Astragaloside II triggers T cell activation through regulation of CD45 protein tyrosine phosphatase activity

Aim： To investigate the immunomodulating activity of astragalosides, the active compounds from a traditional tonic herb Astragalus membranaceus Bge, and to explore the molecular mechanisms underlying the actions, focusing on CD45 protein tyrosine phosphatase （CD45 PTPase）, which plays a critical role in T lymphocyte activation. Methods： Primary splenocytes and T cells were prepared from mice. CD45 PTPase activity was assessed using a colorimetric assay. Cell proliferation was measured using a [3H]-thymidine incorporation assay. Cytokine proteins and mRNAs were examined with ELISA and RT-PCR, respectively. Activation markers, including CD25 and CD69, were analyzed using flow cytometry. Activation of LCK （Tyr505） was detected using Western blot analysis. Mice were injected with the immunosuppressant cyclophosphamide （CTX, 80 mg/kg）, and administered astragaloside II （50 mg/kg）. Results： Astragaloside I, II, III, and IV concentration-dependently increased the CD45-mediated of pNPP/OMFP hydrolysis with the ECso values ranged from 3.33 to 10.42 pg/mL. Astragaloside II （10 and 30 IJg/mL） significantly enhanced the proliferation of primary splenocytes induced by ConA, alloantigen or anti-CD3. Astragaloside II （30 pg/mL） significantly increased IL-2 and IFN-y secretion, upregulated the mRNA levels of IFN-~ and T-bet in primary splenocytes, and promoted CD25 and CD69 expression on primary CD4~ T cells upon TCR stimulation. Furthermore, astragaloside II （100 ng/mL） promoted CD45-mediated dephosphorylation of LCK （Tyr505） in primary T cells, which could be blocked by a specific CD45 PTPase inhibitor. In CTX-induced immunosuppressed mice, oral administration of astragaloside II restored the proliferation of splenic T cells and the production of IFN-γ and IL-2. However, astragaloside II had no apparent effects on B cell proliferation. Conclusion： Astragaloside II enhances T cell activation by regulating the activity of CD45 PTPase, which may explain why Astragalus membranaceus Bge is used as a tonic herb in treating immunosuppressive diseases.