Study On The Effect Of Peptide Intervention On μ-opioid Receptor Phosphorylation On Morphine Analgesia And Morphine Tolerance

Objective:Morphine,as a classic opioid,is widely used in clinical practice and has good analgesic effect.This phenomenon may be closely related to the C-terminal phosphorylation ofμopioid receptors.The purpose of this study is to find the key phosphorylation sites at the C-terminus ofμ-opioid receptors that morphine acts,and to synthesize the polypeptide which covered sites to competitively prevent receptor phosphorylation,so as to explore the analgesic effects and morphine tolerance ofμ-opioid receptor phosphorylation on morphine.To explore the role of different phosphorylation sites in morphine analgesia and morphine tolerance,in order to provide new ideas for enhancing morphine analgesia and delaying tolerance.Methods:Experiments are divided into peptide synthesis experiments,cell experiments and animal experiments.Polypeptide synthesis experiment:Six peptides were synthesized by standard peptide solid-phase synthesis method,and the purity of the peptide was detected by high performance liquid chromatography（HPLC）,and the molecular weight of the peptide was detected by high resolution electrospray mass spectrometry（HRESI-MS）.Peptides with purity>95%and molecular weight consistent with the theoretical value are qualified and can be used in subsequent experiments.Cell experiments:routine cell culture,plasmid extraction and transfection;CCK-8method was used to detect the effect of different concentrations of peptides on cell viability,so as to judge the toxicity of peptides.Animal experiments:1.To explore the effect of different concentrations of peptide pretreatment on the analgesic effect of morphine:66 C57BL/6JNifdc mice were randomly divided into 6 groups:group A,control group（saline group）,mice were subcutaneously injected with normal saline 10m L/kg on the back of the neck.Group B,morphine group,mice were subcutaneously injected with morphine 10mg/kg on the back of the neck,group C,simple polypeptide group,mice were injected intraperitoneally with polypeptide 5mg/kg,D、E、F groups were pretreated with different concentrations of peptides,mice were injected intraperitoneally with different concentrations of peptides（1、5、10 mg/kg）,1 hour later,subcutaneously inject with morphine 10 mg/kg on the back of the neck.Heat pain was measured at 15min,30min,45min,60min,90min,120min,180min and 240min after the last administration,and the MPE（%）value was calculated.2.To explore the effect of different peptide pretreatment on the analgesic effect of morphine:96 C57BL/6JNifdc mice were randomly divided into 8 groups:group A,control group（normal saline group）,mice were subcutaneously injected with normal saline 10m L/kg on the back of the neck;group B,morphine group,mice were subcutaneously injected with morphine 10 mg/kg on the back of the neck,group C、D、E、F、G、H,peptide pretreatment groups,1 hour after intraperitoneal injection of different peptides（1-6#）,mice were subcutaneously injected with morphine 10mg/kg on the back of the neck,15min,30min,45min,60min,90min,120min,180min,240min after the last administration to measure the heat Pain,calculate MPE（%）value.3.To explore the effect of different peptide pretreatments on morphine tolerance:96 C57BL/6JNifdc mice were randomly divided into 8 groups:group A,control group（saline group）,mice were subcutaneously injected with normal saline 10m L/kg on the back of the neck;in group B,morphine group mice received subcutaneous injection of morphine 10 mg/kg on the back of the neck,group C、D、E、F、G、H are peptide pretreatment groups,mice were injected intraperitoneally with different peptides（1-6#）,then subcutaneously injected with morphine 10 mg/kg on the back of the neck 1 hour later,for seven consecutive days,no peptide was given on the eighth day,only morphine was injected,heat pain was measured 30 minutes after administration,and the MPE（%）value was calculated.Results:In the peptide synthesis experiment,the HPLC results of the six peptides were all above 95%,and the molecular weights measured by HRESI-MS were all in line with the theoretical value.In the cell experiment,the results of cck-8 showed that compared with the blank control group,there was no significant difference in the level of cell viability（%）between the groups treated with different concentrations of polypeptides 1,2,3,and 5（P>0.05）.Only the high concentration（100μmol/L）group of polypeptide 4、6 had a statistically significant difference in cell viability（%）compared with the control group,so the safe dose range of the polypeptide was considered to be large.In the animal experiment,part 1:The MPE（%）level of the mice in the peptide group was consistent with the control group（normal saline group）,so the peptide alone had no analgesic effect.Compared with the morphine group,the MPE（%）of the mice in the 1#and 3#polypeptide pretreatment groups were significantly increased,and the effect was dose-related.The MPE（%）of the5#polypeptide pretreated mice was slightly higher than that in the morphine group.The MPE（%）of mice pretreated with 2#and 4#polypeptides increased less than that of morphine group.Part II:The MPE（%）of the 1#-6#g high-dose（10mg/kg）peptide pretreatment group increased at different levels compared with the morphine group.Among them,1#,3#,5#,and 6#have long-lasting effects,while 2#and 4#have limited effects.The third part:MPE（%）of mice in morphine group decreased from 3-4 days,and decreased to the level of control group（normal saline group）from 6-7 days.The morphine tolerance model was successfully established.Compared with the morphine group,the downward trend of the MPE（%）in the 3#,5#and 6#polypeptide pretreatment groups is significantly reduced,these peptides reduce the morphine tolerance.Compared with the morphine group,the MPE（%）of the 1#polypeptide pretreatment group had the same downward trend,and the tolerance was not relieved.The decreasing trend of MPE（%）in the 2#and 4#polypeptide pretreatment groups was slightly slower than that in the morphine group,which may partially delay tolerance.Conclusion:Phosphorylation of the C-terminal ofμ-opioid receptor is closely related to morphine analgesia and morphine tolerance.Phosphorylation at different sites plays different roles in morphine analgesia and morphine tolerance.Among them,the phosphorylation sites of³⁵⁴TSST³⁵⁷,³⁷⁵STANT³⁷⁹ and Thr394 play an important role in the process of morphine analgesia,and the phosphorylation sites of Ser363 and Thr384 are related to morphine analgesia to a certain extent but do not play a key role.Ser363,Thr370,³⁷⁵STANT³⁷⁹,Thr384,Thr394 phosphorylation sites are closely related to morphine tolerance,while³⁵⁴TSST³⁵⁷ does not play a key role in this process.By designing a combination of peptides and morphine to competitively bind to key sites and intervene in the corresponding phosphorylation sites,it can effectively prolong the analgesic effect of morphine,delay morphine tolerance,and provide new ideas for morphine medication and pain treatment.