Protective Effect Of Cholecystokinin-8 On In Vitro Cultured Rat Cortical Neurons

Objective:To investigate the protective effect of cholecystokinin-8(CCK-8) on in vitro cultured rat cortical neurons against injury induced by glutamate, and explore its effects on expression of Bcl-2, Bax and Caspase-3 in the neurons during injury.Methods:Primary cultured cortical neurons from Sprague Dawley rats of 0-24 h-old were cultured for 8 d. The cortical neurons were divided randomly into three groups: control group, Glu group and CCK group. In control group, cells were not treated with glutamate or CCK; Neurons in Glu group were incubated with 50μmol/L glutamate for 30 min; In CCK group, CCK-8(0.01μmol/L, 0.1μmol/L, 1μmol/L) was added to the neurons 24 h prior to incubation with glutamate. After injuried by glutamate, cells in all the groups were incubated with normal medium for 24 h and fixed respectively for experiment. Cell viability were determined by the colorimetric MTT assay. The morphological features of the neurons were observed under fluorescence microscope with AO/EB and DAPI immunofluorescence staining. And the protein expression of Bcl-2, Bax and Caspase-3 in the neurons were determined by immunocytochemistry techniques.Resulits:(1) At the 8th day, the primary cortical neurons grew well, there were plump and solid bodies, nervous processus contacted with each other and a dense network formed under inverted phase contrast microscope.(2) Pretreatment with CCK-8 for 24 h significantly improved glutamate-induced suppression of cell viability(P<0.05), and there was positive correlation between the cell viability and the concentration of CCK-8.(3) Morphological changes of neuronal apoptosis could be observed under fluorescence microscope (AO/EB and DAPI staining) in the Glu group. The patterns of damaged cortical neurons, such as membrane shrinkage, neurite fragmentation, karyopyknosis, and apoptotic body were observed. In the CCK group. Neuronal morphology was similar to those uninjuried neurons . Compared with the Glu groups, the injury was alleviated.(4) Pretreatment with CCK-8 also completely reversed the suppression of Bcl-2 expression, and significantly inhibited Bax over expression and Caspase-3 activition induced by glutamate(P<0.05).Conclusions:(1) On the basis of newborn rat primary cortical neuron cultured, we successfully established the modle of glutamate-induced cortical neuron injury in vitro.(2) Pretreatment with CCK-8 for 24 h significantly improved glutamate-induced suppression of cell viability. The neuroprotective mechanisms of CCK-8 against glutamate-induced injury in cultured cortical neurons may be associated with up-regulation of Bcl-2/Bax ratio and down-regulation of Caspase-3.