Effect of cholecystokinin-8 on in vitro cultured rat cortical neurons against apoptosis

Ying Liu; Jiangbao Zhou

摘要：BACKGROUND: Cholecystokinin (CCK-8) can regulate the synthesis of NO, release of amino acid substance and suppress Ca2+ inflow. It is unknown about neuroprotection of CCK-8 on neuronal apoptosis and its relationship with nerve growth factor (NGF).OBJECTryE: To investigate the protective effect of CCK-8 on in vitro cultured rat cortical neurons against apoptosis induced by glutamate, and explore its effect on expression of NGF in the neurons during apoptosis.DESIGN: Randomized controlled experiment on the basis of cells.SETTING: Children's Research Institute Affiliated to Children Hospital of Chongqing Medical University.MATERIALS: Eighty SD rats of 1-day old; DMEM/F12 culture medium (Biochrom Company, Germany);Fetal bovine serum (TBD Company, Tianjin); CCK-8 (Sigma Company, USA). Glutamate (Bioengineering Company, Shanghai); TUNEL kit and NGF- in situ hybridization kit (Boster Bioengineering Company,Wuhan); anti-NGF polyclonal antibody (Santa-Cluz Company); NGF immunocytochemistry kit (Zhongshan Company, Beijing).METHODS: The experiments were carried out in Children's Research Institute Affiliated to Children Hospital of Chongqing Medical University from December 2004 to September 2005. Primary cultured cortical neurons from SD rats of 1-day oldwere incubated for 7 days. The cultured cells were divided randomly into 3 groups:experimental group, model group and control group. Neurons in experimental groups were added CCK-8 of 1 ×10-6, 1 ×10-7, 1 ×10-8 μ mol/L respectively, and then added 50 μmol/L glutamate solution a hour later. Neurons in model groups were treated with 50 μ mol/L glutamate solution. In the control group, cells were treated with normal medium. Apoptosis of cultured cortical neurons were observed by fluorescent microscope, the expression of NGF protein and mRNA were determined respectively by immunocytochemistry and in situ hybridization, and apoptosis of cortical neurons was detected with terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL).MAIN OUTCOME MEASURES: Morphological results of neuronal apoptosis, apoptosis rates of neurons, NGF protein and mRNA expression in each groups.RESULTS: ① Morphological results: In the model group, the patterns of damaged cortical neurons, such as membrane shrinkage, neurite fragmentation, and karyopyknosis were observed. In the experimental group,configuration of the neurons was similar to those unlesioned neurons. Compared with the model groups, the lesion induced by Glutamate was alleviated. ② The apoptosis rate: The apoptosis rate of the models group were obviously higher than the controls, [(17.53±6.93)%, (8.49±4.54)%, P ＜ 0.05], the apoptosis rate of the neurons treated with 1 ×10-6 and 1 ×10-7 μ mol/L CCK-8 were significantly decreased [(3.87±5.64)%,(7.84±4.19)%, P ＜ 0.05], and the apoptosis rate of the neurons treated with 1 ×10-8μmol/L of CCK-8 was not different statistically from the models [(13.24±3.41)%, P ＞ 0.055]. ③ The NGF protein and mRNA expressions: NGF protein and mRNA expressions of the neurons treated with 1 ×10-6 and 1 ×10-7 μ mol/L CCK-8were significantly up-regulated (P＜ 0.055). (NGF protein: 0.343 2±0.006 81, 0.3301 2±0.001 87, 0.283 77±0.007 69;NGF mRNA: 0.347 4±0.010 04, 0.329 4±0.019 22, 0.301 2±0.001 87; P ＜ 0.055), and the expressions of the neurons treated with 1 ×10-8 μmol/L of CCK-8 was not different statistically from the models (NGF protein:0.2755 2±0.003 73, NGF mRNA: 0.296 8±0.003 78, P＞ 0.055).CONCLUSION: Exogenous CCK-8 can suppress cortical neurons apoptosis induced by glutamate, and alleviate the damage to the neurons, the neuroprotective mechanisms of which may be associated with the up-regulated expression of NGF, and these effects depend on the dosage of CCK-8.