Methods: The HMC-1 (human mast cell line) was cultured with or without SIRT1 activator and inhibitor and stimulated with calcium ionophore and PMA. Four hours after the stimulation, mRNA was extracted from these cells for IL-4 real time PCR. Quantitative measurement of IL-4 in cell supernatant was performed using the human IL-4 ELISA kit. To clarify the direct function of SIRT1, we used small interference RNA against SIRT1. Two kinds of SIRT1 siRNA and negative control siRNA were transfected into HMC-1 cells by electroporation.
Results: HMC-1 cells were expressing IL-4 mRNA in unstimulated state. Stimulation with A23187 and PMA increased the expression of IL-4 mRNA at basal level. Expressions of IL-4 mRNA were enhanced in the presence of splitomysin with dose dependent manner. On the other hand, the resveratrol suppressed IL-4 mRNA expression in dose dependent manner. Quantitative measurement of IL-4 in cell supernatant revealed that IL-4 protein production was enhanced in the presence of splitomysin and suppressed in the presence of resveratrol. SIRT1 knock down up regulates IL-4 mRNA expression.
Conclusions: SIRT1 may play some role in developing allergic diseases via control IL-4 gene expression.