Abstract:

Objective　To investigate the effects of Danggui Shaoyao San (DSS) on the uterine immune microenvironment and the balance of T helper 17 (Th17) cells and regulatory T (Treg) cells in a mouse model of recurrent abortion (RA). Methods　CBA/J female mice were paired with DBA/2 male mice (8-10 weeks old) to establish the RA model. Successful model mice were divided into the RA group and three treatment groups: L-DSS, M-DSS, and H-DSS , with 10 mice in each group. Another CBA / J female mice were paired with BALB / c male mice ( 8-10 weeks old ) to establish a normal pregnancy model, and 10 were taken as the control group. L-DSS, M-DSS and H-DSS groups were given 6.11, 12.22 and 24.44 g/kg DSS by gavage once a day for 14 consecutive days, respectively. Control group and RA group were given the same amount of distilled water by gavage. Pregnancy outcomes were observed in all groups; organ coefficients of the mice's uteri were calculated; HE staining was performed to observe pathological changes in the uterine tissues; levels of interleukin (IL)-17A and tumor necrosis factor alpha (TNF-α) in the blood were detected using ELISA; and the proportions of Th17 and Treg cells, as well as the Th17/Treg ratio, were analyzed using flow cytometry. Statistical methods included Fisher's exact probability method, one-way ANOVA, and SNK-q test. Results　The control group exhibited intact and normal morphology of the endometrium, the number of decidual cells is large; the RA group showed thinning and rupture of the endometrium, with reduced numbers of decidual cells, abnormal vascularity, and evidence of inflammatory cell infiltration. The L-DSS, M-DSS, and H-DSS groups exhibited sequential thickening of the endometrium, increased numbers of decidual cells, and decreased inflammatory cells. The embryo loss rate, IL-17A, TNF-α, Th17 cell proportion, and Th17/Treg ratio in the RA group were all higher than those in the Control group [48.61% (35/72) vs. 6.90% (6/87), (186.92±19.05) ng/L vs. (74.25±15.40) ng/L, (270.57±28.37) ng/L vs. (153.28±24.29) ng/L, (1.19±0.14)% vs. (0.36±0.10)%, 1.64±0.17 vs. 0.10±0.03], while the uterine organ coefficient and Treg cell proportion were lower than those in the Control group [(5.87±1.44)% vs. (10.74±1.83)%, (0.72±0.19)% vs. (3.25±0.33)%], with all differences being statistically significant (all P<0.05). The embryo loss rate, IL-17A, TNF-α, Th17 cell proportion, and Th17/Treg ratio in the L-DSS, M-DSS, and H-DSS groups were lower than those in the RA group, while the uterine organ coefficients and Treg cell proportions were higher than those in the RA group (all P<0.05). Conclusion　DSS can modulate the uterine immune microenvironment and the Th17/Treg cell balance in RA mice, improving pregnancy outcomes.

Key words:

Recurrent abortion, Danggui Shaoyao San, Immune microenvironment, Helper T cells 17, Regulatory T cells

摘要：

目的　探讨当归芍药散（DSS）对复发性流产（RA）小鼠子宫免疫微环境及辅助性T细胞17（Th17）/调节性T细胞（Treg）细胞平衡的影响。方法　CBA/J雌鼠与DBA/2雄鼠（8～10周龄）配对建立RA模型，将建模成功的小鼠分为RA组和L-DSS、M-DSS、H-DSS组，每组10只。另选择CBA/J雌鼠与BALB/c雄鼠（8～10周龄）配对，建立正常妊娠模型，取10只为Control组。L-DSS、M-DSS、H-DSS组分别灌胃6.11、12.22、24.44 g/kg DSS，每天1次，连续14 d。Control组和RA组灌胃等量蒸馏水。观察各组小鼠妊娠结局；统计各组小鼠子宫脏器系数；HE染色观察小鼠子宫组织病理改变；ELISA检测小鼠血液中白细胞介素（IL）-17A和肿瘤坏死因子α（TNF-α）水平；流式细胞仪检测小鼠脾脏细胞中Th17、Treg细胞比例及Th17/Treg比值。统计学方法采用Fisher确切概率法、单因素方差分析、SNK-q检验。结果　Control组子宫内膜形态结构完整正常，蜕膜细胞数量多；RA组子宫内膜变薄断裂，蜕膜细胞数量少，可见血管异常出血，存在炎症细胞浸润；L-DSS、M-DSS、H-DSS组子宫内膜依次增厚，蜕膜细胞数量增多，炎症细胞减少。RA组的胚胎丢失率、IL-17A、TNF-α、Th17细胞比例、Th17/Treg均高于Control组［48.61%（35/72）比6.90%（6/87）、（186.92±19.05）ng/L比（74.25±15.40）ng/L、（270.57±28.37）ng/L比（153.28±24.29）ng/L、（1.19±0.14）%比（0.36±0.10）%、1.64±0.17比0.10±0.03］，子宫脏器系数、Treg细胞比例均低于Control组［（5.87±1.44）%比（10.74±1.83）%、（0.72±0.19）%比（3.25±0.33）%］，差异均有统计学意义（均P<0.05）；L-DSS、M-DSS、H-DSS组的胚胎丢失率、IL-17A、TNF-α、Th17细胞比例、Th17/Treg均低于RA组，子宫脏器系数、Treg细胞比例均高于RA组（均P<0.05）。结论　DSS可以调整RA小鼠子宫免疫微环境及Th17/Treg细胞平衡，改善妊娠结局。

关键词:

复发性流产, 当归芍药散, 免疫微环境, 辅助性T细胞17, 调节性T细胞