Assessment of PI3K/AKT signaling pathway in activated human T lymphocytes in vitro

Background: Silymarin is a complex flavonolignan from milk thistle (Silybum marianum) plant. It exhibits cytoprotective, anticarcinogenic, and anti-inflammatory effects. Although its hepatoprotective effect has been well documented, its effects on T cells have not been thoroughly investigated. In this study, the effects of silymarin on phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway of human activated T lymphocytes were investigated in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated and activated with phytohaemagglutinin (PHA) and 2 μg/ml of monoclonal antibody [anti-cluster of differentiation 28 (CD28)]. Cells were incubated for 72 hours in a Roswell Park Memorial Institute (RPMI-1640) complete medium with 100 mM silymarin and dimethyl sulfoxide (DMSO) as control. Then, cell lysate was prepared with lysis buffer and cell signaling was assessed using enzyme-linked immunosorbent assay (PathScan® Cell Growth Multi-Target Sandwich ELISA Kit, Cell Signaling Technology, USA). Findings: Treatment with 100 μM silymarin for 72 hours could partially decrease the cellular levels of phosphorylated-AKT (Ser473/Thr308) and AKT. In addition, silymarin significantly decreased the cellular expression of phosphorylated-S6 (Ser235/236). Conclusion: Silymarin significantly decreased the cellular level of Ser235/236 but had no significant effects on cellular levels of other molecules such as AKT and Ser473/Thr308. Therefore, the inhibitory effects of silymarin on T lymphocytes proliferation, that have been observed in previous studies, might have been the result of inhibition of some molecules in the PI3K/AKT pathway which are necessary for cell cycle entrance.