SiRNA-mediated Silencing Of Phosphodiesterase 4B Expression Affect The Production Of Cytokines In The Endotoxin-stimulated Primary Cultured Micoglia

Objective:To investigate the effect of phosphodiesterase 4B siRNA on the production of cytokines in the endotoxin-stimulated primary cultured micoglia.Methods:The primary cultured microglia cells from brains of new-born rats were embedded in the 24 pore plate before using. In Part Ⅰ,the expression of PDE4 subtypes (A to D) protein were assessed by Western bolt. In Part Ⅱ, Primary microglia cells were randomly divided into control group(no LPS, no transfection), LPS group(no transfection),and transfection groups, including vehicle group(transfected with LipofectaminTM RNAiMAX),mismatch group(transfected with mismatch siRNA),PDE4-siRNA group A to D(transfected with PDE4-siRNA, A to D respectively). The duration for transfection was 48h. In each group, the mRNA expression of PDE4 A to D was assessed by real-time quantitative PCR, the cAMP level was determined by ELISA. After 48h, except in the control group, cells were simulated with LPS(100 ng·ml-1,30 minutes)and intracellular cAMP levels were measured again. In Part Ⅲ, four groups, including LPS group, vehicle group, mismatch group and PDE4B-siRNA group, were simulated with LPS(100 ng·ml-1,24h). Then TNF-α and IL-1β in the supernatant were measured by ELISA. The expression of ERK and p-ERK were tested by western blot.Result:The primary microglia cells expressed PDE4A to D at protein levels. Compared with control group, siRNAs inhibited the mRNA expression of PDE4A to D respectively(P<0.05). The intracellular cAMP level were not significant different among the transfection groups(P>0.05). LPS stimulation significantly increased the cAMP level in all groups, compared to control. However, only cells treated with PDE4B-siRNA showed intracellular cAMP level significant higher than the LPS group(P<0.05). After LPS stimulation, cells in PDE4B-siRNA group effectively reduced the expression of TNF-a, IL-1β and p-ERK compared with LPS group(P<0.05).The expression of ERK showed no significant difference among the five groups (P>0.05).Conclusions:PDE4B-siRNA can effectively inhibit the expression of PDE4B mRNA, increase intracellular cAMP concentration and reduce the expression of inflammatory cytokines after LPS stimulation, potentially through regulating the expression of p-ERK.