垂体腺苷酸环化酶激活肽拮抗lactacystin细胞毒性的作用

Mechanism of pituitary adenylate cyclase-activating polypeptide against the cytotoxicity of lactacystin

背景:帕金森病的发病机制未明确,目前尚没有有效的治疗方法能从根本上阻止其病程进展。目的:分析垂体腺苷酸环化酶激活肽对lactacystin诱导的帕金森病多巴胺能PC12细胞凋亡的影响及其分子机制。方法:用神经生长因子将PC12细胞诱导分化成神经元的细胞模型,经不同浓度泛素-蛋白酶体抑制剂lactacystin处理,分别作用不同时间,取细胞存活约为50%的lactacystin作用浓度与时间,建立帕金森病细胞实验模型。实验分组:对照组、lactacystin组、垂体腺苷酸环化酶激活肽1-27干预组(干预1组)、垂体腺苷酸环化酶激活肽1-27和垂体腺苷酸环化酶激活肽6-27共同干预组(干预2组)。观察各组细胞形态变化;MTT法检测细胞活力;免疫印迹(Western blot)法检测内质网应激特异性蛋白caspase-12的表达情况。并观察垂体腺苷酸环化酶激活肽1-26及垂体腺苷酸环化酶激活肽6-27对lactacystin毒性作用的影响。结果与结论:不同浓度及作用时间的lactacystin处理PC12细胞后,细胞活力呈浓度及时间依赖性下降,其中lactacystin 20μmol/L作用24 h使细胞活力下降约50%。在相同的lactacystin作用条件下(20μmol/L,24 h),与对照组比较,lactacystin组细胞发生损伤性改变,细胞活力降低,caspase-12活性明显升高(P<0.01);与lactacystin组比较,干预1组细胞损伤性改变明显好转,细胞活力增强,下调凋亡蛋白caspase-12的表达(P<0.01)。干预2组细胞状态则明显不如干预1组,与lactacystin组相差不大。结果提示泛素-蛋白酶体抑制剂lactacystin引起内质网应激导致细胞损伤;垂体腺苷酸环化酶激活肽1-27通过调节上述信号通路发挥保护作用。而作为垂体腺苷酸环化酶激活肽1-27的受体拮抗剂,垂体腺苷酸环化酶激活肽6-27则减弱了垂体腺苷酸环化酶激活肽1-27的这一作用。

BACKGROUND：Pathogenesis of Parkinson’s disease is not completely understood, and there is yet no effective therapy that can prevent the neurodegenerative process of the disease fundamentaly. OBJECTIVE：To explore the effects of pituitary adenylate cyclase-activating polypeptide （PACAP） on lactacystin-induced Parkinson’s disease dopaminergic PC12 cel apoptosis and its molecular mechanism. METHODS： Under induction by nerve growth factors, PC12 cels differentiated into dopaminergic neurons, and then were treated with different concentrations of lactacystin for different time. When the cel survival rate was about 50%,the concentration and action time oflactacystin were selected to establish cel models of Parkinson’s disease. In the study, there were control group, lactacystin group, PACAP1-27 group （intervention group 1） and PACAP1-27＋PACAP6-27 co-intervention group （intervention group 2）. Changes of cel morphology were observed under inverted microscope; cel viability was detected with MTT method; the expression of endoplasmic reticulum stress specific protein caspase-12 was detected by western blot. Then the action of PACAP1-27 and PACAP6-27 to the cytoxicity of lactacystin was observed. RESULTS AND CONCLUSION： With different concentrations and action time of lactacystin, the viability of PC12 cels presented a concentration- and time-dependent decline. When the lactacystin at 20μmol/L acted for 24 hours, the cel viability was declined by about 50%. Under same conditions of lactacystin concentration and action time （20 μmol/L, 24 hours）, the cels in the lactacystin group appeared to have damaged changes, declined cel viability, and increased caspase-12 activity in comparison with the control group （P〈 0.01）. Compared with the lactacystin group, the cel damage was relieved and cel viability was increased significantly in the intervention group 1 as wel as the expression of caspase-12 was decreased （P 〈 0.01）. Experimental findings in the intervention group 2 were similar to those in the lactacystin group. These results suggest that lactacystin, an ubiquitin proteasome inhibitor, can lead to cel damage; PACAP1-27 plays a protective role by regulating the above-mentioned signal pathway. As one PACAP1-27 receptor antagonist, PACAP6-27 can attenuate this effect of PACAP1-27.