title: The Th1/Th2/Th17/Treg paradigm induced by stachydrine hydrochloride reduces uterine bleeding in RU486-induced abortion mice.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
  
  
  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 (1.5mg/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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Xia
authors.initials: X
authors.lastname: Li
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.forename: Yuzhu
authors.initials: Y
authors.lastname: Li
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Xiangzhong
authors.initials: X
authors.lastname: Zhao
authors.forename: Xianbin
authors.initials: X
authors.lastname: Zhou
authors.forename: Yuqi
authors.initials: Y
authors.lastname: Guo
authors.forename: Guosheng
authors.initials: G
authors.lastname: Jiang
authors.forename: Chengfang
authors.initials: C
authors.lastname: Yao
mesh_terms: D000028:Abortion, Induced / Q000009:adverse effects*; D000818:Animals; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D015735:Mifepristone / Q000009:adverse effects* / Q000627:therapeutic use; D058990:Molecular Targeted Therapy / Q000379:methods; D011247:Pregnancy; D011374:Progesterone / Q000378:metabolism; D011392:Proline / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D013154:Spleen / Q000187:drug effects / Q000276:immunology; D050378:T-Lymphocytes, Regulatory / Q000378:metabolism; D018417:Th1 Cells / Q000378:metabolism; D058504:Th17 Cells / Q000378:metabolism*; D018418:Th2 Cells / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014592:Uterine Hemorrhage / Q000097:blood / Q000139:chemically induced / Q000188:drug therapy* / Q000276:immunology
chemical_list: D016207:Cytokines; D014157:Transcription Factors; D015735:Mifepristone; D011374:Progesterone; D011392:Proline; C003342:stachydrine