CCK-8抑制LPS作用下大鼠肺间质巨噬细胞NF-κB活性的cAMP-PKA通路研究

目的： 用cAMP激动剂forskolin和PKA抑制剂H-89，探讨八肽胆囊收缩素（CCK-8）抑制LPS作用下大鼠肺间质巨噬细胞（PIMs）核因子-κB （NF-κB）活性的cAMP-PKA信号通路机制。 方法： 分离纯化大鼠PIMs。用电泳迁移率改变分析（EMSA）法检测大鼠PIMs中NF-κB活性，用Western blotting分析IκB-α蛋白水平。 结果： 正常对照组大鼠PIMs核内未检测到与特异性寡核苷酸探针相结合的NF-κB，LPS组细胞内NF-κB活性明显高于正常对照组（P<0.01），胞浆中IκB-α水平显著低于正常对照组（P<0.01）。CCK组和Fsk组细胞内NF-κB活性和胞浆中IκB-α含量均无明显差异（P>0.05）。CCK+LPS组和Fsk+LPS组，细胞内NF-κB活性均低于LPS组（P<0.05），IκB-α含量均高于LPS组（P<0.01）。LPS+CCK+H-89组NF-κB活性高于CCK+LPS组（P<0.01），而IκB-α蛋白水平低于CCK+LPS组（P<0.01）。 结论： cAMP-PKA信号通路的活化可抑制LPS诱导的大鼠PIMs细胞内NF-κB活性升高和IκB-α蛋白水平的降低，CCK-8的抗炎作用是通过激活cAMP-PKA信号通路进而抑制NF-κB活性来实现的。

Inhibitory effects of CCK -8 on NF - κB activities stimulated by LPS in rat PIMs

AIM: To investigate the inhibitory effects of cholecystokinin octapeptide (CCK-8) on nuclear factor-κB (NF-κB) activities stimulated by lipopolysaccharide (LPS) by using forskolin, the activator of adenylate cyclase, and PKA inhibitor H-89 in rat pulmonary interstitial macrophages (PIMs).METHODS: PIMs were isolated and purified.EMDA was applied to detect NF-κB activities and Western blotting was used to analyze the IκB-α protein level in rat PIMs.RESULTS: The NF-κB activity was not detected in normal control rat PIMs.The NF-κB activity in LPS-treated rat PIMs was obviously higher than that in control group (P<0.01).The IκB-α protein level in endochylema decreased obviously compared to control group (P<0.01).No obvious change of NF-κB activity and IκB-α protein level in CCK or Fsk treated rat PIMs was observed (P>0.05).The NF-κB activity in CCK＋LPS group and LPS＋Fsk group were obviously lower than that in LPS group (P<0.05).The IκB-α protein level was obviously higher (P<0.01).In LPS＋CCK＋H-89 group, the NF-κB activity in rat PIMs was obviously higher than that in CCK＋LPS group (P<0.01), while the IκB-α protein level decreased (P<0.01).CONCLUSIONS: The activation of cAMP-PKA signaling pathway inhibits the increase in NF-κB activity and the decrease in IκB-α protein level stimulated by LPS in rat PIMs.The anti-inflammatory effects of CCK-8 were taken effect by activating cAMP-PKA signaling pathway and further inhibiting the NF-κB activity.