Key Laboratory for Tumor Immunology and Traditional Chinese Medicine Immunology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, 18877 Jingshi Road, Jinan 250062, China.
J Ethnopharmacol. 2013 Jan 9;145(1):241-53. doi: 10.1016/j.jep.2012.10.059. Epub 2012 Nov 22.
The Th1/Th2/Th17/Treg paradigm plays an important role in achieving maternal-fetal immunotolerance and participates in RU486-induced abortion. Excessive uterine bleeding is the most common side effect of RU486-induced abortion; however, its etiopathogenesis has not been fully understood. Therefore, elucidating the correlation between the Th1/Th2/Th17/Treg paradigm and the volume of uterine bleeding may offer novel therapeutic target for reducing uterine bleeding in RU486-induced abortion. Leonurus sibiricus has been used in clinics to reduce postpartum hemorrhage with low toxicity and high efficiency; however, the effective constituents and therapeutic mechanism have not been described. Stachydrine hydrochloride is the main constituent of L. sibiricus, therefore L. sibiricus is regarded as a candidate for reducing uterine bleeding in RU486-induced abortion mice by regulating the Th1/Th2/Th17/Treg paradigm.
The purpose of this study was to determine the Th1/Th2/Th17/Treg paradigm in uterine bleeding of RU486-induced abortion mice and to elucidate the immunopharmacologic effects of stachydrine hydrochloride on inducing the Th1/Th2/Th17/Treg paradigm in reducing the uterine bleeding volume in RU486-induced abortion mice.
To investigate the Th1/Th2/Th17/Treg paradigm in uterine bleeding during RU486-induced abortion mice, pregnant BALB/c mice were treated with high- and low-dose RU486 (1.5mg/kg and 0.9 mg/kg, respectively), and the serum progesterone (P(4)) protein level, uterine bleeding volume, and proportions of Th1/Th2/Th17/Treg cells in mice at the maternal-fetal interface were detected by ELISA assay, alkaline hematin photometric assay, and flow cytometry, respectively. To determine the regulatory effect of stachydrine hydrochloride on the Th1/Th2/Th17/Treg paradigm in vitro, splenocytes of non-pregnant mice were separated and treated with P(4,) RU486, and/or stachydrine hydrochloride (10(-5)M, 10(-4)M, and 10(-3)M, respectively). The proportions of Th1/Th2/Th17/Treg cells were analyzed using flow cytometry. To evaluate the effect of stachydrine hydrochloride in reducing uterine bleeding via regulation of the Th1/Th2/Th17/Treg paradigm, pregnant mice were treated with RU486 (1.5mg/kg) and/or stachydrine hydrochloride (2.5mg/kg, 5mg/kg, and 10mg/kg). The serum P(4) level, uterine bleeding volume, and proportions of Th1/Th2/Th17/Treg cells at the mice maternal-fetal interface were detected. Moreover, the protein levels of cytokines (IL-12 and IL-6) and the cytokine soluble receptors were analyzed by ELISA assay, and the mRNA expression of transcription factors (T-bet, GATA-3, RORγt, and Foxp3) were detected by RT-PCR assay.
Th1- and Th17-biased immunity was observed in RU486-induced abortion mice. The volume of uterine bleeding during RU486-induced abortion was negatively related to the proportions of Th1 and Th17 cells, as well as the ratios of Th1:Th2 cells and Th17:Treg cells, and positively related to the proportions of Th2 and Treg cells. Stachydrine hydrochloride promoted the protein expression of IL-12 and IL-6, as well as the mRNA expression of T-bet and RORγt, while inhibiting the mRNA expression of GATA-3 and Foxp3. Therefore, the Th1/Th2/Th17/Treg paradigm in RU486-induced abortion mice shifted to Th1 and Th17 after stachydrine hydrochloride administration. The volume of uterine bleeding during RU486-induced abortion was reduced significantly after stachydrine hydrochloride administration.
The Th1/Th2/Th17/Treg paradigm is closely related to the volume of uterine bleeding in RU486-induced abortion mice. The Th1/Th2/Th17/Treg paradigm induced by stachydrine hydrochloride contributed to the reduction in uterine bleeding in RU486-induced abortion mice.
ETHNOPHARMACOLOGICAL 相关性：Th1/Th2/Th17/Treg 范式在实现母婴免疫耐受方面发挥着重要作用，并参与 RU486 诱导的流产。过度的子宫出血是 RU486 诱导流产最常见的副作用；然而，其发病机制尚未完全阐明。因此，阐明 Th1/Th2/Th17/Treg 范式与子宫出血量之间的相关性，可能为减少 RU486 诱导流产中的子宫出血提供新的治疗靶点。益母草已在临床上用于减少产后出血，具有低毒性和高效率；然而，其有效成分和治疗机制尚未描述。盐酸水苏碱是益母草的主要成分，因此益母草被认为是通过调节 Th1/Th2/Th17/Treg 范式来减少 RU486 诱导流产小鼠子宫出血的候选药物。
本研究旨在确定 RU486 诱导流产小鼠子宫出血中的 Th1/Th2/Th17/Treg 范式，并阐明盐酸水苏碱对诱导 Th1/Th2/Th17/Treg 范式在减少 RU486 诱导流产小鼠子宫出血量中的免疫药理学作用。
为了研究 RU486 诱导流产小鼠子宫出血中的 Th1/Th2/Th17/Treg 范式，用高剂量和低剂量 RU486（1.5mg/kg 和 0.9mg/kg）处理妊娠 BALB/c 小鼠，通过 ELISA 测定法、碱性血红素比色法和流式细胞术分别检测母胎界面小鼠血清孕激素（P4）蛋白水平、子宫出血量以及 Th1/Th2/Th17/Treg 细胞的比例。为了确定盐酸水苏碱对体外 Th1/Th2/Th17/Treg 范式的调节作用，分离非妊娠小鼠的脾细胞，并分别用 P4、RU486 和/或盐酸水苏碱（10-5M、10-4M 和 10-3M）处理，通过流式细胞术分析 Th1/Th2/Th17/Treg 细胞的比例。为了评估盐酸水苏碱通过调节 Th1/Th2/Th17/Treg 范式减少子宫出血的效果，用 RU486（1.5mg/kg）和/或盐酸水苏碱（2.5mg/kg、5mg/kg 和 10mg/kg）处理妊娠小鼠。检测母胎界面小鼠血清 P4 水平、子宫出血量和 Th1/Th2/Th17/Treg 细胞比例。此外，通过 ELISA 测定法分析细胞因子（IL-12 和 IL-6）及其可溶性受体的蛋白水平，并通过 RT-PCR 检测转录因子（T-bet、GATA-3、RORγt 和 Foxp3）的 mRNA 表达。
在 RU486 诱导流产小鼠中观察到 Th1 和 Th17 偏向性免疫。RU486 诱导流产小鼠的子宫出血量与 Th1 和 Th17 细胞的比例以及 Th1:Th2 细胞和 Th17:Treg 细胞的比值呈负相关，与 Th2 和 Treg 细胞的比例呈正相关。盐酸水苏碱促进 IL-12 和 IL-6 蛋白的表达，以及 T-bet 和 RORγt 的 mRNA 表达，同时抑制 GATA-3 和 Foxp3 的 mRNA 表达。因此，盐酸水苏碱给药后，RU486 诱导流产小鼠的 Th1/Th2/Th17/Treg 范式向 Th1 和 Th17 倾斜。RU486 诱导流产小鼠的子宫出血量明显减少。
Th1/Th2/Th17/Treg 范式与 RU486 诱导流产小鼠的子宫出血量密切相关。盐酸水苏碱诱导的 Th1/Th2/Th17/Treg 范式有助于减少 RU486 诱导流产小鼠的子宫出血。
