补肾活血方对抗心磷脂抗体致复发性流产大鼠的治疗效果

Therapeutic effect of Bushen Huoxuetfang on recurrent abortion induced by cardiolipin antibody in rats

目的探讨补肾活血方对抗心磷脂抗体(aCL)致复发性流产(RSA)大鼠的治疗效果及对血小板/嘌呤能离子通道型7(P2X7)/Nod样受体蛋白3(NLRP3)炎症小体通路的影响.方法随机将受孕大鼠24只,分为aCL-RSA、aCL-RSA+低分子肝素组、aCL-RSA+丹寿汤组、模型组,每组6只;建立aCL-RSA孕鼠模型,给予补肾活血方干预,观察16天孕鼠体质量,孕鼠胚胎吸收率,胎鼠体质量;检测大鼠血浆血小板活化因子受体(PAFR)、血小板第4因子(PF4)含量,大鼠胎盘组织中白细胞介素-1β(IL-1β)、IL-18含量,HE染色检测胎盘组织病理学改变,TUNEL染色法检测胎盘组织中细胞凋亡;Western Blot检测cleaved caspase-1、人细胞凋亡相关斑点样蛋白(ASC)、NLRP3、Bcl2、Bax、cleaved caspase-3、嘌呤能离子通道型7(P2X7)蛋白的表达,探讨aPL致复发性流产与血小板活化、NLRP3炎症小体通路及P2X7的相关性,及丹寿汤对aCL-RSA孕鼠的影响及对血小板活化/P2X7/NLRP3通路的影响.结果与模型组比较,aCL-RSA组能明显降低孕鼠体质量,增加孕鼠胚胎吸收率,降低胎鼠体质量,增加血浆PAFR、PF4含量(P<0.05);aCL-RSA组胎盘组织迷宫区母性红细胞较少,较多血栓形成,胎盘血窦空间明显减小;胎盘组织中凋亡细胞数目明显增加,胎盘组织中IL-1β、IL-18增加,胎盘组织中cleaved caspase-1、ASC、NLRP3、Bax、cleaved caspase-3、P2X7蛋白表达增加(P<0.05),Bcl2蛋白水平降低(P<0.05).结论补肾活血方能抑制血小板活化,下调P2X7的表达,抑制NLRP3的激活,减轻胎盘的高凝、炎症状态进而减少aCL-RSA的发生.

OBJECTIVE To investigate the therapeutic effect of Bushen Huoxuefang on recurrent abortion induced by cardiolipin antibody(CL)in rats and its effect on the platelet/purinergic2X7(P2X7)/Nod-like receptor protein 3(NLRP3)inflammatory vesicle pathway.METHODS Twenty-four pregnant rats were randomly divided into aCL-RSA group,aCL-RSA+LMWH group,aCL-RSA+Danshou tang group,and NH-IgG group,with 6 rats in each group.The aCL-RSA pregnant mouse model was established,and Bushen Huoxuefang was given intervene,and the body mass of the pregnant rats,embryo absorption rate and fetal body mass were observed in E16 days.The contents of platelet-activating factor receptor(PAFR)and platelet factor 4(PF4)in rat plasma,interleukin-1β(IL-1p)and IL-18 in rat placental tissue were detected by ELISA,the histopathological changes of placental tissue were detected by HE staining,and apoptosis in placental tissue was detected by TUNEL staining.Western Blot analysis was performed to detect the expression of cleaved caspase-l,human apoptosis-associated mottle protein(ASC),NLRP3,Bcl2,Bax,cleaved caspase-3,and P2X7 proteins,and the correlation between APL-induced recurrent abortion and platelet activation,NLRP3 inflammasome pathway,and purinergic2X7(P2X7),as well as the effect of Danshou decoction on aCL-RSA pregnant mice and platelet activation/P2X7/NLRP3 pathway,were investigated.RESULTS Compared with NH-IgG group,the aCL-RSA group could significantly reduce the body mass of pregnant rats,increase embryo absorption rate of pregnant rats,reduce the body mass of fetal rats,and increase the plasma PAFR and PF4 contents of pregnant mice(P<0.05).The aCL-RSA group had fewer maternal erythrocytes more thrombosis in the labyrinthine area of placental tissue,and a significantly reduced placental blood sinus space.There was a significant increase in the number of apoptotic cells,an increased in IL-1βand IL-18,and an increase in the expressions of cleaved caspase-1,ASC,NLRP3,Bax,cleaved caspase-3,and P2X7 in placenta tissues(P<0.05),and a decrease in the level of Bcl2 protein(P<0.05).CONCLUSION Bushen Huoxuefang could inhibit platelet activation,down-regulate the expression of P2X7,inhibit the activation of NLRP3,elevate the hypercoagulability and inflammation of placenta,and then reduce the occurrence of aCL-RSA.