中医杂志

目的 从滤泡辅助性T (Tfh)细胞和Janus激酶/信号转导与转录激活因子（JAK/STAT）通路探讨活血消异方治疗卵巢型子宫内膜异位症的可能作用机制。方法 使用GEO数据库下载人类增殖期子宫内膜异位症异位组织与正常女性增殖期的子宫组织测序数据。将具有正常动情周期的45只雌性SD大鼠随机分为活血消异方组、模型组和正常组，每组15只。采用异位组织插入法建立卵巢型子宫内膜异位症大鼠模型，造模成功后活血消异方组予活血消异方溶液5.85 g/（kg·d）灌胃，模型组和正常组予生理盐水1 ml/d灌胃，均连续14天。采用转录组学技术测序分析各组大鼠卵巢异位组织和正常子宫组织的基因表达情况，与人类数据进行比较，以探讨增殖期异位组织中JAK/STAT通路激活情况，使用免疫组化法检测大鼠卵巢异位组织和正常子宫组织中CXC趋化因子受体5(CXCR5)、白细胞介素21(IL-21)阳性表达，Western Blot法检测IL-21、白细胞介素21受体（IL-21R）、Janus激酶1 (JAK1)、信号转导与转录激活因子6 (STAT6)、B淋巴细胞瘤2(Bcl-2)蛋白水平，免疫浸润法分析滤泡辅助性T (Tfh)细胞情况。结果 通过基因集富集分析发现，与正常子宫组织比较，人类增殖期子宫内膜异位症异位组织JAK/STAT通路被明显激活。与正常组比较，模型组大鼠卵巢异位组织中JAK/STAT通路被明显激活；与模型组比较，活血消异方组大鼠卵巢异位组织中JAK/STAT通路被抑制。与正常组子宫组织比较，模型组卵巢异位组织中Tfh细胞组分评分，IL-21及CXCR5阳性表达，IL-21、IL-21R、JAK1、STAT6及Bcl-2蛋白水平均升高（P<0.05）；与模型组比较，活血消异方组卵巢异位组织中IL-21及CXCR5阳性表达，IL-21、IL-21R、JAK1、STAT6及Bcl-2蛋白水平均降低（P<0.05）。结论 活血消异方可能通过抑制Tfh细胞分泌IL-21来抑制卵巢型子宫内膜异位症模型大鼠异位组织中JAK/STAT通路的激活，从而发挥治疗卵巢型子宫内膜异位症的作用。

Objective To explore the potential mechanism of Huoxue Xiaoyi Formula （活血消异方, HXF） in treating ovarian endometriosis(OEM) from the perspective of T follicular helper(Tfh) cells and the Janus kinase/signal transducer and activator of transcription(JAK/STAT) signaling pathway. Methods Forty-five female SD rats with normal estrous cycles were randomly divided into three groups, HXF group, model group, and normal group, with 15 rats in each group. A rat model of OEM was established by autologous endometrial tissue implantation.After successful modeling, the treatment group received HXF at 5. 85 g/（kg·d） by gavage for 14 consecutive days.The model group and normal group received 1 mL/d of normal saline by gavage. RNA-sequencing data from human proliferative-phase endometriotic and normal endometrial tissues were downloaded from the GEO database. Transcriptomic sequencing was used to analyze gene expression in rat ovarian ectopic tissues and normal uterine tissues, and comparisons were made with human data to verify JAK/STAT pathway activation in proliferative-phase ectopic tissues.Immunohistochemistry was used to detect the positive expression of CXC chemokine receptor 5(CXCR5) and interleukin-21(IL-21) in rat ovarian ectopic and normal uterine tissues. Western Blotting was performed to detect the protein levels of IL-21, IL-21 receptor(IL-21R), Janus kinase 1(JAK1), signal transducer and activator of transcription 6(STAT6), and B-cell lymphoma 2(Bcl-2). Tfh cell infiltration was analyzed using immune cell infiltration methods. Results Gene set enrichment analysis showed that the JAK/STAT pathway was significantly activated in human proliferativephase endometriotic tissues compared to normal endometrial tissues. Similarly, the JAK/STAT pathway was markedly activated in rat ovarian ectopic tissues in the model group compared to the normal group, but suppressed in the HXF group compared to the model group. Compared with normal uterine tissues, ovarian ectopic tissues in the model group showed increased Tfh cell infiltration scores, higher CXCR5 and IL-21 expression, and elevated levels of IL-21, IL-21R, JAK1, STAT6, and Bcl-2 proteins. Compared with the model group, HXF group showed reduced CXCR5and IL-21 expression and decreased protein levels of IL-21, IL-21R, JAK1, STAT6, and Bcl-2. Conclusion HXF may suppress activation of the JAK/STAT signaling pathway in ovarian endometriotic tissues by inhibiting IL-21 secretion from Tfh cells.