Huang Cong, Song Kunkun, Ma Wenwen, Ding Jiahui, Chen Zhuo, Zhang Mingmin
Institute of Integrated Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical Collage, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.
J Ethnopharmacol. 2017 Aug 17;208:44-56. doi: 10.1016/j.jep.2017.06.022. Epub 2017 Jun 21.
Bushen Huoxue recipe (BHR) is a Chinese herbal prescription composed of ten herbs and it is widely used for the treatment of diminished ovarian reserve (DOR). This study investigates the potentially beneficial effects and underlying mechanism of BHR on a cyclophosphamide (CTX) induced model of DOR in mice.
Granules of BHR were first subjected to high performance liquid chromatography (HPLC) to determine the exact ingredients within the mixture. We then induced DOR in mice by injecting them with 90mg/kg of CTX. Following the single intraperitoneal injection, mice then received either saline or BHR for 21 days. To assess the effects of BHR on DOR, we examined splenic and ovarian morphology, estrous cycle duration, ovarian index, follicle number, body weight, and concentration of serum E and FSH. To explore the immunological mechanism behind the effects, mouse splenocytes were isolated in order to analyze the proportion of CD4, CD8 T cells and Th1, Th17 and Treg subsets by flow cytometry. The serum levels of IFN-γ, TNF-α, IL-4, IL-17A, IL-6 and IL-10 were detected using Cytometric Bead Array (CBA). Additionally, the mRNA expression levels of T-bet, RORγt and Foxp3 were measured with quantitative real-time PCR.
Our results show that following treatment with BHR in DOR mice, several measures showed significant improvement. The morphology of the ovary and spleen, estrous cycle duration, body weight, ovarian index, and serum levels of E and FSH recovered to approximately normal levels and the loss of follicles at all stages was significantly attenuated. Furthermore, the elevated proportions of CD4 T cells, Th1, Th17, Treg subsets and the increased serum levels of IFN-γ, TNF-α, IL-17A, IL-6 and IL-10 as well as the mRNA expressions of T-bet, RORγt and Foxp3 in DOR mice were significantly decreased.
Our results show that BHR is a promising candidate to treat DOR mice and this beneficial effect may be mediated through the downregulation of augmented autoimmunity.
补肾活血方（BHR）是一种由十味草药组成的中药方剂，广泛用于治疗卵巢储备功能下降（DOR）。本研究探讨BHR对环磷酰胺（CTX）诱导的小鼠DOR模型的潜在有益作用及潜在机制。
首先对BHR颗粒进行高效液相色谱（HPLC）分析，以确定混合物中的具体成分。然后通过给小鼠注射90mg/kg的CTX诱导其发生DOR。单次腹腔注射后，小鼠接受生理盐水或BHR治疗21天。为评估BHR对DOR的影响，我们检查了脾脏和卵巢形态、发情周期持续时间、卵巢指数、卵泡数量、体重以及血清E和FSH浓度。为探究其作用背后的免疫机制，分离小鼠脾细胞，通过流式细胞术分析CD4、CD8 T细胞以及Th1、Th17和Treg亚群的比例。使用细胞因子珠阵列（CBA）检测血清中IFN-γ、TNF-α、IL-4、IL-17A、IL-6和IL-10的水平。此外，用定量实时PCR检测T-bet、RORγt和Foxp3的mRNA表达水平。
我们的结果表明，DOR小鼠经BHR治疗后，多项指标有显著改善。卵巢和脾脏形态、发情周期持续时间、体重、卵巢指数以及血清E和FSH水平恢复至大致正常水平，各阶段卵泡丢失明显减轻。此外，DOR小鼠中升高的CD4 T细胞、Th1、Th17、Treg亚群比例以及升高的血清IFN-γ、TNF-α、IL-17A、IL-6和IL-10水平以及T-bet、RORγt和Foxp3的mRNA表达均显著降低。
我们的结果表明，BHR是治疗DOR小鼠的一个有前景的候选药物；这种有益作用可能是通过下调增强的自身免疫介导的。
