补肾助孕方对黄体功能不全性不孕症大鼠Treg、Th17及其相关因子的影响

阮芳; 周惠芳

doi:10.14148/j.issn.1672-0482.2024.0129

补肾助孕方对黄体功能不全性不孕症大鼠Treg、Th17及其相关因子的影响

doi: 10.14148/j.issn.1672-0482.2024.0129

阮芳^1,,
周惠芳^2, ,

1.
南京中医药大学第一临床医学院, 江苏南京 210023
2.
南京中医药大学附属医院, 江苏南京 210029

基金项目:

国家自然科学基金面上项目 81973898

江苏省社会发展重大项目 BE2021726

江苏省中医院高峰学术人才项目 y2021rc11

江苏省中医药领军人才培养项目 SLJ0202

详细信息

作者简介:
阮芳, 女, 硕士研究生, E-mail: 1274610801@qq.com

通讯作者:
周惠芳, 女, 教授，博士生导师, 主要从事月经不调、不孕症等基础研究, E-mail: yfy0005@njucm.edu.cn

中图分类号: R285.5
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- 文章访问数: 93
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- 被引次数: 0
出版历程
- 收稿日期: 2023-08-28
- 网络出版日期: 2024-02-26
- 发布日期: 2024-02-10

The Effect of Bushen Zhuyun Decoction on Treg, Th17 and Their Related Factors in Luteal Phase Defect Infertility Rats

RUAN Fang^1
,,
ZHOU Huifang^{2
, ,}

1.
The First School of Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China

摘要

摘要: 目的探讨补肾助孕方对黄体功能不全(Luteal phase defect, LPD)性不孕症大鼠模型调节性T细胞(Treg)、辅助性T细胞17(Th17)及相关因子的影响。方法采用大鼠灌服米非司酮混合液(10 mL·kg^-1)构建LPD性不孕症模型, 分别给予阳性药地屈孕酮混合液(0.02 g·kg^-1)和补肾助孕低(4.5 g·kg^-1)、中(9 g·kg^-1)、高(18 g·kg^-1)剂量组干预。通过苏木精-伊红(Hematoxylin-eosin, HE)染色观察子宫组织病理形态, 流式细胞法和ELISA法检测大鼠外周血Treg、Th17细胞百分比和相关因子含量, Western blot和qPCR法检测大鼠Treg、Th17细胞转录因子蛋白水平及mRNA的表达。结果与空白组相比, 模型组子宫内膜厚度明显变薄(P＜0.01), Treg细胞百分比及抗炎因子白介素-10(IL-10)含量显著下降(P＜0.01), Th17细胞百分比及炎症因子白介素-17A(IL-17A)明显升高(P＜0.01), FOXP3蛋白及mRNA表达被抑制(P＜0.05，P＜0.001), RORγT蛋白及mRNA水平呈现高表达(P＜0.01, P＜0.001);与模型组相比, 地屈孕酮及补肾助孕中、高剂量组能增加子宫内膜增厚(P＜0.05, P＜0.01), 促进子宫内膜腺体的增生和发育; Treg细胞百分比及IL-10含量显著增加(P＜0.01, P＜0.001), Th17细胞百分比明显下降(P＜0.01, P＜0.001), FOXP3蛋白表达上调(P＜0.01, P＜0.001);补肾助孕中、高剂量组IL-17A含量及RORγT蛋白表达降低(P＜0.05, P＜0.01);地屈孕酮组和补肾助孕高剂量组FOXP3 mRNA水平显著升高(P＜0.01, P＜0.001), RORγT mRNA表达下调(P＜0.001)。结论补肾助孕方可调节LPD性不孕症大鼠的免疫平衡, 提高子宫内膜容受性。
- 补肾助孕方 /
- 调节性T细胞 /
- 辅助性T细胞17 /
- 黄体功能不全性不孕症
Abstract: OBJECTIVE To investigate the effects of Bushen Zhuyun Decoction (BSZYD) on regulatory T cells (Treg), helper T cell 17 (Th17) and related factors in rat with luteal phase defect (LPD) infertility. METHODS Rats were administered mifepristone mixture (10 mL·kg-1) to construct the LPD-induced infertility model. The positive drug dydrogesterone mixture (0.02 g·kg^-1) and low (4.5 g·kg^-1), medium (9 g·kg^-1), and high (18 g · kg^-1) doses of BSZYD were given for intervention. Uterine tissue morphology of the rat was evaluated by HE staining; the ratio of Treg and Th17 and related factor contents in rat peripheral blood were detected by flow cytometry and ELISA; the protein and mRNA expression levels of Treg and Th17 cell transcription factors were detected by Western blot and qPCR. RESULTS Compared with the blank group, the endometrial thickness of the model group significantly decreased (P < 0.01), the proportion of Treg and the content of the anti-inflammatory factor interleukin-10 (IL-10) reduced significantly (P < 0.01), the percentage of Th17 cells and the inflammatory factor interleukin-17A (IL-17A) were significantly increased (P < 0.01), the protein and mRNA expression levels of FOXP3 were inhibited (P < 0.05, P < 0.001), the RORγT protein and mRNA levels were higher (P < 0.01, P < 0.001). Compared with the model group, the dydrogesterone and middle- and high-dose BSZYD could increase endometrial thickness (P < 0.05, P < 0.01), promoting the proliferation and development of endometrial glands; the percentage of Treg cells and IL-10 content increased significantly (P < 0.01, P < 0.001), the percentage of Th17 cells decreased significantly (P < 0.01, P < 0.001), and FOXP3 protein expression was increased (P < 0.01, P < 0.001); IL-17A content and RORγT protein expression were decreased in the middle and high-dose BSZYD groups (P < 0.05, P < 0.01); the FOXP3 mRNA levels in the dydrogesterone group and the high-dose BSZYD group were significantly increased (P < 0.01, P < 0.001), and the expression of RORγT mRNA was down-regulated (P < 0.001). CONCLUSION BSZYD can regulate the immune balance of Treg and Th17 cells in LPD-infertility rats and improve endometrial receptivity.
- Bushen Zhuyun Decoction /
- regulatory T cells /
- helper T cells 17 /
- luteal phase defect infertility

HTML全文

图 1 黄体功能不全性不孕症大鼠造模方法

Figure 1. The modeling method for infertile rats with luteal phase defect

下载: 全尺寸图片幻灯片

图 2 各组大鼠子宫组织病理表现(HE, ×200)

注：红色箭头指示血管; 黑色箭头指示腺体。

Figure 2. Pathological manifestations of uterine tissue in each group (HE, ×200)

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图 3 大鼠外周血中Th17、Treg细胞占比变化情况

注: 与空白组比较, ^##P＜0.01, ^###P＜0.001;与模型组相比, ^{* *}P＜0.01, ^{* * *}P＜0.001。x±s, n=3。

Figure 3. Changes of Th17 and Treg cell ratios in peripheral blood of rats

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图 4 各组大鼠外周血IL-10、IL-17A含量比较

注: 与空白组比较, ^##P＜0.01;与模型组相比, ^{* *}P＜0.01。x±s, n=6。

Figure 4. Comparison of blood IL-10 and IL-17A in each group

下载: 全尺寸图片幻灯片

图 5 大鼠子宫组织FOXP3、RORγT蛋白水平比较

注: A.空白组; B.模型组; C.地屈孕酮组; D.补肾助孕低剂量组; E.补肾助孕中剂量组; F.补肾助孕高剂量组; 与空白组比较, ^##P＜0.01，^###P＜0.001;与模型组相比, ^*P＜0.05, ^{* *}P＜0.01, ^{* * *}P＜0.001。x±s, n=6。

Figure 5. Comparison of the protein expression of FOXP3 and RORγT in uterine tissue of rats

下载: 全尺寸图片幻灯片

图 6 大鼠子宫组织FOXP3、RORγT mRNA水平比较

注: 与空白组比较, ^#P＜0.05, ^###P＜0.001;与模型组相比, ^**P＜0.01, ^{* * *}P＜0.001。x±s, n=6。

Figure 6. Comparison of the mRNA expression of FOXP3 and RORγT in uterine tissue of rats

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequences

基因	前引(5'→3')	后引(5'→3')	引物长度/bp
FOXP3	CAGTACCCCCAAATTCCTGC	GCTGAAGACGTGTGCATCCTA	192
RORγT	CCTGGGCTCCTCGCCTGACC	TCTCTCTGCCCTCAGCCTTGC	170
GAPDH	GGTTGTCTCCTGCGACTTCA	TGGTCCAGGGTTTCTTACTCC	183

下载: 导出CSV

表 2 子宫内膜厚度比较(x±s, μm, n=6)

Table 2. Endometrial thickness comparison (x±s, μm, n=6)

组别	内膜厚度
空白组	228.72±14.64
模型组	165.16±16.95^##
地屈孕酮组	207.43±5.22^*
补肾助孕低剂量组	162.90±16.91
补肾助孕中剂量组	212.90±16.47^**
补肾助孕高剂量组	218.99±9.33^**
注: 与空白组比较, ^##P＜0.01;与模型组相比, ^P＜0.05, ^{ *}P＜0.01。

下载: 导出CSV

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