磷酸二酯酶4B在脂多糖诱导大鼠小胶质细胞炎性因子释放中的作用

Role of phosphodiesterase 4B in lipopolysaccharide-induced release of inflammatory factors in rat microglias

目的 评价磷酸二酯酶4B（PDE4B）在脂多糖诱导大鼠小胶质细胞炎性因子释放中的作用.方法 采用随机数字表法,将原代大鼠小胶质细胞分为5组（n=24）：对照组、脂多糖组、单纯载体组、错配小干扰RNA组（siRNA）组和PDE4B-siRNA组.对照组和脂多糖组不转染;单纯载体组给予脂质载体1 μl;错配siRNA组和PDE4B-siRNA组分别加入错配siRNA 2μl、PDE4B-siRNA 2 μl与脂质载体1μl（加入100μl无血清培养基中,siRNA终浓度为20 nmol/L）,各组转染或培养48 h后,采用Western blot法和RT-PCR法分别测定PDE4B蛋白与mRNA的表达,除对照组外其余各组加入含有脂多糖100 ng/ml的无血清培养基,培养24 h后采用ELISA法测定TNF-α及IL-1β释放水平,采用Western blot 法检测小胶质细胞细胞外调节蛋白激酶（ERK）、磷酸化ERK（p-ERK）的蛋白表达.结果 与对照组相比,其余4组TNF-α和IL-1β释放水平升高,小胶质细胞p-ERK表达上调,PDFB-siRNA组小胶质细胞PDE4B蛋白及其mRNA表达下调（P＜0.05）,脂多糖组、单纯载体组和错配siRNA组差异无统计学意义（P＞0.05）;与脂多糖组相比,PDE4B-siRNA组TNF-α和IL-1β释放水平降低,p-ERK表达下调（P＜0.05）,单纯载体组和错配siRNA组差异无统计学意义（P＞0.05）.5组小胶质细胞ERK表达水平差异无统计学意义（P＞0.05）.结论 PDE4B可促进脂多糖诱导的小胶质细胞炎性因子释放,机制与激活ERK有关.

Objective To evaluate the role of phosphodiesterase 4B （PDE4B） in lipopolysaccharide （LPS）-induced release of inflammatory factors in rat microglias.Methods The pri mary cultured microglial cells were randomly divided into 5 groups （n =24 each） using a random number table：control group （group C）,LPS group,vehicle group,mismatch small interfering RNA （siRNA） group and PDE4B-siRNA group.The cells were incubated for 48 h in C and LPS groups.The cells were transfected with lipofectaminTM RNAiMAX 1 μl for 48 h in vehicle group.In mismatch siRNA and PDE4B-siRNA groups,mismatch siRNA 2 μl and PDE4B-siRNA 2 μl were added to 100μl serum-free culture medium （final concentration of siRNA 20 nmol/L）,respectively,lipofectaminTM RNAiMAX 1 μl was added simultaneously and the cells were then transfected for 48 h.The expression of PDE4B protein and mRNA was determined by Western blot and real-time PCR,respectively.The cells were cultured for 24 h in serum-free culture medium containing LPS 100 ng/ml in LPS,vehicle,mismatch siRNA and PDF4B-siRNA groups.Then the release of TNF-α and IL-1β was measured by ELISA and the expression of extracellular signalregulated protein kinase （ERK） and phosphorylated ERK （p-ERK） was detected by Western blot.Results Compared with C group,the expression of PDE4B protein and mRNA was significantly down-regulated in group PDE4BsiRNA （P ＜ 0.05）,no significant changes were found in LPS,vehicle and mismatch siRNA groups （P ＞ 0.05）,and the release of TNF-α and IU1β was increased and the expression of p-ERK was up-regulated in the other four groups （P ＜ 0.05）.Compared with LPS group,the release of TNF-α and IL-1β was decreased and the expression of p-ERK was down-regulated in PDE4B-siRNA group,and no significant changes were found in vehicle and mismatch siRNA groups （P ＞ 0.05）.There was no significant difference in ERK expression between the five groups （P ＞ 0.05）.Conclusion PDF4B can promote LPS-induced release of inflammatory factors in rat microglias and activation of ERK is involved in the mechanism.