文献类型：期刊文献

中文题名：桃红四物汤加味含药血清调控卵巢颗粒细胞分泌雌二醇改善Th17/Treg平衡的机制研究

英文题名：Mechanism of modified Taohong Siwu Decoction-containing serum in improving Th17/Treg balance via regulation of estradiol secretion of ovarian granulosa cells

作者：杨鹏斐[1];李炳男[2];晏蜀云[1];李怡霏[3];杜雪洋[1];周文丽[4];金彩云[1];牛凡琪[5];汤亮[5];李勋济[5];王思农[1]

第一作者：杨鹏斐

机构：[1]甘肃中医药大学,甘肃兰州730000;[2]郑州卫生健康职业学院,河南郑州450000;[3]山西省晋城市人民医院,山西晋城048000;[4]甘肃中医药大学附属医院,甘肃兰州730000;[5]江西省九江市第三人民医院,江西九江332000

第一机构：甘肃中医药大学

年份：2025

卷号：50

期号：24

起止页码：6978

中文期刊名：中国中药杂志

外文期刊名：China Journal of Chinese Materia Medica

收录：;北大核心:【北大核心2023】；

基金：国家重点研发计划“中医药现代化研究”重点专项(2018YFC1705302);全国名老中医药专家王文春传承工作室建设项目(国中医药人教发[2010]59号);甘肃省高等学校科技成果转化项目(2017D-17);甘肃省教育厅-2025年甘肃省高校教师创新基金项目(2025A-120);2025年甘肃省高校产业支撑计划项目(2025CYZC-046);甘肃中医药大学“岐黄英才”导师专项(ZYXKBD-202406)。

语种：中文

中文关键词：桃红四物汤加味含药血清;卵巢颗粒细胞;雌二醇;Th17/Treg平衡

外文关键词：modified Taohong Siwu Decoction-containing serum;ovarian granulosa cell;estradiol;Th17/Treg balance

摘要：探讨桃红四物汤加味含药血清调控卵巢颗粒细胞分泌雌二醇(E_(2))改善辅助性T细胞17(Th17)/调节性T细胞(Treg)平衡的作用机制。采用不同浓度的白细胞介素(IL)-17和桃红四物汤加味含药血清诱导小鼠卵巢颗粒细胞,筛选最佳浓度的IL-17和含药血清进行后续实验。将小鼠卵巢颗粒细胞分别给予空白/含药血清、IL-17+空白/含药血清、IL-17+含药血清+雌激素抑制剂干预,提取细胞培养液,分别加入Th17/Treg失衡模型培养液中。采用CCK-8试剂盒检测小鼠卵巢颗粒细胞活力;流式细胞术检测细胞凋亡;酶联免疫吸附测定(ELISA)检测E_(2)、细胞色素P450胆固醇侧链裂解酶(P450scc)、3β-羟基类固醇脱氢酶(3β-HSD)、17β-羟基类固醇脱氢酶(17β-HSD)水平;实时荧光定量聚合酶链式反应(RT-qPCR)检测P450scc、3β-HSD、17β-HSD mRNA表达水平。采用流式细胞术检测CD4^(+)T细胞中Th17/Treg细胞比例;ELISA检测IL-17、IL-2、IL^(-1)0水平;免疫荧光检测维甲酸相关孤核受体γt(RORγt)、叉头状转录因子3(Foxp3)表达;蛋白免疫印迹法(Western blot)检测雌激素受体(ER)α、ERβ蛋白表达水平;RT-qPCR检测ERα、ERβmRNA表达水平。结果显示,50、100 ng·mL^(-1)IL-17可明显降低小鼠卵巢颗粒细胞E_(2)浓度,升高凋亡率和抑制率(P<0.05);15%、20%桃红四物汤加味含药血清可明显升高小鼠卵巢颗粒细胞E_(2)浓度,降低凋亡率和抑制率(P<0.05)。桃红四物汤加味含药血清可明显升高小鼠卵巢颗粒细胞P450scc、3β-HSD、17β-HSD浓度及基因表达水平(P<0.05)。此外,桃红四物汤加味含药血清还可明显降低CD4^(+)T细胞中IL-17A+、RORγt+细胞阳性率、IL-17浓度、RORγt荧光强度,升高Foxp3+细胞阳性率、IL-2与IL^(-1)0浓度、Foxp3荧光强度、ERα与ERβ蛋白及基因表达水平,降低Th17/Treg细胞比例(P<0.05)。结果表明,桃红四物汤加味含药血清可明显改善卵巢颗粒细胞凋亡,促进细胞活力及E_(2)分泌,进而抑制Th17细胞分化因子表达,促进Treg细胞分化因子表达,起到改善Th17/Treg平衡的作用。
This study aimed to explore the mechanism by which modified Taohong Siwu Decoction-containing serum regulates estradiol(E_(2))secretion in ovarian granulosa cells to improve Th17/Treg balance.Mice ovarian granulosa cells were treated with different concentrations of IL-17 solution and modified Taohong Siwu Decoction-containing serum.Optimal concentrations of IL-17 and medicated serum were selected for subsequent experiments.The cells were then divided into groups treated with blank/medicated serum,IL-17+blank/medicated serum,and IL-17+medicated serum+estrogen inhibitor.The culture media from each group were introduced to a Th17/Treg imbalance model culture medium.The viability of mice ovarian granulosa cells was detected by CCK-8 kit.Apoptosis was detected by flow cytometry.The levels of E_(2),P450scc,3β-HSD and 17β-HSD were detected by ELISA.The expression levels of P450scc,3β-HSD and 17β-HSD mRNA were detected by RT-qPCR.The ratio of Th17/Treg cells in CD4^(+)T cells was detected by flow cytometry.The levels of IL-17,IL-2 and IL^(-1)0 were detected by ELISA.Immunofluorescence was used to detect the expression of RORγt and Foxp3.Western blot was used to detect the expression levels of ERαand ERβprotein.RT-qPCR was used to detect the expression level of ERαand ERβmRNA.The results showed that IL-17 at 50 and 100 ng·mL^(-1)significantly decreased the E_(2)concentrations in mice ovarian granulosa cells while increasing their apoptosis and inhibition rates(P<0.05).Modified Taohong Siwu Decoction-containing serum at 15%and 20%significantly increased the E_(2)concentrations in granulosa cells while reducing the apoptosis and inhibition rates(P<0.05).The medicated serum significantly increased the concentrations of P450scc,3β-HSD,and 17β-HSD,and gene expression levels in mice ovarian granulosa cells(P<0.05).Additionally,it significantly decreased the positive rates of IL-17A+and RORγt+cells,IL-17 concentration,RORγt fluorescence intensity,and the Th17/Treg ratio in CD4^(+)T cells,while significantly enhancing the positive rates of Foxp3+cells,IL-2 and IL^(-1)0 concentrations,the fluorescence intensity of Foxp3,and the protein and gene expression levels of ERαand ERβ(P<0.05).In conclusion,modified Taohong Siwu Decoction-containing serum can improve ovarian granulosa cell apoptosis and increase cell viability and E_(2)secretion,which leads to the suppression of factors that promote Th17 cell differentiation,while upregulating those that promote Treg cell differentiation,thereby improving the Th17/Treg balance.