The Mechanism Of Low-Dose Ketamine Inhibiting Morphine Tolerance

Part1Effects of ketamine on cAMP concentration and receptorexpression in a cellular model of morphine toleranceObjective: To explore the receptor mechanism underlying low-doseketamine preventing morphine tolerance, we observed the effects of low-doseketamine on the expression of δ-opioid receptor-1(DOR1) andN-methl-D-aspartic acid receptor2B subunit (NR2B) in a cellular model ofmorphine tolerance.Methods: The PC12cell was seeded in six well plates with density of5×105cells/ml (2ml/well), and divided into five groups randomly (n=5):0μmol/L ketamine group (group K0),0.5μmol/L ketamine group (group K0.5),5μmol/L ketamine group (group K5),50μmol/L ketamine group (group K50)and500μmol/L ketamine group (group K500). Incubated with serum-freemedium containing ketamine, and the concentration of ketamine werefollowed by0and0.5,5,50,500μmol/L. The time of incubation was48h.The concentration of cAMP was tested by time-resolved fluorescenceimmunoassay.The PC12cell was seeded in six well plates with density of5×105cells/ml (2ml/well), and divided into five groups randomly (n=5): controlgroup (group Con), morphine group (group Mor), morphine+0.5μmol/Lketamine group (group M+K0.5), morphine+5μmol/L ketamine group (groupM+K5) and morphine+50μmol/L ketamine group (group M+K50). GroupCon: incubated with serum-free DMEM/High glucose medium; Group Mor,group M+K0.5, group M+K5and group M+K50: incubated with serum-freemedium containing10μmol/L morphine and ketamine, while theconcentration of ketamine followed by0.5,5,50μmol/L. The time of incubation was48h. The concentration of cAMP was tested by time-resolvedfluorescence immunoassay.The PC12cell was seeded in six well plates with density of5×10⁵cells/ml (2ml/well), and divided into four groups randomly (n=5): controlgroup (group C), morphine group (group M), ketamine group (group K) andmorphine+ketamine group (group M+K). Group C: incubated withserum-free DMEM/High glucose medium; group M: incubated withserum-free medium containing10μmol/L morphine; groupK: incubated withserum-free medium containing0.5μmol/L ketamine; group M+K: incubatedwith serum-free medium containing10μmol/L morphine and0.5μmol/Lketamine; The time of incubation was48h. DOR1and NR2B expression weretested by Real-Time PCR.Results:①Compared with group K₀, there was no significant differencein concentration of cAMP in group K₀.5, group K5, and group K₅0(P>0.05).The concentration of cAMP were significantly higher in group K₅00and ingroup K₀(P<0.05).②The concentration of cAMP was significantly lower ingroup M+K₀.5, group M+K5and group M+K₅0than in group Mor. Theconcentration of cAMP was significantly lower in group M+K₀.5and incontrol group (P<0.05).③The expression of DOR1was lower and theexpression of NR2B was higher in group M than than in control group,respectively (P<0.05). There was no significant difference in the expression ofDOR1and NR2B between group K and group C (P>0.05). The expression ofDOR1was higher and the expression of NR2B was lower in group M+K thanin control, respectively (P<0.05).Conclusion:①Low-dose ketamine could prevent morphine tolerance atcell level.②Low-dose ketamine could prevent morphine tolerance byinhibiting the down-regulation of DOR1and the up-regulation of NR2B inPC12cells. Objective: Observation of low-dose ketamine on morphine tolerance inPC12cells Ca²⁺concentration, calcium/calmodulin-dependent protein kinaseII (CaMK II) expression of cAMP response element binding protein (CREB)levels in the general level of its phosphorylation (pCREB), to exploresmalldoses of ketamine to inhibit the molecular mechanisms of morphine tolerance.Methods: The PC12cell was seeded in six well plates with density of5×105cells/ml (2ml/well), and divided into five groups randomly (n=5):Control group (group C), morphine group (group M+K). Incubated withserum-free medium. Group C: medecine free; group M: the concentration ofmorphine was10μmol/L; group K: the concentration of ketamine was0.5μmol/L; group M+K: the concentration of morphine and ketamine was10μmol/L and0.5μmol/L,seperately. The time of incubation was48h.Theconcentration of Ca²⁺was tested by fluorescence.The expression of CaMKⅡ,CREB and pCREB were tested by wetern blotting.Results:①Compared with group C, the concentration of Ca²⁺and theexpression of CaMKⅡ were significantly higher in group M (P<0.05), whilelower in group M+K (P<0.05).②There was no significant difference in theexpression level of CREB among the four group (P>0.05). Compared withgroup C, the expression level of pCREB were significantly higher in group M(P<0.05), while lower in group M+K (P<0.05).Conclusion: Low-dose ketamine prevent morphine tolerance byinhibiting the up-regulation of the concentration of Ca²⁺, expression level ofCaMKⅡ and pCREB in PC12cells.