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Objective To evaluate the role of μ opioid receptor in morphine preconditioning-induced reduction of myocardial ischemia-reperfusion (I/R) injury in rats with chronic heart failure.Methods Adult male Sprague-Dawley rats,weighing 170-230 g,in which chronic heart failure was induced by injecting doxorubicin via the tail vein,were studied.The rats were sacrificed and their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95％ O2-5％ CO2 at 37 ℃.Forty isolated rat hearts with I/R injury were randomly divided into 4 groups (n=10 each):group I/R,morphine preconditioning group (group MP),μ opioid receptor antagonist CTOP plus morphine preconditioning group (group CTOP+MP) and CTOP group.Myocardial I/R was induced by occlusion of the left coronary artery for 30 min followed by 120 min of reperfusion.In group MP,the hearts were perfused with K-H solution for 15 min,with K-H solution containing 1 μmol/L morphine for 5 min and with K-H solution for 5 min,3 cycles in total,and then the model of myocardial I/R was established.The hearts were perfused with K-H solution containing 1 μmol/L CTOP starting from 10 min before morphine preconditioning until 5 min of ischemia in group CTOP + MP.The hearts were perfused with K-H solution containing 1 μmol/L CTOP starting from 40 min before ischemia until 5 min of ischemia in group CTOP.The coronary effluent was collected at 15 min of equilibration (baseline) and 5 and 10 min of reperfusion to detect the activity of lactate dehydrogenase (LDH).Myocardial infarct size (IS) and the area at risk (AAR) were measured by 2,3,5-triphenyl-tetrazolium staining,and IS/AAR percentage was calculated.The expression of Bcl-2 and Bax mRNA was determined using uantitative real-time polymerase chain reaction,and the ratio of Bcl-2/Bax was calculated.Results Compared with group I/R,the IS and IS/AAR percentage were significantly decreased,the activity of LDH in coronary effluent was decreased,the expression of Bax mRNA was downregulated,the expression of Bcl-2 mRNA was up-regulated,and the Bcl-2/Bax ratio was increased in group MP (P＜0.05),and no significant change was found in the IS or IS/AAR percentage in CTOP and CTOP+ MP groups (P＞0.05).Compared with group MP,the IS and IS/AAR percentage were significantly increased,the activity of LDH in coronary effluent was increased,the expression of Bax mRNA was up-regulated,the expression of Bcl-2 mRNA was down-regulated,and the Bcl-2/Bax ratio was decreased in group CTOP+MP (P＜0.05).Conclusion The mechanism by which morphine preconditioning reduces myocardial I/R injury may be related to activating μ opioid receptors and thus maintaining the balance between Bcl2 and Bax gene expression in the rats with chronic heart failure.
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Objective To evaluate the effect of oxycodone on migration of human colon cancer cells and the role of μ and κ receptors.Methods The human colon cancer HCT116 cells at the logarithmic growth phase were seeded in 24-well or in 6-well plates at a density of 1 × 106 cells/mnl (0.5 ml/well or 2 ml/well,144 wells in total).The cells were divided into 6 groups (n=24 each) using a random number table:control group (group C),1,5 and 10 μmol/L oxycodone groups (group O1,group O2 and group O3),oxycodone plus μ receptor antagonist CTOP group (group O2+CTOP) and oxycodone plus κ receptor antagonist nor-binaltorphimine group (group O2+BNI).The cells were incubated for 24 h with oxycodone 1,5 and 10 μmol/L in O1,O2 and O3 groups,respectively.The cells were incubated for 24 h with 5 μmol/L oxycodone plus 20 μmol/L CTOP and 5 μmol/L oxycodone plus nor-binahorphimin 20 μmol/L in O2+CTOP and O2+BNI groups,respectively.The invaded and migrated cells were counted,and the levels of Ras homolog gene family member A (RhoA),Rho-associated protein kinase 1 (ROCK1),matrix metalloproteinase-2 (MMP-2) and MMP-9 were detected.Results Compared with group C,the number of invaded and migrated cells was gradually decreased,and the levels of RhoA,ROCK1,MMP-2 and MMP9 were gradually decreased in O1,O2 and O3 groups (P＜0.05),and no significant change was found in the parameters mentioned above in group O2+BNI (P＞0.05).Compared with group O2,the number of invaded and migrated cells was significantly increased,and the levels of RhoA,ROCK1,MMP-2 and MMP9 were increased in group O2 + BNI (P＜0.05),and no significant change was found in the parameters mentioned above in group O2+CTOP (P＞0.05).Conclusion Oxyc odone can inhibit the migration of human colon cancer cells,and the mechanism is totally related to inhibition of RhoA/ROCKl signaling pathway activation after activating κ receptors,but not related to μ receptors.
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Objective To evaluate the effect of the opioid switch from morphine to sufentanil on the expression of μ-opioid receptors in the midbrain periaqueductal gray (PAG) of rats.Methods Forty healthy male Wistar rats,aged 8 weeks,weighing 250-290 g,were randomly assigned into 5 groups (n=8 each) using a random number table:control group (group C),7 day sufentanil group (group S),7 day morphine group (group M),14 day morphine group (group MM),and 14 day alternate administration of morphine and sufentanil group (group MS).Normal saline 2 ml/kg,sufentanil 0.01 mg/kg and morphine 10 mg/kg were injected subcutaneously in the cervical region twice a day for 7 consecutive days in C,S and M groups,respectively.In group MM,morphine 10 mg/kg was injected subcutaneously in the cervical region twice a day for 14 consecutive days.In group MS,morphine 10 mg/kg was injected subcutaneously in the cervical region twice a day for 7 consecutive days (1st-7th days),and sufentanil 0.01 mg/kg was then injected subcutaneously in the cervical region twice a day for 7 consecutive days (8th-14th days).The tail flick latency (TFL) to a thermal nociceptive stimulus was measured at 15 and 30 min after the initial administration every day.After the last administration,the rats were sacrificed,and the midbrain PAG was isolated for determination of the expression of the μ-opioid receptor and μ-opioid receptor mRNA using Western blot and real-time reverse transcriptase polymerase chain reaction,respectively.Results Compared with group C,the TFL was significantly prolonged on 1st-6th days after the beginning of administration in M,MM and MS groups,the TFL was significantly prolonged on 1st-7th days after the beginning of administration in group S,and the expression of the μ-opioid receptor and μ-opioid receptor mRNA in the midbrain PAG was significantly down-regulated in M,MM and MS groups (P＜0.05).Compared with group MM,the TFL was significantly prolonged on 8th-14th days after the beginning of administration,and the expression of the μ-opioid receptor and μ-opioid receptor mRNA in the midbrain PAG was significantly up-regulated in group MS (P＜0.05).Conclusion The mechanism by which the opioid switch from morphine to sufentanil reduces morphine tolerance is related to enhanced activity of μ-opioid receptors in the midbrain PAG of rats.
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Objective To evaluate the role of μ opioid receptor exon 7 in the analgesic efficacy of endomorphin-2 in rats.Methods Twenty-four male Sprague-Dawley rats in which IT catheters were successfully implanted,weighing 220-260 g,were randomly divided into 3 groups (n =8 each) using a random number table:normal saline control group (group C),negative siRNA control group (group N-siRNA) andμ opioid receptor exon 7 siRNA group (group E7-siRNA).In C,N-siRNA and E7-siRNA groups,30μl saline solution,negative siRNA plasmid 20 μl + lipofectamine 2000 (10 μl),and μ opioid receptor siRNA plasmid 20μ1 + lipofectamine 2000 (10 μl) were intrathecally injected once a day for 3 consecutive days.The mechanical pain threshold was measured on 4th day (baseline).Endomorphin-2 10 μg was injected intrathecally at 1 h after measurement of the pain threshold.The mechanical pain threshold was measured at 5,20,40 and 60 min after endomorphin-2 injection,and the analgesic efficacy was calculated.Results There was no significant difference in the baseline pain threshold among the three groups.Compared with group C,no significant difference was found in the analgesic efficacy at each time point after endomorphin-2 injection in group N-siRNA,and the analgesic efficacy was significantly decreased at 5 and 20 min after endomorphin-2 injection in group E7-siRNA.Conclusion μ opioid receptor exon 7 is involved in the analgesic efficacy of endomorphin-2 in rats.
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Objective To investigate the effects of intrathecal(IT)CX3 CR1 neutralizing antibody(antiFKR)on morphine tolerance in rats with bone cancer pain(BCP)and the unlerlying mechanism.Methods Forty-eight adult female SD rats aged 3 months weighing 180-200 g were randomized into 4 groups(n =12 each):group I sham operation(S); group Ⅱ BCP + normal saline(NS); group Ⅲ BCP + IgG(IgG)and group ⅣBCP + anti-FKR.Bone cancer pain(BCP)was induced by injecting Walker 256 cancer cells 10 μl(400 cells/ μl)into the medullary cavity of right tibia in groups Ⅱ,Ⅲ and Ⅳ.Ten days later morphine 20 μg/kg was administered IT twice a day for 7 consecutive days.Starting from the 8th day NS,IgG and anti-KFR 10 μl was administered IT once a day for 3 consecutive days in groups Ⅱ,Ⅲ and ⅣⅣ respectively.Paw withdrawal threshold to yon Frey filament stimulation(MWT)and paw withdrawal duration(MWD)were determined bcfore(To,baseline)and at 3,6,9 day after intra-tibial cancer cell inoculation(T1.2,3),on the 3rd and 7th day of IT morphine(T4.5)and on the 3rd day of IT NS/lgG/anti-KFR(T6).The animals were killed at T6 after last pain behavior assessment.The lumbar segment(L4-6)of the spinal cord was removed for determination of the expression of CX3 CR1 protein(by Western blot),μ-opioid receptor and TRPV1 receptor(by immuno-histochemistry)in the dorsal horn of spinal cord.Results IT morphine significantly eased BCP at T4,but morphine analgesia was significantly reduced on the 7th day of IT morphine in the 3 groups indicating morphine tolerance which was significantly relieved by anti-KFR in group Ⅳ.IT anti-KFR significantly down-regulated CX3CR1 prolein and TRPVI receptor expression and up-regulated μ-opioid receptor in group Ⅳ as compared with IT NS and lgG in groups Ⅱ and Ⅲ.Conctusion IT anti-KFR can relieve morphine tolerance in the rats with bone cancer pain by up-regulating μ-opioid receptor and down-regulating CX3 CR1 protein and TRPVI receptor expression.
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Objective To establish an automatic synthesis method for 11C-carfentanil (CFN) as an novel opiate receptor radioligand and study its biodistribution in rats. Methods 11C-Triflate-CH3 was bubbled into 0.5 mg precursor desmethyl-CFN (which was dissolved in 0.15 ml DMSO) to generate 11C-CFN in a V-tube at room temperature. Sep-Pak C2 column was used for purification of 11C-CFN, which was eluted by 3ml binary system aqueous solution, 10 ml water thrice, and then I ml ethanol. The biodistribution (% ID/g) of 11C-CFN in SD rats was studied. SPSS 13.0 was used for statistical analysis. Non-normal distribution data were analyzed using nonparametric test. Results The synthesis time for 11C-CFN was 20 min (end of bombardment, EOB). The synthesis yield was (35.5 ± 2.2) % on average (n = 12, uncorrected)with the radiochemical purity over 98%. Biodistribution study in rats showed that the tracer had a high brain uptake, rapid blood clearance, and a metabolic pathway via liver and kidney. The highest tracer uptake was in thalamus (4.26 ± 0.89) % ID/g and striatum (4.05 ± 1.08) % ID/g at 5 min after injection, followed by cerebral cortex (2.63±0.89) %ID/g, pons (2.26 ±0.57) % ID/g, hippocampus (2. 17 ±0.55) %ID/g and cerebellum (2. 15 ±0.39) %ID/g. Conclusions The automatic synthesis of 11C-CFN is fast and reliable, and this radioligand can be used for opiate receptor imaging.
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Objective To investigate the expression of μ-opioid system in the epidermis of patients with atopic dermatitis and its role in the pathogenesis of atopic dermatitis. Methods Thirty-two mice were equally divided into 4 groups, negative control group, pre-treatment group, naloxone group, and physiological saline group. Ovalbumin was used to sensitize mice in pretreatment group, naloxone group, and physiological saline group for 7 weeks, then, mice in naloxone group and physiological saline group were treated with intracutaneous naloxone or physiological saline solution for 1 week, respectively. Mice were killed in negative control group and pre-treatment group at the end of sensitization, and in naloxone group and physiological saline group after 1-week injection with naloxone or physiological saline, skin tissues were obtained from the back of killed mice and subjected to histological examination with HE staining and quantitative fluorescent PCR for the detection of mRNA expression of μ-opioid receptor (MOR) and its ligand (β-endorphin) in epidermis. The atopic dermatitis severity index of lesions and histological changes were assessed before and after the treatment. Results In comparison with the negative control mice, the epidermal expression level of MOR was signifieantly decreased (t = 2.549, P ＜ 0.05 ) in pre-treatment group, but increased in naloxone group and showed no statistical difference from the negative control group (t = 0.671, P ＞ 0.05). No significant difference was observed in the epidermal β-endorphin mRNA expression between negative control group and pre-treatment group or naloxone group (both P ＞ 0.05 ). The improvement of lesions could be visualized after treatment with naloxone (t = 8.338, P ＜ 0.01 ), which was concordant with the histological changes in naloxone group. Conchusions As an antagonist of MOR, naloxone can restore the expression of epidermal MOR in mice model for atopic dermatitis, and shows a certain efficacy in the treatment of atopic dermatitis, which proves that μ-opioid system is somewhat associated with the pathogenesis of atopic dermatitis.
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Objective To investigate the role of hippocampal mu-opioid receptors (MORs) in the formation of seizure susceptibility induced by kainic acid (KA). Methods The SD rats were injected subcutaneously with a convulsive dose of KA (10 mg/kg). Then mini-osmotic pumps were used for intrahippocampal injection with a selective MOR antagonist ?-FNA, and a selective MOR agonist PL017 in these rats. 7 days later, the seizure susceptibility was checked by a subthreshold dose of KA (5 mg/kg). Results All animals showed motor seizures over Stage 4 after a convulsive dose KA injection. 7 days later, 100% animals showed seizures over Stage 4 in KA, CSF + KA and PL017+KA groups after injection of subthreshold dose of KA, but only 28.5% in PL017+KA group. Mean latency of PL017+KA group was (10.1?3.0) minutes, shorter significantly than that of KA and CSF+KA groups (0.010.05). The mean stage of ?-FNA + KA group was 1.7?0.1, being reduced significantly as compared with the stages of other three groups ( P
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Objective To investigate the different changes in the regulation and gene expression of mu opioid receptor (MOR) in rat brain after acute and chronic morphine dependence.Methods Forty male SD rats weighing (210?35)g were randomly divided into five equal groups of eight animals each: (1) control; (2) acute dependence: (3) chronic dependence;(4) acute abstinence; (5) chronic abstinence. In acute dependence group rats received eight consecutive subcutaneous injection of morphine 5mg?kg-1 at 2h interval. In chronic dependence group morphine was injected subcutaneously three times a day(8:00, 15: 00, 22:00) for six days. The doses of morphine were 5, 10, 20, 40, 50, 60 mg? kg-1?day-1 from the 1st day to the 6th day respectively. In the two abstinence groups, the withdrawal syndromes were induced by intraperitoneal naloxone 5 mg ? kg-1. The rats in control group received saline. 30 min after the end of all procedures the animals were decapitated on ice. Brain was removed immediately and kept in liquid nitrogen. The Bmax and Kd values of 3H-DAMGO saturation binding to MOR were measured by Scatchard analysis. The gene expression of MOR was appraised by RT-PCR. Results (1) In the acute dependence group the Bmax value(the specific binding capacity of MOR) significantly increased and the affinity decreased. After abstinence the Bmax value returned to normal, but the affinity was still low. In chronic dependence and abstinence groups Bmax value decreased significantly and there was no change in Kd value. (2) The level of MOR mRNA increased significantly in acute dependence group and returned rapidly to normal after abstinence . In chronic dependence and abstinence groups the transcription of MOR was significantly lower than in control group. Conclusions The modulation of MOR in rat brain is different between acute and chronic dependence and there must be similar post-receptor mechnism involved.