The Th1/Th2/Th17/Treg paradigm induced by stachydrine hydrochloride reduces uterine bleeding in RU486-induced abortion mice
The Th1/Th2/Th17/Treg paradigm induced by stachydrine hydrochloride reduces uterine bleeding in RU486-induced abortion mice
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盐酸水苏碱诱导的 Th1/Th2/Th17/Treg 范式可减少 RU486 诱导流产小鼠的子宫出血
DOI：
10.1016/j.jep.2012.10.059
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发表时间：
2013-01-09
影响因子：
5.4
通讯作者：
Yao, Chengfang
中科院分区：
文献类型：
--
作者：
Li, Xia;Wang, Bin;Yao, Chengfang
民族药理学相关性：Th 1/Th 2/Th 17/Treg在母胎免疫耐受中起重要作用，并参与RU 486诱导的流产。子宫过度出血是RU 486人工流产最常见的副作用，然而，其发病机制尚未完全了解。因此，阐明Th 1/Th 2/Th 17/Treg范式与子宫出血量的相关性，可能为减少RU 486流产后子宫出血提供新的治疗靶点。益母草具有低毒、高效的优点，已被临床应用于产后出血的防治，但其有效成分及作用机制尚未见报道。盐酸水苏碱是西伯利亚甘草的主要成分，因此西伯利亚甘草被认为是通过调节Th 1/Th 2/Th 17/Treg范式来减少RU 486诱导流产小鼠子宫出血的候选药物。本研究的目的是确定RU 486 - 1子宫出血的Th 1/Th 2/Th 17/Treg模式。目的：观察盐酸水苏碱对RU 486流产小鼠子宫出血量的影响，并探讨水苏碱在减少RU 486流产小鼠子宫出血量中诱导Th 1/Th 2/Th 17/Treg范式的免疫药理学作用。为了研究RU 486诱导流产小鼠子宫出血中的Th 1/Th 2/Th 17/Treg模式，用高剂量和低剂量RU 486处理妊娠BALB/c小鼠（分别为1.5 mg/kg和0.9 mg/kg），血清孕酮（P-4）蛋白水平，子宫出血量，分别采用ELISA法、碱性高铁血红素光度法和流式细胞术检测母胎界面Th 1/Th 2/Th 17/Treg细胞比例。为了确定盐酸水苏碱对体外Th 1/Th 2/Th 17/Treg范例的调节作用，分离非妊娠小鼠的脾细胞并用P-4、RU 486和/或盐酸水苏碱（分别为10（-5）M、10（-4）M和10（-3）M）处理。采用流式细胞术分析Th 1/Th 2/Th 17/Treg细胞比例。为了评估盐酸水苏碱通过调节Th 1/Th 2/Th 17/Treg范式减少子宫出血的作用，用RU 486（1.5 mg/kg）和/或盐酸水苏碱（2.5 mg/kg、5 mg/kg和10 mg/kg）处理妊娠小鼠。检测血清P-4水平、子宫出血量及母胎界面Th 1/Th 2/Th 17/Treg细胞比例。此外，通过ELISA分析细胞因子（IL-12和IL-6）和细胞因子可溶性受体的蛋白水平。结果：RU 486诱导流产小鼠产生了Th 1和Th 17偏向的免疫应答，并通过RT-PCR检测了T-bet、加塔-3、ROR γ t和Foxp 3等转录因子的mRNA表达。RU 486流产时子宫出血量与Th 1、Th 17细胞比例、Th 1：Th 2细胞比例、Th 17：Treg细胞比例呈负相关，与Th 2、Treg细胞比例呈正相关。盐酸水苏碱促进IL-12和IL-6蛋白表达，以及T-bet和ROR γ t mRNA表达，而抑制加塔-3和Foxp 3 mRNA表达。因此，在RU 486诱导的流产小鼠中，Th 1/Th 2/Th 17/Treg范式在盐酸水苏碱给药后转移到Th 1和Th 17。结论：Th 1/Th 2/Th 17/Treg细胞亚群与RU 486流产小鼠子宫出血量密切相关。盐酸水苏碱诱导的Th 1/Th 2/Th 17/Treg范式有助于减少RU 486诱导流产小鼠的子宫出血。(C)2012爱思唯尔爱尔兰有限公司保留所有权利。
Ethnopharmacological relevance: 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.Aim of the study: 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.Materials and Methods: 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 (1.5 mg/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 cytomety, 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 (1.5 mg/kg) and/or stachydrine hydrochloride (2.5 mg/kg, 5 mg/kg, and 10 mg/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, ROR gamma t, and Foxp3) were detected by RT-PCR assay.Result: 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 ROR gamma t, 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.Conclusion: 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. (C) 2012 Elsevier Ireland Ltd. All rights reserved.