The Study On Alleviating Morphine Tolerance Symptom Of Rats And Related Mechanism With CRE-decoy ODN

Chronic opiates can lead to tolerance and dependence. Tolerance is described as the need for an increasing dose of opiate to achieve the same effect; this term was based originally on the tolerance that develops to many of the acute behavioral actions of opiates (e.g., analgesia, autonomic inhibition, and "high"). Dependence is described an altered physiological state caused by repeated opiate exposure such that cessation of drug administration leads to a withdrawal syndrome characterized by serious physiological disturbances. This term was used originally to describe physical opiate dependence as characterized by classical opiate withdrawal. More recently, it has become clear that opiates also result in a psychological form of dependence, which results in emotional or motivational symptoms upon drug withdrawal. This latter can be defined as compulsive drug craving and administration despite horrendous adverse consequences. Tolerance and dependence posed serious health, social, and economic problems around the world. The neurobiological and molecular mechanism of them is still unclarified, there is no effective protocol in clinical practice. Thus, modern medical science has the challenging task to find a safe and effective drug to treat them.The discovery of endogenous opioid peptides and opioid receptors over twenty years ago led to the expectation that adaptations in these proteins in response to repeated opiate exposure underlie many features of opiate addiction. While investigation of the opioid peptides enkephalin, endorphin, and dynorphin has revealed a great deal about peptide neurotransmitters in general and a role for the opioid peptides in stress-related phenomena, adaptations in the expression of peptides themselves have not provided compelling models for opiate tolerance or dependence. Similarly, early studies on opiate regulation of the three types of opioid receptor, μ,δ, and κ, by useof radioligand binding assays failed to explain opiate tolerance and dependence, at least in in vivo systems. The recent cloning of these receptors has enabled more penetrating analyses, which could yet reveal alterations related to opiate addiction. For example, exposure to agonist in vitro elicits receptor desensitization and down-regulation, which could be related to aspects of tolerance in vivo.In contrast with the lack of evidence for a role of adaptations in opioid peptides and receptors in opiate addiction, considerable evidence has implicated a role for postreceptor, intracellular messenger pathways. Chronic exposure to opiates has been shown to elicit adaptations in some of the same intracellular pathways that mediate the acute actions of the drugs. Moreover, such adaptations have been related to tolerance and dependence phenomena that can be demonstrated at the level of individual neurons. We now know that these changes are mediated in part by an up-regulation of the cAMP pathway induced by chronic exposure to opiates.The mechanism by which chronic opiate exposure leads to up-regulation of the cAMP pathway in LC neurons is the focus of ongoing investigation, transcription factor cAMP response element-binding protein (CREB) is being given more attention. Levels of CREB are increased in the LC and other brain regions following chronic morphine administration. This raises the possibility that increases in CREB contribute to up-regulation of the other cAMP pathway proteins and thereby to the effects of chronic opiates on the electrophysiological state of LC neurons and their behavioral manifestations. And this possibility has been proved by intra-LC administration of antisense oligonucleotides to CREB, or observation in genetic mutant mice deficient in CREB.Opiate-induced adaptations in neuronal function involve molecular adaptations in components of signaling pathways that go far beyond regulation of endogenous opioid peptides and opioid receptors or other neurotransmitter receptor systems. Increasing evidence supports a role for adaptations in the cAMP pathway as one important molecular mechanism of opiate tolerance anddependence. Transcription factors CREB activates target genes through binding with specific DNA motifs CRE in the promoter regions. It has been shown that short sequences of DNA containing the consensus binding site, even in the absence of surrounding DNA, can bind transcription protein in a highly specific manner. Specific DNA sequences have been used successfully as "decoys" to bind specific TFs in cultured cells or in vivo, rendering the TFs incapable of subsequent binding to the promoter region of target genes.This approach has been shown to be effective in modulating gene expression in vitro and in vivo.So in this experiment, we synthesized CRE-decoy ODN, and study its alleviativing effect on morphine tolerance rats, and explore related mechanism. 1 The effect of CRE-decoy ODN on morphine analgesia tolerance in rats(1) The effect of CRE-decoy ODN on morphine analgesia tolerance in rats. Sixty Wistar rats, weighting between 200-220g were used. The rats were randomly divided into six groups: NS group, Mor group, Mor+NS group, Mor+mismatch ODN group, Mor+nonsense ODN group, Mor+CRE-decoy ODN group. The models of morphine tolerance were established by subcutaneous injection of morphine in gradually increasing doses for six days in the latter five groups; the rats in the four latter groups were catheterized, and given 10 U 1 NS or ODN intracerebroventricularly before morphine injection at noon during the experiment, the dose of ODN was 33 U g. TFL were measured by warm-water tail-flick test after morphine injection at noon. Data were presented as x + s and analyzed by one way ANOVA and least significant difference test using SPSS statistical program. A level of P<0.05 was considered statistically significant. Results: the TFL of Mor group was (7.35+0.53) s in dl, and decreased to (3.55+0.46) s and (3.59+0.38) s in d3 and d6 separately, the values in d3 and d6 had no significant difference compare with NS group (P>0.05) ; the TFL of Mor+CRE-decoy ODN group in d3 and d6 were (6.15+0.36)s and (6.39+0.28) s separately, the values had significant difference compare with Mor group in same time (PO.05) .There were no significant difference between Mor+NS group, Mori-mismatch ODN group, Mor+nonsense ODN group and Mor group CP>0.05 ). (2) The effect of CRE-decoy ODN on pain threshold in rats. Fifty rats were randomly divided into five groups: Negative group, NS group, Mismatch ODN group, Nonsense ODN group, CRE-decoy ODN group. The rats in the latter four groups were catheterized, and given 10 P 1 NS or ODN intracerebroventricularly at noon every day during six days experiment process, the TFL were measured in dl, d3, and d6 after injection. Results: the TFL in five groups had no significant difference. These results demonstrated that the injection of CRE-decoy ODN intracerebroventricularly could alleviate morphine tolerance, and had no analgesic effect alone. 2 Effects of CRE-decoy ODN on />CREB-1 protein and CREB/DNAbinding activity in brain stem and spinal cord of chronic morphinetolerance ratsChronic morphine leads to compensatory up-regulation of cAMP signaling pathways in numerous brain regions. Transcription factor CREB may regulate neuroadaptation and alter gene expression that may play an important role in chronic morphine tolerance and dependence. In this part, changes ofpCREB-1 protein and CREB/DNA binding activity of brain stem and spinal cord in chronic morphine tolerance rats and the effect of CRE-decoy ODN on them were examined using Western blot and EMSA. Twenty four Wistar rats were randomly divided into eight groups: NS group, Mor group, Mismatch ODN group, Nonsense ODN group, CRE-decoy ODN group, Mor + mismatch ODN group, Mor +nonsense ODN group, Mor + CRE-decoy ODN group. The administration of morphine and ODN may be seen in the second part. Data were presented as x + s and analyzed by one way ANOVA and least significant difference test using SPSS statistical program. A level of.PO.05 was considered statistically significant. Results: (1) pCREB-1 of brain stem and spinal cord increased in chronic morphine tolerance rats compared with NS group (PO.01) , CRE-decoy ODN inhibited this increasement (PO.01) . There were no statistical difference betweenMismatch ODN group, Nonsense ODN group, and NS group. There were also no statistical difference between Mor + mismatch ODN group , Mor + nonsense ODN group, and Mor group (P>0.05) . (2) CREB/DNA binding activity of brain stem and spinal cord increased in chronic morphine tolerance rats compared with NS group (PO.Ol) , CRE-decoy ODN inhibited this increasement (P<0.01) . There were no statistical difference between Mismatch ODN group, Nonsense ODN group, and NS group. There were also no statistical difference between Mor + mismatch ODN group , Mor + nonsense ODN group, and Mor group (.P>0.05) . These results demonstrated that CRE-decoy ODN can inhibit the increase of pCREB-1 and CREB/DNA binding activity of brain stem and spinal cord in chronic morphine tolerance rats.3 The effects of CRE-decoy ODN on the expression of CCK and receptor gene expresion in SK-N-SH cells incubated by chronic morphineNeuropeptide cholecystokinin is a physiological antagonist of opiate analgesia. There may exist regulatory loops between CCK and opioids systems. Accelerated expression of CCK gene in some brain regions was seen and it rendered tolerant to morphine. There is CRE sequence in cholecystokinin gene, accelerated expression of CCK gene may be related to activation of CREB. In this part, the effects of CRE-decoy ODN on the expression of CCK and receptor gene expresion were examined in chronic morphine incubated SK-N-SH cells. This part contains ten groups: NS group, Mor group, DOTAP group, Mor+ DOTAP group, Mismatch ODN group, Mor+ mismatch ODN group, Nonsense ODN group, Mor+nonsense group, CRE-decoy ODN group, Mor +CRE-decoy ODN group. When the adherent cells yielded approximately 60% confluency, the medium was replaced, CRE-decoy ODN and control ODNs at concentration of 150 nmol/L in the presense of cationic lipid (DOTAP) was added in groups containing them, after one hour, morphine was added at final concentration of 100 umol/L in groups containing morphine. The period of morphine act on the cells was forty eight hours. Cellular uptake, stability, and specificity of ODN were alsoexamined in this part. Data were presented as jc±s and analyzed by one way ANOVA and least significant difference test using SPSS statistical program. A level of P<0.05 was considered statistically significant. Results: (1) Cellular uptake examined by [y-32P] labeled ODNs and distribution of ODN examined by confocal fluorescence microscopy exhibited that ODN can penetrate into the cell successfully. There was no obvious morphological changes when the cells were incubated with ODNs, cell numbers in different groups had no significant difference. (2) Electrophoresis, silver stain, and autoradiography proved stability of cell-incorporated CRE-decoy ODN. (3) EMS A testing of binding site specificity of CRE-decoy ODN to CREB: The retarded band was effectively competed by the addition 50-fold excess of a cold CREB binding double-stranded probe or the CRE-decoy ODN respectively, and the retarded band was not competed by the addition 50-fold excess of a cold AP-2 binding double-stranded probe, Mismatch ODN, or Nonsense ODN containing no CRE respectively. Supershift of the retarded band was caused by an antibody to CREB. (4) cAMP level in Mor group was increased significantly compared with NS group(1446±138.7 pmol.g"1 vs 1135±243.5 pmol.g"1 , P< 0.05), Mor + CRE-decoy ODN group decreased compared with Mor group (P< 0.05). (5) CCK mRNA and protein level in Mor group was increased significantly compared with NS group, Mor + CRE-decoy ODN group decreased compared with Mor group (P< 0.05 ) . (6) CCK-AR and CCK-BR protein were found in extraction protein of SK-N-SH cells, CCK-BR protein level increased when the cells were incubated by chronic morphine, and CRE-decoy ODN could inhibit this increase.CONCLUSIONSl.The injection of CRE-decoy ODN intracerebroventricularly could inhibit TFL decrease in morphine tolerance rats, and this action was in a specific sequence manner. The injection of CRE-decoy ODN alone did not affect the pain threshold, which indicated that CRE-decoy ODN had no...