Inhibitory Effect of CCK-8 on Methamphetamine-Induced Apoptosis / 法医学杂志

OBJECTIVES@#To investigate the inhibitory effect of cholecystokinin octapeptide (CCK-8) binding to cholecystokinin 2 receptor (CCK2R) on methamphetamine (METH)-induced neuronal apoptosis, and to explore the signal transduction mechanism of β-arrestin 2 in CCK-8 inhibiting METH-induced neuronal apoptosis.@*METHODS@#SH-SY5Y cell line was cultured, and HEK293-CCK1R and HEK293-CCK2R cell line were constructed by lentivirus transfection. Small interfering RNA (siRNA) was used to knockdown the expression of β-arrestin 2. Annexin Ⅴ-FITC/PI staining and flow cytometry were used to detect the apoptotic rate of cells, and Western blotting was used to detect the expression of apoptosis-related proteins.@*RESULTS@#The apoptosis of SH-SY5Y cells was induced by 1 mmol/L and 2 mmol/L METH treatment, the number of nuclear fragmentation and pyknotic cells was significantly increased, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were increased. CCK-8 pre-treatment at the dose of 0.1 mmol/L and 1 mmol/L significantly reversed METH-induced apoptosis in SH-SY5Y cells, and inhibited cell nuclear fragmentation, pyknosis and the changes of apoptosis-related proteins induced by METH. In lentivirus transfected HEK293-CCK1R and HEK293-CCK2R cells, the results revealed that CCK-8 had no significant effect on METH-induced changes of apoptosis-related proteins in HEK293-CCK1R cells, but it could inhibit the expression level of apoptosis-related proteins in HEK293-CCK2R cells induced by METH. The inhibitory effect of CCK-8 on METH-induced apoptosis was blocked by the knockdown of β-arrestin 2 expression in SH-SY5Y cells.@*CONCLUSIONS@#CCK-8 can bind to CCK2R and exert an inhibitory effect on METH-induced apoptosis by activating the β-arrestin 2 signal.

Effect of CCK-8 on expression of MMPs/TIMP-1 in TNF-α-induced RSC-364 / 中国药理学通报

Aim To observe the influence of CCK-8 on expression of MMPs/TIMP-1 in TNF-α-induced rat fibroblast-like synovial cell line RSC-364.Methods The secretion levels of MMP-1, MMP-3, MMP-9 and TIMP-1 were determined using ELISA;MMP-3 and MMP-9 mRNA expressions were detected by RT-PCR.Results MMP-3 and MMP-9 could not be examined in RSC-364 incubated with CCK-8 and unstimulated RSC-364, which was able to product a little MMP-1, TIMP-1 and express even less MMP-3,-9 mRNA.CCK-8 inhibited the increase in MMP-1, MMP-3, MMP-9 secretion and MMP-3,-9 mRNA expression in TNF-α-induced RSC-364.TIMP-1 production was also increased in TNF-α-induced RSC-364.CCK-8 had no effect on TIMP-1 production in TNF-α-induced RSC-364, but was able to reduce the ratios of MMP-1, MMP-3, MMP-9 to TIMP-1.Conclusion The inhibitory effect of CCK-8 on MMPs activity may be related to the decrease of MMPs mRNA expression, MMPs secretion and the ratios of MMPs to TIMP-1 in TNF-α-induced RSC-364, which indicates that CCK-8 might be a possible regulator in the pathogenesis of rheumatoid arthritis.

Effects of CCK-8 and its receptor antagonists on expression of CREB and pCREB in prefrontal cortex and hippocampus of morphine withdrawal rats / 中国病理生理杂志

AIM:To observe the effects of cholecystokinin octapeptide (CCK-8) and its receptor antagonists on cAMP response element binding protein ( CREB) and phosphorylated CREB ( pCREB) expression in frontal cortex and hippocampus of morphine withdrawal rats , which aim to explore the post-receptor mechanism through which CCK-8 regu-lates morphine withdrawal .METHODS: After the morphine dependence and naloxone-precipitated withdrawal animal models were established, the effects of CCK-8, L-364718 (CCK1 receptor antagonist) and LY-288513 (CCK2 receptor an-tagonist) pretreatment on CREB and pCREB expression in frontal cortex and hippocampus were observed by Western blot -ting and immunohistochemistry .RESULTS:In rat frontal cortex neuron , CREB was expressed in both cytoplasm and nu-cleus, but pCREB was only highly expressed in the nucleus .In the pyramidal cell layer of hippocampal CA 1 region, CREB showed high expression in the cytoplasm and low expression in the nucleus , while pCREB was only expressed in the nu-cleus.No obvious change of CREB was observed after either chronic morphine treatment or naloxone withdrawal .The pCREB expression was increased after chronic morphine treatment and further increased after naloxone withdrawal .Com-pared with the withdrawal group , chronic pretreatment with CCK-8, L-364718 and LY-288513 had no effect on CREB expression in the frontal cortex , but obviously decreased the pCREB expression .In the hippocampus , pretreatment with L-364718 and LY-288513 decreased CREB and pCREB expression , but only the pCREB expression was decreased after CCK-8 treatment.CONCLUSION:CCK-8 and CCK receptor antagonists may alleviate morphine withdrawal symptoms by regulating CREB , with specificity in different brain regions .

CCK-8 prevents glutamate-induced apoptosis in cultured cortical neurons via up-regulation of bcl-2/bax ratio and down-regulation of caspase-3 / 重庆医科大学学报

Objective: To investigate the mechanisms responsible for the neuroprotection by cholecystokinin octapeptide against glutamate-induced apoptosis in vitro cultured cortical neurons. Methods: Primary cultured corticaI neurons from SD rats of 0～24 hold were incubated for 8 days. The cultured cells were divided randomly into three groups: control group,glutamate group and CCK group. In the controI group,cells were not treated with glutamate orCCK;Neurons in glutamate group were incubated with 50?mol/Lglutamate for 30 min;In CCK group,CCK-8 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 0,6,12,24 h and 48 h. At the five time points,cells were fixed respectively for experiment. Cell viability were determined by the colorimetric MTT assay;The protein expression of Bcl-2,Bax and Caspase-3 were determined by immunocytochemistry techniques. Results:Pretreatment with CCK for 24 h significantly improved glutamate-induced suppression of cell viability. Pretreatment with CCK also completely reversed the suppression of Bcl-2 expression,and significantly inhibited Bax overexpression and Caspase-3 activition induced by glutamate. Conclusion:Theneuroprotective mechanisms of CCK against glutamate-induced apoptosis in cultured cortical neurons may be associated with up-regulation of Bcl-2/Bax ratio and down-regulation of Caspase-3.

CCK-8 decreases RSC-364 proliferation and p38 MAPK activation induced by TNF-? in rats / 基础医学与临床

Objective To study the effect of cholecystokinin-octapeptide(CCK-8) on the proliferation of fibroblast-like synovial cell line RSC-364 and p38 MAPK activity induced by TNF-? in rat.Methods The proliferation of RSC-364 cells was measured by monotetrazolium(MTT) colourmetric assay and the level of activation of p38 MAPK was deteced by Western blot.Results An increase in p38 MAPK phosphorylation was detected 5 min after TNF-?((50 ?g/L))addition,and reached a plateau at(15 min),finally returned to the basic level at(2 h).TNF-?(10,25,(50 ?g/L)) increased p38 MAPK phosphorylation in a dose dependent manner at 15 min.CCK-8((10~(-10))～(10~(-6)mol/L))could inhibit the proliferation and the level of phosphorylation of p38 MAPK in a dose dependent manner.Moreover the inhibitory effects were partly reversed by CCK-A receptor specific antagonist CR1409 or CCK-B receptor specific antagonist CR2945.SB203580 inhibited TNF-?-stimulated RSC-364 proliferation.Conclusion CCK-8 inhibited TNF-?-stimulated proliferation by decreasing p38 MAPK phosphorylation in RSC-364 cells,which was mediated through CCK-A receptor or CCK-B receptor.

Inhibitory effect of cholecystokinin octapeptide on carotid sinus nerve afferent discharge in anesthetized rats / 中国药理学与毒理学杂志

AIM To study if cholecystokinin octapeptide (CCK-8) alter cardiovascular functions by its direct inhibitory effect on carotid sinus baroreceptor (CSB) activity. METHODS The functional curve of carotid baroreceptor (FCCB) was constructed and the functional parameters of carotid baroreceptor were measured by recording sinus nerve afferent discharge in anesthetized male rats with perfused isolated carotid sinus. RESULTS ① CCK-8 0.1, 0.5 and 1.0 μmol·L-1 shifted FCCB to the right and downward, with a marked decrease in peak slope and peak integral value of carotid sinus nerve discharge in a concentration-dependent manner, indicating the inhibitory effect of CCK-8 on CSB activity. ② Pretreatment with proglumide (100 μmol·L-1), a nonselective CCK receptor antagonist, or Bay K8644 (0.5 μmol·L-1), an agonist of calcium channel, partially attenuated the inhibitory effect of CCK-8 (0.5 μmol·L-1) on CSB activity. Pretreatment with L-NAME (100 μmol·L-1), an inhibitor of NO synthase, did not affect the inhibitory action of CCK-8. CONCLUSION CCK-8 inhibits CSB activity, which may be mediated by activating CCK receptors in the carotid sinus area and thereby resulting in an inhibition of stretch-sensitive channels and decrease in Ca2+ influx.

Effects of CCK-8 on the withdrawal symptoms and expression of c-jun protein in Cd of morphinomania rats / 中国药理学通报

Aim To investigate the effects of cholecys-tokinin octapeptide(CCK-8)on the withdrawal symp-toms of morphinistic rats by the observation of praxiology,and change of expression of proto-oncogene c-jun in caudate nucleus(Cd) after CCK-8 injected by immunohistochemistry.Methods Animal praxiology evaluation and immunohistochemistry were used.The influence of CCK-8(10 mg?L-1,1 ?l) on the withdrawal symptoms and the expression of c-jun of morphinistic rats was observed.Results Morphinistic rats had obvious praxiological changes in the withdrawal symptoms compared with normal saline rats;the scores of withdrawal symptoms decreased most obviously at withdrawal 40 h after Cd injection of CCK-8;the expression of c-jun protein in Cd decreased in morphinistic rats,but increased after Cd injection of CCK-8.Conclusions Models of morphinomania rats were successfully built.The Cd injection of CCK-8 could decrease the withdrawal symptoms of morphinomania rats.The CCK-8 could increase the expression of c-jun protein in Cd.These findings suggested that CCK-8 could regulate the production of withdrawal symptoms and the expression of c-jun protein in Cd of morphinomania rats.