环磷腺苷通路对内毒素刺激的小胶质细胞分泌细胞因子的影响

目的观察环磷腺苷下游主要效应分子PKA和Epac对内毒素刺激的原代培养小胶质细胞分泌炎症细胞因子的影响。方法 24孔板接种原代培养的新生大鼠脑小胶质细胞,按不同药物处理分为6组:组1,先用DMSO及HBSS分别孵育30 min,然后HBSS孵育24 h;组2,先用DMSO及HBSS分别孵育30 min;组3,先用DMSO及6-Bnz-cAMP 100nmol分别孵育30 min;组4,先用DMSO及8-pCPT-2'-O-Me-cAMP 50 nmol分别孵育30 min;组5,先用50nmol的H-89及6-Bnz-cAMP 100 nmol分别孵育30 min;组6,先用50 nmol的H-89及8-pCPT-2'-O-Me-cAMP 50 nmol分别孵育30 min,最后组2～组6内毒素0.5μg孵育24 h。内毒素处理后0、3、6、24 h后,倒置显微镜下观察细胞形态改变,并计算6 h时长短轴比值。24 h时收集各组上清液,用ELISA法测定TNF-α、IL-1β及IL-10浓度。结果内毒素刺激后3 h,细胞成双极杆状细胞,6 h时最为明显,24 h细胞形态又恢复至椭圆或阿米巴样。仅6-Bnz-cAMP能明显抑制6 h时细胞形态的改变,预给予H-89,能完全逆转6-Bnz-cAMP对细胞形态学的影响。内毒素刺激24 h后,6-Bnz-cAMP能明显减少TNF-α、IL-1β的分泌,促进IL-10的释放,H-89能完全逆转该效应。给予50 nmol的8-pCPT-2'-O-Me-cAMP仅减少TNF-α的分泌,H-89不影响8-pCPT-2'-O-Me-cAMP的作用。结论较Epac,PKA对内毒素刺激的小胶质细胞释放炎症细胞因子发挥主要的作用。

The effect of cAMP signaling pathway on the expression of cytokines by the microglia stimulated with endotoxin

Aim To observe the effect of protein kinase A(PKA) and Epac(exchange proteins directly activated by cAMP)-the downstream effectors of cAMP on the expression of inflammatory cytokines by the primary cultured microglia stimulated with endotoxin.MethodsThe primary cultured microglia cells from brains of new-born rats were embedded in the 24 pore plate then treated with different drugs as the following: group 1 in which microglias were incubated with 10% DMSO for 30 min,then HBSS for 30 min,finally HBSS for 24 h;group 2 with 10% DMSO and HBSS for 30 min respectively,group 3 with 10% DMSO and 100 nmol 6-Bnz-cAMP for 30 min respectively,group 4 with 10% DMSO and 50 nmol 8-pCPT-2′-O-Me-cAMP for 30 min respectively,group 5 with 50 nmol H-89 and 100 nmol 6-Bnz-cAMP for 30 min respectively,group 6 with 50 nmol H-89 and 50 nmol 8-pCPT-2′-O-Me-cAMP for 30 min respectively,finally 0.5 μg endotoxin for 24 h for group 2 to 6.The morphologic changes were observed with an inverted microscope 0,3 h,6 h and 24 h after endotoxin stimluation and the ratio of long axis of microglia over short axis at 6 h was calcuated.The supernate was collected at 24 h after endotoxin chanllenge and the levels of TNF-α,IL-1β and IL-10 were measured by ELISA.Results The morphology of microglia changed to bipolar rod shapes 3 h after endotoxin challenge,reached peak at 6 h and then returned to oval or amoeboid at 24 h.Only treatment with 6-Bnz-cAMP could significantly inhibit the morphologic change at 6 h,but pretreatment with H-89 completely reversed the effect of 6-Bnz-cAMP.Treatment with 6-Bnz-cAMP could significantly inhibited the overexpression of TNF-α and IL-1β and enhance the secretion of IL-10,while these effects were completely reversed by H-89.Treatment with 8-pCPT-2′-O-Me-cAMP only slightly reduced the expression of TNF-α,meanwhile this effect was not influenced by H-89.Conclusion Compared with Epac,PKA shows more important effects on the expression of inflammatory cytokines by the primary cultured microglia stimulated with endotoxin.