二氢小檗碱通过抑制 Rheb/mTOR 信号通路缓解卵巢早衰小鼠的 Th17/Treg 失衡。

Dihydroberberine alleviates Th17/Treg imbalance in premature ovarian insufficiency mice via inhibiting Rheb/mTOR signaling.

机构信息

Department of Gynaecology, Hospital of Chengdu University of Traditional Chinese Medicine, No.39-41, Shijiqiao Road, Jinniu District, Chengdu, 610075, Sichuan Province, China.

State Key Laboratory of Southwestern Chinese Medicine Resources, Chengdu University of Traditional Chinese Medicine, No.1166, Liutai Avenue, Wenjiang District, Chengdu, 611137, Sichuan Province, China.

出版信息

Mol Med. 2024 Oct 29;30(1):194. doi: 10.1186/s10020-024-00971-z.

DOI:10.1186/s10020-024-00971-z

PMID:39472803

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11523677/

Abstract

BACKGROUND

Premature ovarian insufficiency (POI) is an immune-related condition. Dihydroberberine (dhBBR) plays a regulatory role in maintaining the T-helper 17 (Th17)/regulatory T (Treg) cell balance. This study aimed to explore the action mechanisms of dhBBR on POI.

METHODS

In vivo, female BALB/c mice were used as POI models, treated with dhBBR, or injected with recombinant interleukin (rIL)-17 and anti-CD25 monoclonal antibody. Hematoxylin and eosin staining was used to validate the model and assess the therapeutic effects of dhBBR. mRNA expression levels of cytochrome P450 (Cyp)-17a1, Cyp19a1, Cyp11a1, steroidogenic acute regulatory protein, and luteinizing hormone receptor in mouse ovaries were quantified via quantitative polymerase chain reaction (qPCR). Enzyme-linked immunosorbent assay was used to determine the cytokine and sex hormone levels. Immunohistochemical staining for cleaved-caspase 3 and Ki-67 were performed to assess ovarian cell apoptosis and proliferation. Flow cytometry was used to analyze the Th17/Treg cell balance in the ovary and spleen. In vitro cytotoxicity of dhBBR was measured using the cell counting kit-8 assay. GTP-Ras homolog enriched in brain (Rheb) activity was determined via immunofluorescence assay. Co-immunoprecipitation was performed to assess Rheb activity, Th17 or Treg induction, and binding between Rheb and mammalian target of rapamycin (mTOR) after dhBBR treatment. Flow cytometry and qPCR assays were used to verify the effect of dhBBR on CD4 + cell differentiation. Finally, Rheb/mTOR pathway activation was confirmed via western blotting of proteins, including mTOR, p-mTOR, p70S6K, p-p70S6K, 4E-BP1, and p-4E-BP1.

RESULTS

dhBBR improved the ovarian function in a dose-dependent manner. It also decreased ovarian cell apoptosis and increased cell proliferation. It decreased Th1 and Th17 cell proportions but increased Treg cell proportions in the ovaries and spleens of POI model mice. Cell experiments revealed that dhBBR promoted CD4 + cell differentiation into Treg cells. Co-immunoprecipitation revealed Rheb as the dhBBR target that bound to mTOR. However, MHY1485 restored dhBBR-induced changes in forkhead box P3, IL-10, transforming growth factor-β1, IL-17, IL-22, retinoic acid-related orphan receptor-γt and p-mTOR levels in Th17- and Treg-induced CD4 + cells.

CONCLUSION

Overall, dhBBR targeted the Rheb/mTOR pathway to promote CD4 + cell differentiation into Treg cells and alleviate POI.

摘要

背景

卵巢早衰（POI）是一种与免疫相关的疾病。二氢小檗碱（dhBBR）在维持辅助性 T 细胞 17（Th17）/调节性 T（Treg）细胞平衡方面发挥着调节作用。本研究旨在探讨 dhBBR 对 POI 的作用机制。

方法

在体内，使用 BALB/c 雌性小鼠作为 POI 模型，用 dhBBR 处理，或注射重组白细胞介素（rIL）-17 和抗 CD25 单克隆抗体。用苏木精和伊红染色验证模型，并评估 dhBBR 的治疗效果。通过定量聚合酶链反应（qPCR）定量检测小鼠卵巢中细胞色素 P450（Cyp）-17a1、Cyp19a1、Cyp11a1、类固醇急性调节蛋白和黄体生成素受体的 mRNA 表达水平。酶联免疫吸附测定用于测定细胞因子和性激素水平。用免疫组化染色检测卵巢细胞凋亡和增殖的 cleaved-caspase 3 和 Ki-67。用流式细胞术分析卵巢和脾脏中 Th17/Treg 细胞的平衡。用细胞计数试剂盒-8 法测定 dhBBR 的细胞毒性。用免疫荧光法测定 GTP-Ras 同源物富集在脑中（Rheb）的活性。用免疫共沉淀法评估 Rheb 活性、Th17 或 Treg 诱导以及 dhBBR 处理后 Rheb 与哺乳动物雷帕霉素靶蛋白（mTOR）之间的结合。用流式细胞术和 qPCR 检测 dhBBR 对 CD4+细胞分化的影响。最后，通过 Western blot 检测蛋白，包括 mTOR、p-mTOR、p70S6K、p-p70S6K、4E-BP1 和 p-4E-BP1，证实了 Rheb/mTOR 通路的激活。

结果

dhBBR 以剂量依赖性方式改善卵巢功能。它还降低了卵巢细胞凋亡，增加了细胞增殖。它降低了 POI 模型小鼠卵巢和脾脏中 Th1 和 Th17 细胞的比例，但增加了 Treg 细胞的比例。细胞实验表明，dhBBR 促进 CD4+细胞分化为 Treg 细胞。免疫共沉淀显示 Rheb 是与 mTOR 结合的 dhBBR 靶标。然而，MHY1485 恢复了 dhBBR 诱导的 Th17 和 Treg 诱导的 CD4+细胞中叉头框 P3、IL-10、转化生长因子-β1、IL-17、IL-22、维甲酸相关孤儿受体-γt 和 p-mTOR 水平的变化。