Effect Of CCK-8 And Its Recepetor Antagonists On CREB And PCREB In Different Brain Regions Of Morphine Withdrawal Rats

Objective: For the moment, drug abuse become more and more severe which restrict social and economic development. Opioid(such as morphine and heroin) is the most commonly abused drug in our country, characterized by compulsive drug use, but the mechanism is not indentified yet. Morphine addiction involve many kinds of neurotransmitters, neuro-modulators and neuropeptide. Cholecystokinin (CCK) is a neuropeptide found in varieties systems, which has a borad distribution in the central system and takes part in regulation of mang physiological functions. Study reported that CCK-8 is the most potent endogenous anti-opioid peptides and there is a CCK-8 up-regulation in muti-brain region with morphine dependence, therefore CCK-8 participates in the morphine dependence by combining with its receptors.Long-term application of opium results in neural adaptive and plasticity change in many diffirent brain regions, such as caudate putamen, nucleus accumbens, amygdaloid nucleus, ventral tegmental area, substantia nigra, periaou- eductal gray matter, locus caeruleus and so on. Morphine addiction involves temporary protein and long-term gene expression changes in these regions, such as opioid receptor, G-protein, second messenger synthetase, protein kinase, transcription factor. CREB, as a nuclear factor, plays a important role in this process.In our study, morphine dependent and withdrawal models were established by subcutaneous injection of morphine in gradually increased dose. CCK-8 and its recepter antagoists were injected by i.p and i.c.v to observe their effect on morphine withdrawal. Then, immunohistochemical method was used to measure the change of CREB and pCREB in the brain region concerned to explore the cellular signal transduction mechanism of CCK on morphine dependent and withdrawal.Method: Adult male Wistar rats were used(200-220g) which were provided by Experimental Animal Center of Hebei Province.The morphine dependent model was established by subcutaneous injectiong of morphine. The daily dose gradually increased as following: 10, 20, 30, 40, 50mg·kg-1 twice a day, at 8:00 and 20:00, for 5 days. On the 6th day, 50mg·kg-1 morphine was given at 8:00, withdrawal syndrome was precipitated by intraperitoneal injection of naloxone 5 mg·kg-1 two hours later.Two ways, i.p and i.c.v, were used in our research to observe the effect of CCK on morphine withdrawal syndrome, CCK-8(50μg·kg-1, i.p.; 0.1μg/rat, i.c.v), L-364,718(1mg·kg-1, i.p.; 1μg/rat, i.c.v), LY-288,513(1mg·kg-1, i.p.; 1μg/rat,i.c.v) were given 30min before morphine injection every day. At the last day, naloxone was used two hours after morphine given. Then, the morphine withdrawal syndrome was observed and estimated by Gellert-Holtzman scale. The change of CREB and pCREB were measured by immunohistochemical method in the related brain regionsResult:1 CCK-8 and its antagosists on morphine dependent and morphine withdrawal syndrome. Morphine dependent and withdrawal models were successfully established. After chronic treatment with CCK-8 and its receptor antagosists by i.p or i.v.c, development of morphine dependence was inhibited and morphine withdrawal syndrome was alleviated.2 Effect of CCk-8 and its receptor antagosists on CREB and pCREB in caudate putamen, nucleus accumbens, amygdaloid nucleus, ventral tegmental area, substantia nigra, periaoueductal gray matterand locus caeruleus of morphine withdrawal rats.The change of CREB and pCREB were measured in caudate putamen, nucleus accumbens,amygdaloid nucleus, ventral tegmental area, substantia nigra,periaoueductal gray matter,and locus caeruleus to observe the effect of CCK-8 and its receptor antagosists on morphine dependent and withdrawal by immunohistochemical method. The results showed that in the AcbSH there was a similar tendency of CREB and pCREB. Chornic morphine treatment increased CREB and pCREB expression(P<0.01) and they further increased after naloxone precipitation(P<0.01). CCK-8(i.p.;i.c.v), L-364,718(i.p.; i.c.v), LY-288,513(i.p.; i.c.v)could decrease their expression(P<0.01). In the LSI, chornic morphine treatment incressed CREB and pCREB (P<0.01). Naloxone treatment increased CREB (P<0.01) but had no effect on pCREB (P>0.05). CCK-8 (i.c.v), L-364,718(i.p.; i.c.v), LY-288,513(i.p.;i.c.v)treatment decreased the CREB and pCREB expression(P<0.01), but CCK-8 (i.p.) had no effect (P>0.05). In the CPu , there was a similar tendency of CREB and pCREB. Chornic morphine treatment increased CREB and pCREB expression(P<0.01) and they further increased after naloxone precipitation(P<0.01). CCK-8(i.p.;i.c.v), L-364,718 (i.p.; i.c.v), LY-288,513(i.p.; i.c.v)could decrease their expression(P<0.01). In amygdaloid nucleus, chornic morphine treatment increased pCREB(P<0.01)but had no effect on CREB. Naloxone treatment increased CREB and pCREB(P<0.01). LY-288,513(i.p.; i.c.v)decreased CREB expression(P<0.01), but CCK-8 (i.p.; i.c.v),L-364,718(i.p.; i.c.v)had no effect on CREB(P>0.05). CCK-8 (i.c.v),L-364,718(i.p.;i.c.v),LY-288,513(i.p. ;i.c.v)decreased pCREB(P<0.01), but CCK-8 (i.p.)had no effect on pCREB(P>0.05).In the VTA, there was no distinct difference of CREB among each group.Chornic morphine treatment increased pCREB(P<0.01). Naloxone treatment had no effect on pCREB (P>0.05). CCK-8 (i.p; i.c.v), L-364,718(i.p.;i.c.v), LY-288,513(i.p.;i.c.v) had no effect on pCREB (P>0.05).In the substantia nigra, there was no obvious difference of CREB among each group. Chornic morphine treatment increased pCREB (P<0.01).Naloxone treatment has no effect on pCREB(P>0.05).CCK-8 (i.c.v)decreased pCREB (P<0.01), but CCK-8 (i.p.),L-364,718(i.p.;i.c.v), LY-288,513(i.p.;i.c.v)had no effect on pCREB (P>0.05). In PAG, there was no distinct difference of CREB among each group. Chornic morphine treatment increased pCREB (P<0.01). Naloxone treatment decreased pCREB (P < 0.01), CCK-8 (i.p.;i.c.v ) , L-364,718(i.p.;i.c.v), LY-288,513(i.p.;i.c.v)increased pCREB(P<0.01).In LC, there was no obvious difference of CREB among each group. Chornic morphine treatment increased pCREB ( P < 0.01 ) .Naloxone treatment increased pCREB (P<0.01), L-364,718(i.p.;i.c.v), LY-288,513(i.p.;i.c.v)decreased pCREB (P<0.01), but CCK-8 (i.p. ;i.c.v)had no effect on pCREB.Conclusion:Our findings suggested that caudate putamen, nucleus accumbens, amygdaloid nucleus, ventral tegmental area, substantia nigra, periaoueductal gray matter and locus caeruleus were involved in the morphine dependent and withdrawal process. CCK-8,L-364,718,LY-288,513 could alleviate morphine withdrawal syndrome through regulating CREB and/or pCREB expression in different brain regions.