cAMP/Epac/Rap1信号通路调控NG2细胞分泌IL-1β、TNF-α、BDNF及酸枣仁皂苷A的作用研究

The role of cAMP/Epac/Rap1 signaling pathway regulate the secretion of IL-1β,TNF-α,BDNF from NG2 cell and the effect of Jujuboside A

目的研究环磷酸腺苷(cAMP)/环磷酸腺苷结合蛋白(Epac)/Ras相关蛋白1(Rap1)信号通路是否参与调控NG2细胞分泌白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、脑源性神经营养因子(BDNF)及酸枣仁皂苷A(JuA)的作用。方法体外培养NG2细胞,分为对照组、百日咳毒素(PTX)组、非环状核苷酸Epac拮抗剂(ESI-09)组、酸枣仁皂苷A(JuA)组、艾司唑仑(阳性药)组。采用CCK-8法检测不同浓度JuA对NG2细胞存活率的影响,RT-PCR法和Western blot法检测各组细胞IL-1β、TNF-α、BDNF、cAMP、Epac、Rap1 mRNA及蛋白的表达情况。结果与对照组比较,PTX组降低IL-1β和TNF-αmRNA和蛋白的表达(P<0.01),增加BDNF和cAMP mRNA和蛋白的表达(P<0.01);ESI-09组增加IL-1β和TNF-αmRNA和蛋白的表达(P<0.05),降低BDNF、Epac和Rap1 mRNA和蛋白的表达(P<0.01);JuA组、阳性药组均增加IL-1β、TNF-α、BDNF、cAMP、Epac、Rap1 mRNA和蛋白的表达(P<0.05)。结论cAMP/Epac/Rap1信号通路可介导NG2细胞分泌IL-1β、TNF-α、BDNF,JuA可能作用于cAMP/Epac/Rap1信号通路影响NG2细胞分泌BDNF。

Objective To investigate whether the cyclic adenosine monophosphate(cAMP)/exchange proteins directly activated by cAMP(Epac)/ras-related protein 1(Rap1)signalling pathway is involved in the intervening mechanisms of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),brain-derived neurotrophic factor(BDNF)and Jujuboside A(JuA)secretion by NG2 cells.Methods NG2 cells were cultured in vitro and the experiment was divided into control group,pertussis toxin(PTX)group,ESI-09 group,JuA group and positive drug group.The effect of different concentrations of JuA on the survival rate of NG2 cells was detected by CCK-8 method,and the expression of IL-1β,TNF-α,BDNF,cAMP,Epac,Rap1 mRNA and protein in each group was detected by RT-PCR and Western blot.Results Compared with the control group,the PTX group decreased the expression of IL-1βand TNF-αmRNA and protein(P<0.01)and increased the expression of cAMP and BDNF mRNA and protein(P<0.01);the ESI-09 group increased the expression of IL-1βand TNF-αmRNA and protein(P<0.05)and decreased the expression of BDNF,Epac and Rap1 mRNA and protein expression(P<0.01);the JuA group and positive drug group increased IL-1β,TNF-α,BDNF,cAMP,Epac,Rap1 mRNA and protein expression(P<0.01).Conclusion The cAMP/Epac/Rap1 signaling pathway is involved in the secretion of IL-1β,TNF-α,and BDNF by NG2 cells.JuA may act on cAMP/Epac/Rap1 signaling pathway to affect the secretion of BDNF by NG2 cells.