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

Abstract

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.

Keywords

• Dihydroberberine
• Premature ovarian insufficiency
• Th17/Treg immune imbalance
• Rheb/mTOR signaling pathway
• CD4 + T cell differentiation