siRNA as a tool to delineate pathway channelization in H2O2 induced apoptosis of primary Leydig cells in vitro

Using siRNA as a tool, the channelization of pathway in H₂O₂ induced apoptosis of primary Leydig cells was investigated in vitro. Exposure (4?h) to H₂O₂ (250?μM) induced maximum apoptosis but affected Leydig cell viability significantly. Therefore, expression of apoptotic marker genes, caspase-8, -9, -3 and polyadenosine ribose polymerase was subsequently investigated using the same concentration post 1?h exposure. Incubation with siRNA (20?nM) either for caspase-8 or -9, inhibited their individual expressions by 55–60?% and activity, 50–55?%. The inhibition efficiency using siRNA was comparable with post- or pre-H₂O₂ treatment of cells. Like siRNA, Eugenia jambolana (100?μg/ml) plant extract too, effectively countered over-expression of all apoptotic marker proteins. Silencing expressions of caspase 8 but not 9 through siRNA leads to a profound inhibition of caspase 3 implying that H₂O₂ induced Leydig cell apoptosis is preferably channeled through extrinsic and later extending to other pathways.