Experimental Study Of The Mechanism Of Alleviating Morphine Dependence By A CRE-decoy Oligodeoxynucleotide Competing Against The CREB Binding Site

Part 1 The inhibitory effects of CRE-decoy ODN on the CREB DNA-binding activity in morphine-dependent SK-N-SH cells in vitroObjective: To investigate the effects of a synthetic phosphorothioate CRE-transcription factor decoy oligodeoxynucleotide (CRE-decoy ODN) targeting the cAMP response element binding protein (CREB) on the CREB DNA-binding activity in morphine-dependent SK-N-SH cells in vitro.Methods: (1) Synthesis of the 24-mer phosphorothioate ODNs: CRE-decoy and control ODNs used in the present studies were phosphorothioate oligonucleotides. Their sequences are as follows: 24-mer CRE palindrome, 5'-TGACGTCA TGACGTCA TGACGTCA-3'; 24-mer CRE mismatch control, 5'-TGTGGTCA TGTGGTCA TGTGGTCA-3'; and 24-mer nonsense-sequence palindrome, 5'-CTAGCTAG CTAGCTAG CTAGCTAG-3'. The CRE cis-element, TGACGTCA, is palindromic, a synthetic single-stranded phosphorothioate oligodeoxynucleotide composed of the CRE sequence self-hybridizes to form a duplex/hairpin. Cationic lipid N-[ 1 -(2,3-dioleoyloxy)propyl]-N, N,N-trimethylammonium methylsulfate (DOTAP) was used as transfection reagent. Dilute ODN and DOTAP in HBS buffer according to ODN : HBS = 1:10 (μg/μl) and DOTAP:HBS = 1:2.3 (μg/μl) respectively. Transfer the nucleic acid solution to the reaction tube already containing the DOTAP in HBS buffer according to ODN: DOTAP = 1:6 (μg/μg) and carefully mix, then incubate the transfection mixture for 10 15 min at 15²5℃. (2) Cell culture and experimental groups : human neuroblastoma cell line SK-N-SH was propagated in MEM medium containing 0.1 mmol/L non-essential amino acids, 10 % fetal bovine serum, and 2 mmol/L glutamine at 37℃ in a humidified 5 % CO₂ incubator. Whenthe adherent cells yielded approximately 50 % confluency, CRE-decoy ODN and control ODNs in the presense of cationic lipid DOTAP was added at 1 h before exposure to 100 umol/L morphine hydrochloride in fresh culture medium (1 d after seeding) at final concentration 150 nmol/L. At 48 h of incubation with morphine, 10 umol/L naloxone hydrochloride was added to the medium for 15 min. All five groups were separately designated as: (D untreated group: containing saline-treated control, chronic morphine administration (M), and morphine+naloxone (M+N) subgroups; ? CRE-decoy ODN-treated group: containing CRE-decoy ODN-treated C, M, and M+N subgroups; (3) mismstch ODN-treated group: containing mismstch ODN-treated C, M, and M+N subgroups;? nonsense ODN-treated group: containing nonsense ODN-treated C, M, and M+N subgroups; (§) DOTAP-treated group: containing DOTAP-treated C, M, and M+N subgroups. (3) Cellular morphology and number: SK-N-SH cellular morphology and number was determined by a inverted microcope. (4) Electrophoresis mobility shift assay (EMSA): (D Control binding reactions: including negative and positive controls; (2) In vitro competitive binding assays: 50-fold excess of unlabeled CREB binding double-stranded probe, AP-2 binding double-stranded probe, CRE-decoy ODN, mismatch ODN, or nonsense ODN was used as a cold competitor respectively, and the effects of the above-mentioned probes on the retarded bands were observed; (D Supershift experiment: supershift was done with an antibody against CREB; ? The effects of ODNs on the CREB DNA-binding activity activated by chronic morphine: 5 ug of nuclear protein extracts was used for each lane. Samples of nuclear protein extracts were incubated with 1 ul 32P-labeled CREB binding double-stranded probe. (§) Electrophoresis and analysis of the results: the reaction mixtures were separated on a 6 % nondenaturing polyacrylamide gel at 350 V for 45 min, then the gel was dried and visualized by autoradiography for 48 h at -70°C. Semi-quantitative analysis of the CREB DNA-binding activity was measured by Gel-Pro analyzer version 3.0 software, and the results were expressed as AU (^(area-lvalue). (5) Cellular uptake ofODNs: CRE-decoy ODN, mismatch ODN and nonsense ODN was separately labeled in 5'-endwith [y-32P]ATP by T4 polynucleotide kinase. Three groups were used in the experiment: saline-treated control group, chronic morphine administration group, and morphine-f-naloxone group. 0.5 ul 32P-labeled CRE-decoy ODN in the presense of cationic lipid DOTAP was added at 1 h before exposure to 100 umol/L morphine. At 6 h, 12 h, 24 h, and 48 h, cells were separately washed four times with phosphate-buffered saline, and radioactivities in the cell pellets and culture media were determined. The cell uptake of oligonucleotides was calculated as percent radioactivity in cell pellets relative to the total radioactivity in cell pellet plus culture medium. Cellular uptake of other two ODNs was also examined. (6) Stability of cell-incorporated CRE-decoy ODN: morphine-dependenct cells were incubated in growth medium containing 32P-labeled CRE-decoy ODN in the presence of DOTAP for (24⁴8) h. Cell-incorporated CRE-decoy ODNwas extracted with phenol:chloroform, precipitated with ethanol, and subjected together with 32P-labeled CRE-decoy ODN, 10 bp DNA step marker and unlabelled ODNs to 20 % nondenaturing polyacrylamide gel electrophoresis at 150 V for 2.5 h. Then the gel was divided into two parts for autoradiography and silver staining respectively.Rsults: (1) Cellular morphology and number: cells in all five groups did not exhibit any changes in cellular morphology and growth. Furthermore, the cellular number within all groups had no statistical significance (P > 0.05). (2) Binding site specificity of CRE-decoy ODN to CREB: the results obtained by a positive control for CREB of EMSA revealed that the specific CREB-DNA binding complex could be detected using the present method. In vitro competitive binding assays were performed to examine sequence-specific interactions between CRE-decoy ODN and CREB. The retarded band was effectively competed for 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 for by the addition 50-fold excess of a cold AP-2 binding double-stranded probe, mismatch ODN, or nonsense ODNcontaining no CRE respectively. Supershift of the retarded band was caused by an antibody to CREB. (3) The effects of CRE-decoy ODN on the CREB DNA-binding activity: comparing with saline-treated SK-N-SH cells, EMSA demonstrated a 3.71-fold (P < 0.01) and 2.81-fold (P < 0.01) increase from 191.60 + 13.52 to 710.00 ±11.60 and 538.00+11.51 in the CREB DNA-binding activity in morphine-dependent and naloxone-precipitated withdrawn SK-N-SH cells respectively. The nuclear extracts from cells additionally treated with the 24-mer CRE-decoy ODN showed a marked reduction in formation of the CREB-DNA binding complex in morphine-dependent and naloxone-precipitated withdrawn SK-N-SH cells approximately 45.9 % (P < 0.01) and 46.7 % (P < 0.01) inhibition respectively (P < 0.01). Furthermore, the CRE-decoy ODN also had little inhibitory effect on the basal CREB-DNA binding complex approximately 15.2 % (P < 0.01). The suppression was CRE sequence-specific as the two-base mismatched control ODN or the nonsense-sequence palindromic ODN that contains no CRE sequence and DOTAP had no inhibitory effects. (4) Cellular uptake of ODNs: the amount and the rate of the incorporation of the oligonucleotides were similar at indicated times among saline-treated control group, chronic morphine administration group, and morphine+naloxone group. At 6 h, about 10 % of the total input ODN accumulated in SK-N-SH cells and the incorporation continued to rise thereafter, reaching 22 % at 12 h, 38 % at 24 h, and reaching 45 % maximum levels at 48 h. (5) Stability of cell-incorporated CRE-decoy ODN: silver staining showed that the location of the fragment of mismatch ODN was 30 bp, CRE-decoy ODN and nonsense ODN was 18 bp respectively. The result of autoradiography showed that up to 24 h and 48 h of examination the fragment of 18-bp 32P-labeled CRE-decoy ODN accumulated in morphine-dependent SK-N-SH cells.Conclusoins: CRE-decoy ODN shows marked inhibitory effects on the induced CREB DNA-binding activity in morphine-dependent SK-N-SH cells.Part 2 The inhibitory effects of CRE-decoy ODN on the upregulated nNOS expression in morphine-dependent SK-N-SH cells in vitroObjective: To investigate the effects of CRE-decoy ODN targeting transcription factor CREB on nNOS mRNA and protein expression in morphine-dependent SK-N-SH cells in vitro.Methods: (1) Synthesis of the 24-mer phosphorothioate ODNs: see part 1. (2) Cell culture and experimental groups: see part 1. (3) RT-PCR testing nNOS mRNA expression: single stranded cDNA was generated from 5 ug of total cellular RNA, and the resultant cDNA was amplified. 15 ul of the PCR products was detected by electrophoresis on a 1.8 % agarose gel containing ethidium bromide. The intensity of PCR products was measured using Gel-Pro analyzer version 3.0 software, and the results were expressed as AU (^area-lvalue). The signals of nNOS mRNA were expressed as investigated protein/(3-actin ratio. (4) Western blot analysis for nNOS protein expression: aliquots of crude cytosolic extracts containing 100 fig of protein were loaded on 4 % stock gel and separated by 10 % SDS-polyacrylamide gel electrophoresis, followed by transfer on nitrocellulose membrane by electroblotting for 12 h. The membrane was incubated with primary antibody against nNOS overnight at 4°C, and further incubated with a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody at 37°C for 1 h. Then the membrane was developed with DAB. The intensity of Western blot products was measured using Gel-Pro analyzer version 3.0 software, and the results were expressed as AU (^area-lvalue). The signals of nNOS protein were expressed as investigated protein/p-actin ratio.Resuts: (1) Effects of CRE-decoy ODN on the level of nNOS mRNA expression: RT-PCR demonstrated that unstimulated SK-N-SH cells showed a basal nNOS mRNA expression to a lesser extent. The incubation of these cells with 100 umol/L morphine for 48 h and subsequently with 10 umol/L naloxone for 15 min resulted in an increase in nNOS mRNA expression (P < 0.01). Comparing with saline-treated control, nNOS mRNA expression in morphine-dependent and naloxone-precipitated withdrawal SK-N-SH cells increased 1.78-fold (P < 0.01) and 4.04-fold (P < 0.01), from 0.22+0.01 to 0.40 ±0.05 and 0.89 ±0.02 respectively. Pretreatment with 150 nmol/LCRE-decoy ODN (1 h before exposure to morphine) markedly inhibited nNOS mRNA expression in morphine-dependent and naloxone-precipitated withdrawal SK-N-SH cells approximately 33.7 % (P < 0.01) and 60.8 % (P < 0.01) respectively. CRE-decoy ODN also showed little inhibition on the basal nNOS mRNA expression approximately 10.1 % (P < 0.01). The suppression was CRE sequence-specific as the two-base mismatched control ODN or the nonsense-sequence palindromic ODN that contains no CRE sequence and DOTAP had no inhibitory effects. (2) Effects of CRE-decoy ODN on the level of nNOS protein expression: Western blot analysis showed nNOS protein expression in morphine-dependent and naloxone-precipitated withdrawal SK-N-SH cells increased 2.86-fold (P < 0.01) and 5.29-fold (P < 0.01) than saline-treated control, from 0.15 + 0.01 to 0.42 + 0.02 and 0.77 ± 0.01 respectively; induced nNOS protein was suppressed by CRE-decoy ODN approximately 21.7 % (P < 0.01) and 30.0 % (P < 0.01) respectively. CRE-decoy ODN also showed little inhibitory effects on the basal nNOS protein expression approximately 17.2 % (P < 0.01). The suppression was CRE sequence-specific as the two-base mismatched control ODN or the nonsense-sequence palindromic ODN that contains no CRE sequence and DOTAP had no inhibitory effects.Conclusoins: CRE-decoy ODN shows marked inhibitory effects on the upregulated nNOS mRNA and protein expression in morphine-dependent SK-N-SH cells.Part 3 The inhibitory effects of CRE-decoy ODN on the upregulated fosB expression in morphine-dependent SK-N-SH cells in vitroObjective: To investigate the effects of CRE-decoy ODN targeting transcription factor CREB on fosB mRNA and protein expression in morphine-dependent SK-N-SH cells in vitro.Methods: (1) Synthesis of the 24-mer phosphorothioate ODNs: see part 1. (2) Cell culture and experimental groups: see part 1. (3) RT-PCR testing/osï¿¡ mRNA expression: single stranded cDNA was generated from 5 ug of total cellular RNA, and the resultant cDNA was amplified. 15 ul of the PCRproducts was detected by electrophoresis on a 1.8 % agarose gel containing ethidium bromide. The intensity of PCR products was measured using Gel-Pro analyzer version 3.0 software, and the results were expressed as AU (^area-lvalue). The signals of fosB mRNA were expressed as investigated protein/p-actin ratio. (4) Western blot analysis for AFosB protein expression: aliquots of crude nuclear extracts containing 50 ug of protein were loaded on 4 % stock gel and separated by 10 % SDS-polyacrylamide gel electrophoresis, followed by transfer on nitrocellulose membrane by electroblotting for 1 h. The membrane was incubated with primary antibody FosB (against a peptide corresponding to an amino acids 75 150 mapping at the amino terminus of FosB) overnight at 4°C, and further incubated with a goat anti-mouse IgM horseradish peroxidase-conjugated secondary antibody at 37°C for 1 h. Then the membrane was developed with DAB. The intensity of Western blot products was measured using Gel-Pro analyzer version 3.0 software, and the results were expressed as AU (v4areavlvalue). The signals of AFosB were expressed as investigated protein/p-actin ratio.Resuts: (1) Effects of CRE-decoy ODN on the level of fosB mRNA expression: RT-PCR demonstrated that unstimulated SK-N-SH cells showed a basal fosB mRNA expression to a lesser extent. The incubation of these cells with 100 umol/L morphine for 48 h and subsequently with 10 umol/L naloxone for 15 min resulted in an increase infosB mRNA expression (P < 0.01). Comparing with saline-treated control, fosB mRNA expression in morphine-dependent and withdrawal SK-N-SH cells increased 5.15-fold (P < 0.01) and 2.10-fold (P < 0.01), from 0.20+0.03 to 0.98 + 0.04 and 0.43+0.05 respectively. CRE-decoy ODN markedly inhibited fosB mRNA expression upregulated by chronic morphine administration and naloxone-precipitated withdrawal approximately 51.2 % (P < 0.01) and 52.3 % (P < 0.01) respectively. CRE-decoy ODN also showed little inhibitory effects on the basal fosB mRNA expression approximately 16.4 % (P < 0.01). The suppression was CRE sequence-specific as the two-base mismatched control ODN or the nonsense-sequence palindromic ODN that contains no CREsequence and DOTAP had no inhibitory effects. (2) Effects of CRE-decoy ODN on the level of AFosB expression: Western blot analysis showed 35 kDa AFosB in morphine-dependent and withdrawal SK-N-SH cells increased 5.68-fold (P < 0.01) and 3.84-fold (P < 0.01) than saline-treated control, from 0.16 + 0.01 to 0.90 + 0.02 and 0.61+0.03 respectively; whereas 37 kDa A FosB increased 5.45-fold (P < 0.01) and 3.67-fold (P < 0.01), from 0.17 + 0.01 to 0.92 + 0.02 and 0.62 + 0.03 respectively. CRE-decoy ODN markedly inhibited the 35 kDa A FosB in morphine-dependent and withdrawal SK-N-SH cells approximately 48.3 % (P < 0.01) and 47.5 % (P < 0.01) respectively, and inhibited the 37 kDa AFosB approximately 43.0 % (P < 0.01) and 46.3 % (P < 0.01) respectively. The CRE-decoy ODN also showed little inhibitory effects on the basal 35 kDa and 37 kDa AFosB expression approximately 11.1 % (P > 0.05) and 16.7 % (P < 0.01) respectively. The suppression was CRE sequence-specific as the two-base mismatched control ODN or the nonsense-sequence palindromic ODN that contains no CRE sequence and DOTAP had no inhibitory effects.Conclusoins: CRE-decoy ODN shows marked inhibitory effects on the upregulated fosB mRNA and protein expression in morphine-dependent SK-N-SH cells.Part 4 Suppressing morphine withdrawal signs and symptoms in rats by CRE-decoy ODNObjective: To investigate the effects of CRE-decoy ODN targeting transcription factor CREB on morphine withdrawal signs and symptoms in rats.Methods: (1) Synthesis of the 24-mer phosphorothioate ODNs: see part 1.(2) Experimental groups: all five groups were separately designated as ? normal saline-treated control group, (2) i.c.v. saline-injected control group,(3) morphine physical dependence group, @ naloxone-precipitated withdrawal group, and ?CRE-decoy ODN-treated withdrawal group. (3) Establishment of morphine physical dependence model: the rats were rendered dependent on morphine by subcutaneous injection with mophinehydrochloride 10 mg/kg, on day 1, twice, then increased by 10 mg/kg each day. On d 6, 2 h after injection with morphine 50 mg/kg, morphine withdrawal syndrome was precipitated by intraperitoneal injection of naloxone 5 mg/kg. An equal volume of normal saline was administered in the saline-treated control and i.c.v. saline-injected control groups. 20 ug of CRE-decoy ODN per rat was singly injected into the left intracerebroventriculus 30 h before the last injection of morphine. An equal volume of normal saline was injected into the left intraventriculus in every rat among (D^? groups at the same time. (4) Behavioral assessment of morphine withdrawal: withdrawal behaviors were monitored by a blind observer from 15 min to 1 h after naloxone administration at 15 min intervals. Two classes of withdrawal signs were measured: counted signs and observed signs. The absolute frequency of four episodic counted signs was recorded, and an additional score was calculated based on five incidents (0=no incidents; 1=1-—-5 incidents; 2=6^<sup>10 incidents; and 3=11 or more incidents). Behaviors scored in this manner included wet dog shakes, teeth chatter, vacuous chewing, and jumping. Six other observed signs, which could not be defined in discrete episodes, were assessed using predefined anchor points on a four-point scale: 0=absent; l=mild; 2=moderate; and 3=marked. Behaviors scored in this manner included ptosis, lacrimation, salivation, piloerection, irritability, and diarrhea. (5) Assessment of withdrawal scores of body weight loss: the weights of the animals, 1 h before and after precipitation of withdrawal, were obtained. The withdrawal scores of body weight loss were determined by the weight difference (AW) 60 min before and after administration of the naloxone, which were assigned as follows: 0=no change; 1=AW<2 %; 5=AW<4 %; 10=AW<6 %; 15=AW<8 %; 20=AW^8 %oResults: (1) Effects of intracerebroventricular injection of CRE-decoy ODN on the response scores of morphine withdrawal symptoms: withdrawal behaviors were precipitated by administration of naloxone (5 mg/kg, i.p.). No withdrawal behaviors were observed in rats before administration of naloxone. Intracerebroventricular injection of CRE-decoy ODN inhibited most of the...