补肾活血法调控内皮细胞TLR-4/NF-κB通道干预ACA致妊娠丢失的抗凝机制

Recurrent spontaneous abortion (RSA) is a reproductive problem with poorly understood etiology and shown an upward trend in Chinese. It is multifactorial in nature, and several factors are proposed to contribute to its pathogenesis. The coagulation abnormalities caused by autoimmune disorders are gradually being paid attention to RSA. Anticardiolipin antibody (ACA) is a kind of autoimmune antibody. Previous researches have showed that it could cause placental thrombosis and infarction, resulting hypoperfusion of placenta and fetal ischemia and hypoxia. But the mechanism is not clear enough. Our previous clinical studies have demonstrated that DanShou Tang, an experienced prescription of BuShenHuoXue Method could effectively treat ACA induced early threatened abortion and inhibit embryo resorption in ACA induced fetal loss models, down regulate mRNA expression of PAF and VEGF. Based on that, we hypothesize that BuShenHuoXue Method may potentially inhibit the vascular endothelial cell injury and inhibit the formation of local thrombosis. Through the establishment of ACA induced fetal loss pregnant rat model and vascular endothelial cell culture in vitro, we measure the methylation and histone acetylation levels of promoter DNA of vascular endothelial cell signal transduction pathway TLR-4 , NF- kappa B, mRNA and protein expression of downstream cytokines including ICAM-1, VCAM-1, TF and IL-6 and endothelial cell apoptosis after a DanShou Tang intervention, aiming to confirm the hypothesis and explore a new target and method for the drug treatment of RSA.

复发性流产(RSA)在我国发病率呈上升趋势,由免疫功能紊乱导致局部凝血功能异常渐受重视。研究证实抗心磷脂抗体（ACA）可激活血管内皮细胞TLR-4/NF-κB通道，引起母胎界面局部微血栓形成，导致胎盘低灌注与胚胎停育，但机制尚不清楚。课题组前期研究发现补肾活血法经验方丹寿汤能有效治疗ACA阳性者的自然流产，降低ACA阳性孕鼠胎盘组织PAF及VEGF mRNA表达，提出“丹寿汤可能有拮抗内皮细胞损伤，抑制局部血栓形成”假说。通过建立ACA致妊娠丢失孕鼠模型，采用细胞培养、Western-blot 及RT-PCR等技术，观察丹寿汤干预后内皮细胞信号通路TLR-4、NF-κB的启动子DNA甲基化水平，粘附分子ICAM-1、VCAM-1及 TF、 IL-6、TNFα等蛋白及mRNA的表达水平，揭示丹寿汤对内皮细胞的抗凝调控机制，探索补肾活血法治疗RSA新的靶点与途径。

由于环境与社会的多种复杂因素，复发性流产（recurrent spontaneous abortion, RSA）的发病率逐年上升，尤其是不明原因的复发性流产（unexplained spontaneous abortion，URSA）显著增加，约占RSA的50 %。URSA被证实主要是由于自身免疫功能紊乱所致，通过免疫排斥、细胞杀伤、促进滋养细胞凋亡、诱发局部血栓形成等多种方式导致胚胎停育。其中局部血栓形成，既可是单独致病因素，也可是其它途径的并发环节而致病，因此受到更多的关注。中医学“滑胎”类似于现代医学的RSA，认为冲任损伤、肾虚无力固胞为该病之本。肾虚则失温煦，寒则血滞不行或阴虚生热,火热灼精血均可导致血瘀。本课题组将补肾与活血法有机结合，针对本病建立了补肾活血安胎的原则，拟定补肾活血经验方-丹寿汤。本课题组前期结果提示丹寿汤可能通过调控内皮细胞抗凝而达到减少妊娠丢失的作用，因此本项目首先研究丹寿汤对抗心磷脂抗体（anticardiolipin antibody, aCL）引起的内皮细胞损伤的影响，利用丹寿汤大鼠含药血清对人内皮细胞HUVEC进行处理后，添加aCL-IgG构建细胞模型，经细胞生物学及分子生物学实验进行检测，结果显示丹寿汤含药血清可通过降低TLR4/MyD88/NF-κB通路活性，抑制炎性因子的释放对aCL所致HUVEC损伤具有明显保护作用。其次本项目通过注射aCL-IgG复制aCL致妊娠丢失孕鼠模型，采用丹寿汤进行药物干预治疗，经过胚胎吸收率的计算及分子生物学实验检测，结果显示丹寿汤能够抑制胚胎吸收，提高妊娠成功率，其作用机制可能与调节Th17/Treg平衡失调有关。为了进一步了解丹寿汤的起效机制，我们研究丹寿汤中的主要成分之一Hyperoside的作用机制。首先复制aCL致妊娠丢失孕鼠模型，利用Hyperoside进行药物干预治疗，结果显示Hyperoside可通过激活自噬和抑制炎症减轻妊娠丢失。其次，利用Hyperoside对aCL-IgG诱导的细胞损伤模型的HUVEC进行预处理，结果表明Hyperoside可通过激活自噬保护人脐静脉内皮细胞免受抗心磷脂抗体诱导的损伤。本项目的研究结果为中药治疗RSA提供理论依据，为丹寿汤及Hyperoside在临床上用于治疗RSA提供数据支撑，还可为临床开发新的RSA有效治疗药物提供。

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