分子肝素组产妇血清中ET-1、sVCAM-1表达水平明显更高;与PE组相比,低分子肝素组产妇血清中上述细胞因子的表达水平明显更低,差异均有统计学意义(P<0.05).预先使用低分子肝素处理可显著抑制PE血清诱导的内皮细胞凋亡,并改善PE血清对其的通透性影响,从而保护内皮细胞的完整性.Western blot结果表明,低分子肝素能够通过促进抗凋亡相关蛋白Bcl-2的表达,抑制促凋亡蛋白Bax、Cleaved caspase-3的表达来改善PE血清对内皮细胞损伤,而使用Notch信号通路抑制剂MK-0752能显著逆转低分子肝素对凋亡相关蛋白的上述影响.结论联合使用低分子肝素能够通过促进胎盘组织内皮细胞中DLL4的表达,其可能通过Notch/DLL4信号通路抑制内皮细胞的凋亡,从而保护内皮细胞的完整性,改善PE的临床预后.

Objective To explore the effect of low molecular weight heparin(LMWH)on the expression of Notch signaling pathway ligand DLL4 in vascular endothelial cells in the placenta tissue of patients with preeclampsia(PE),and its effect on endothelial cell damage and related mechanisms.Methods Selected 60 patients with early-onset severe PE who were hospitalized in the Department of Obstetrics and Gynecology of the Second Affiliated Hospital of Hainan Medical College from March 2018 to April 2019 were divided into the PE group(using magnesium sulfate alone)and the LMWH group(combined use of magnesium sulfate and LMWH)according to the random number table,with 30 cases in each group.30 cases of normal pregnant women with cesarean section during the same period were selected as the control group.Immunohistochemical method was used to observe and compare the expression level of DLL4 in the placenta tissues of the three groups;ELISA was used to detect the expression of endothelial injury-related cytokine endothelin-1(endothelian-1,ET-1)and soluble vascular cell adhesion molecules-1(sVCAM-1)in the peripheral blood of the three groups.HUVEC was cultured in vitro,pre-intervention with LMWH,the effects of LMWH on endothelial cell apoptosis and integrity induced by PE serum were observed by stimulation,TUNEL staining and endothelial cell permeability test to observe the LMWH induced endothelial cell apoptosis and endothelial cell integrity.Western blot was used to detect the effect of LMWH on the expression of Bcl-2,Bax and cleaved caspase-3 in endothelial cells induced by PE serum,and the Notch signal pathway blocker MK-0752 was used for intervention to observe whether LMWH can inhibit the apoptosis of endothelial cells induced by PE serum through the Notch/D114 signal pathway.Results DLL4 was mainly expressed in the vascular endothelial cells of the placenta tissue,and compared with the control group(63.5%),the positive rate of DLL4 expression in the placenta tissue of the PE group and the LMWH group(14.1%,36.9%,respectively)was significant decreased,and the expression of DLL4 in the LMWH group was significantly higher than that in the PE group,the difference was statistically significant(P<0.05).Compared with the control group,the expression levels of ET-1 and sVCAM-1 in the maternal serum of the PE group and the LMWH group were significantly increased;compared wth the PE group,the expression levels of the above-mentioned cytokines in the maternal serum of the LMWH group were significantly reduced,the differences were statistically significant(P<0.05).Pre-treatment with LMWH can significantly inhibit the apoptosis of endothelial cells induced by PE serum and improve the permeability of PE serum to protect the integrity of endothelial cells.Western blot results show that LMWH can improve the damage of PE serum to endothelial cells by promoting the expression of anti-apoptosis-related protein Bcl-2 and inhibiting the expression of pro-apoptotic proteins Bax and Cleaved caspase-3,while using Notch signaling pathway inhibitor MK-0752 can significantly reverse the above-mentioned effects of LMWH on apoptosis-related proteins.Conclusion The combined use of LMWH can promote the expression of DLL4 in placental endothelial cells,which may inhibit endothelial cell apoptosis through the Notch/DLL4 signaling pathway,thereby protecting the integrity of endothelial cells and improving the clinical prognosis of PE.