Quantitation of Cytokine mRNA Expression in Cobra Snake Venom Stimulated PBMC of Horses Using Real-Time RT-PCR

Characterization and quantification of cytokine production is essential for understanding the immune response.A SYBR Green real-time quantification Reverse Transcriptase-Polymerase Chain Reaction (RT-qPCR) was used to measure mRNA expression levels of the most common cytokines in horses after cobra snake venom stimulated Peripheral Blood Mononuclear Cells (PBMC) carried out by in vitro method.The PBMC from horse was stimulated with cobra snake venom (10 μg mLG 1 ) prior to incubate at 3, 6, 24 and 48 h.Cytokine mRNA expression level, IL-1β, IL-10, IFNγ and TNFα, were measured using SYBR Green real-time RT-PCR.The highest mRNA expression of IL-1β and IL-10 were demonstrated at 3 h after stimulation of cobra snake venom.The levels of IL-1β and IL-10 expression were 0.7 and 2.3 folds.Meanwhile, the mRNA expression of IFNγ and TNFα were peaked at 6 and 24 h with appreciable increment of 13 and 5.4 folds, respectively.However, the mRNA expression of these cytokines was decreased after 24 h.It seems likely that cobra snake venom could stimulate more mRNA expression of IFNγ than IL-1β, IL-10 and TNFα in PBMC of horse.Secretion of IFNγ is likely to be important in early host defense against pro-inflammation and tissue injury from cobra snake venom.Thus, the study of mRNA expression of inflammatory cytokine profiles in this model could provide information useful for understanding the immunopathological mechanism of inflammation from snake venom immunization in horses and led to improve the design and manufacture of snake anitvenom.