Tsumura Kampo Research Laboratories, Kampo Research & Development Division, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki, 300-1192, Japan.
Tsumura Kampo Research Laboratories, Kampo Research & Development Division, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki, 300-1192, Japan.
J Ethnopharmacol. 2021 Jan 30;265:113288. doi: 10.1016/j.jep.2020.113288. Epub 2020 Aug 22.
Tokishakuyakusan (TSS) is a Kampo medicine that is prescribed for the treatment of infertility in Japan. However, its precise mechanism of action remains unclear.
Leukemia inhibitory factor (LIF) in the endometrium plays an indispensable role in embryo implantation and is linked to infertility or implantation failure. Previously, we demonstrated that TSS ameliorated implantation failure induced by mifepristone (RU-486), an antagonist of progesterone, in rats. Herein, we aimed to clarify whether the ameliorating effect of TSS on implantation failure in the rat model involves endometrial LIF. Additionally, we determined whether decidualization, the dysfunction of which is linked to infertility or implantation failure similar to LIF, progesterone, and other implantation-related factors, are involved in the effect of TSS.
The implantation failure rat model was developed via the subcutaneous administration of RU-486 (7 mg/kg) on day 3 post-coitus. Sesame oil was administered as the vehicle control. Rats were fed a diet containing 1% or 3% TSS or a control diet from day 13 pre-coitus. Subsequently, the implantation sites were assessed, and plasma progesterone levels were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on day 8 post-coitus. The LIF mRNA of the endometrial gland, which was segmented via laser-microdissection from the endometrial tissue, was measured, and endometrial LIF immunostaining was carried out on day 5. The gene expression of different factors related to implantation, including decidualization and progesterone-responsiveness on days 5 and 6, were measured. The human endometrial Ishikawa cell line derived from human adenocarcinoma was treated with TSS (30-300 μg/mL) for 24 h, and the LIF concentrations in the cell culture supernatants were measured.
RU-486 decreased the number of implantation sites in the uterus of rats; however, the decrease was significantly alleviated by TSS (3%-diet), which tended to increase plasma progesterone. In rats with RU-486-induced implantation failure, endometrial gland LIF mRNA and endometrial LIF protein were markedly decreased while the gene expression of both decidualization-related factors such as interleukin-11, insulin-like growth factor binding protein-1, and cyclooxygenase-2, and progesterone responsive-related factors such as FK506 binding protein 5, were significantly decreased. These changes in the uterus of rats with implantation failure were significantly alleviated by TSS (3%-diet). Additionally, TSS significantly enhanced LIF protein production and LIF mRNA in Ishikawa cells.
The mechanism whereby TSS ameliorates RU-486-induced implantation failure in rats may involve the alleviation of decreased LIF production derived from the endometrial gland, and a dysfunction of decidualization, including lower progesterone responsiveness in the model. These findings may partly contribute to the interpretation of the beneficial effects of TSS on infertility.
调气活血汤（TSS）是一种汉方药，用于治疗日本的不孕症。然而，其确切的作用机制仍不清楚。
子宫内膜中的白血病抑制因子（LIF）在胚胎植入中起着不可或缺的作用，与不孕或植入失败有关。此前，我们证明 TSS 可改善米非司酮（RU-486）诱导的大鼠植入失败，RU-486 是一种孕激素拮抗剂。在此，我们旨在阐明 TSS 是否通过改善子宫内膜 LIF 来改善大鼠模型中的植入失败。此外，我们还确定了蜕膜化是否参与了 TSS 的作用，蜕膜化与 LIF、孕激素和其他与植入相关的因素一样，与不孕或植入失败有关。
通过在交配后第 3 天皮下注射 RU-486（7mg/kg）建立植入失败大鼠模型。玉米油作为载体对照给药。从交配前 13 天开始，大鼠食用含 1%或 3% TSS 或对照饮食的食物。随后，通过液相色谱-串联质谱法（LC-MS/MS）在交配后第 8 天分析胚胎着床部位和血浆孕激素水平。通过激光显微切割从子宫内膜组织中分离出子宫内膜腺的 LIF mRNA 进行测量，并在第 5 天进行子宫内膜 LIF 免疫染色。测量不同与植入相关的因素（包括蜕膜化和孕激素反应性）的基因表达，第 5 天和第 6 天。用 TSS（30-300μg/ml）处理人子宫内膜 Ishikawa 细胞系 24 小时，测量细胞培养上清液中的 LIF 浓度。
RU-486 降低了大鼠子宫内的着床部位数量；然而，TSS（3%-饮食）显著缓解了这种下降，TSS 倾向于增加血浆孕激素。在 RU-486 诱导的植入失败大鼠中，子宫内膜腺 LIF mRNA 和子宫内膜 LIF 蛋白明显减少，而蜕膜化相关因子如白细胞介素 11、胰岛素样生长因子结合蛋白 1 和环氧化酶 2 的基因表达，以及孕激素反应性相关因子如 FK506 结合蛋白 5 的基因表达均明显减少。TSS（3%-饮食）显著缓解了植入失败大鼠子宫中的这些变化。此外，TSS 显著增强了 Ishikawa 细胞中的 LIF 蛋白产生和 LIF mRNA。
TSS 改善 RU-486 诱导的大鼠植入失败的机制可能涉及缓解子宫内膜腺中 LIF 产生减少，以及蜕膜化功能障碍，包括模型中孕激素反应性降低。这些发现可能部分有助于解释 TSS 对不孕症的有益作用。
