PACAP27对鱼藤酮诱导细胞凋亡抑制作用机制的研究

The mechanism of neuroprotective effect of PACAP27 on rotenone-induced apoptosis in PC12 cells

目的 探讨垂体腺苷酸环化酶激活肽27（PACAP27）对鱼藤酮诱导的PC12细胞损伤保护作用的胞内分子机制。方法 诱导分化后的PC12细胞，经鱼藤酮（250nmol·L^-1）和（或）PACAP27（10^-7～10mol·L^-1）处理后，四甲基偶氮唑盐（MTT）法检测细胞活性及代谢状态；Apo-ONE均质荧光法检测Caspase-3活性变化，流式细胞术检测细胞线粒体膜电位变化。结果PKA刺激剂db-cAMP（10^-4mol·L^-1），Forskolin（10^-6mol·L^-1）可模拟PACAP27的保护作用，而PKC刺激剂TPA（10^-7mol·L^-1）则无此作用（P〉0．05）；PKA抑制剂H-89（10^-6mol·L^-1）可明显削弱PAC-AP27的保护作用，而PKC抑制剂Myr-ΨPKC（10^-6mol·L^-1）则无此作用（P〉0．05）；ERK抑制剂PD98059（2×10^-5mol·L^-1）和p38抑制剂SB203580（5×10^-5mol·L^-1）可削弱PACAP27的保护作用，而JNK抑制剂SP600125（4×10^-5mol·L^-1）则无此作用（P〉0．05）；PACAP27可以明显抑制鱼藤酮诱导的Caspase-3活化；但PACAP27却无法逆转鱼藤酮诱导的线粒体膜电位减低（P〉0．05）．结论PACAP27通过PAC1受体介导，激活了PKA和MAPK信号通路，对鱼藤酮诱导的细胞凋亡进行了有效抑制，同时鱼藤酮诱导的这种细胞凋亡与非线粒体依赖的Caspase-3活化有关，

Abstract：Aim To investigate the mechanism of neuroprotective effect of pituitary adenylate cyclase-activating polypeptide27 （PACAP27） on rotenone-induced cytotoxicity in PC12 cells. Methods Differentiated PC12 cells were treated with rotenone and/or PACAP27, PACAP6-27; cell viability was assessed with MTF and Caspase-3-1ike activity was measured using fluorescence assay with the probe Z-DEVD-rhodamine 110. Mitochondrial membrane potential was detected using ratiometric probe JC-1 with flow cytometry. Resuits db-cAMP and Forskolin, as potent PKA stimulators, mimicked PACAP27 neuroprotection compared to TPA, a potent PKC stimulator. By contrast, the selective PKA inhibitor, H89, ihhitited the neuroprotection of PACAP27 on rotenone-induced cytotoxicity and the selective PKC inhibitor, Myr-ΨPKC did not have such actions. PD98059 and SB203580 showed marked inhibition of this neuroprotection but SP600125 didn＇t. After 24 h exposure to rotenone,the level of Caspase-3-like activity was significantly increased compared to that of control groups, but PACAP27 resulted in a sig- nificant decrease in Caspase-3-like activity induced by rotenone and this effect was also blocked by PACAP6-27. Treatment with rotenone for 24 h caused a marked decrease in mitochondrial potential. However, addition of PACAP27 showed no protection against the impaired conditions. Conclusion These results suggest that PACAP27 attenuates rotenone-induced neurotoxicity by PAC1 receptor involved PKA and MAPK signaling pathway with inhibition of Caspase-3 activity through mitochondrial-independent pathways in PC12 cells.