To investigate the therapeutic effect and primary pharmacological mechanism of Ziyuglycoside I (Ziyu I) on collagen-induced arthritis (CIA) mice. CIA mice were treated with 5, 10, or 20 mg/kg of Ziyu I or 2 mg/kg of methotrexate (MTX), and clinical manifestations, as well as pathological changes, were observed. T cell viability and subset type were determined, and serum levels of transforming growth factor-beta (TGF-β) and interleukin-17 (IL-17) were detected. The mRNA expression of retinoid-related orphan receptor-γt (RORγt) and transcription factor forkhead box protein 3 (Foxp3) in mouse spleen lymphocytes was ascertained by the real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). Molecular docking was used to detect whether there was a molecular interaction between Ziyu I and protein kinase B (Akt). The activation of mechanistic target of rapamycin (mTOR) in T cells was verified by Western blotting or immunofluorescence. Ziyu I treatment effectively alleviated arthritis symptoms of CIA mice, including body weight, global score, arthritis index, and a number of swollen joints. Similarly, pathological changes of joints and spleens in arthritic mice were improved. The thymic index, T cell activity, and RORγt production of Ziyu I-treated mice were significantly reduced. Notably, through molecular docking, western blotting, and immunofluorescence data analysis, it was found that Ziyu I could interact directly with Akt to reduce downstream mTOR activation and inhibit helper T cell 17 (Th17) differentiation, thereby regulating Th17/regulatory T cell (Treg) balance and improving arthritis symptoms. Ziyu I effectively improves arthritic symptoms in CIA mice by inhibiting mTOR activation, thereby affecting Th17 differentiation and regulating Th17/Treg balance.
Keywords: Foxp3; RORγt; Th17/Treg; Ziyuglycoside I; mTOR; rheumatoid arthritis.