| Background and PurposeAs the most widely used analgesic drugs in clinic,opioids have irreplaceable advantages in the treatment of severe pain,but the vast majority of opioids in clinic have side effects such as tolerance,constipation,respiratory depression,immunosuppression,dependence and addiction.These side effects bring great trouble to users and become an urgent problem to be solved in the field of opiates.As the main receptor that binds to opioids,MOR encoding gene OPRM1 can form a wide range of variants through different ways of splice to exert the unique pharmacological effects of opioids.The MOR-1B variants encoded by Exon 5 in OPRM1 are one of the seven transmembrane full-length variants,which are highly expressed in the brain.Previous studies have shown that MOR-1B variants cloned on cells have a stronger affinity with opioids than MOR-1,but what is the function of MOR-1B variants in vivo?This study aims to clarify the effects of MOR-1B variants on the analgesia and side effects of opioid drugs in vivo,so as to find a new target and a new molecular mechanism of traditional opioid drugs,and provide theoretical guidance for the later research and development of opioid drugs with stronger analgesic effects and effective reduction of side effects.Research Contents and ResultsFirstly,we used CRISPR/Cas9 technology to establish the Exon 5 of OPRM1 gene knockout C57BL/6J mice in this research.It was clear that the knockout of the Exon 5 in OPRM1 could completely eliminate the mRNA expression levels of MOR-1B variants,and could not significantly affect the mRNA expression levels of other MOR variants.The C57BL/6J mice model with specific knockout of MOR-1B variants was successfully established and provided a suitable animal model for the further study of the functional significance of MOR-1B variants in vivo.In the C57BL/6J mice model with specific knockout of MOR-1B variants,the mice were divided into wild type(Exon5+/+group),heterozygote(Exon5+/-group)and homozygote(Exon5-/-group)according to their genotypes.Then,the effects of morphine,pethidine and fentanyl on analgesia,tolerance,constipation and acute withdrawal behaviors in these mice were observed.It was showed that the analgesic effects of the first administration of morphine,pethidine or fentanyl in these mice were not significantly different in the cold and hot plate test,thermal radiation tail flick test and mechanical paw withdraw test.After 7 days of continuous injection of morphine,pethidine or fentanyl,the analgesic effects in these mice were significantly reduced,suggesting the formation of tolerance.There was still no significant difference in the analgesic effects in these mice after 7 days of morphine or pethidine injection,but the analgesic effects in the Exon5 +/-group and the Exon5-/-group were significantly weaker than those of the Exon5+/+group after 7 days of fentanyl injection.The dose-response curves of fentanyl on day 1 and day 7 also showed that fentanyl had stronger analgesic tolerance in the Exon5+/-group and the Exon5-/-group than those in the Exon5+/+group.After the first injection of morphine,pethidine or fentanyl,there was no significant difference in the defecation ability of these mice with three genotypes at different time points after administration.This remained the case on day 7 of continuous administration of morphine or pethidine,but the defecation capacities of the Exon5+/-group and the Exon5-/-group were significantly more than that of the Exon5+/+group on day 7 of continuous administration of fentanyl.Acute opioid withdrawal was induced by naloxone after 7 days of morphine,pethidine or fentanyl injection,and there were no significant differences in the number of withdrawal jumps and weight loss among these mice with three genotypes.The results showed that MOR-1B variants reduced the analgesic and constipation effects after chronic fentanyl injection,but had no significant effects on the analgesic,tolerance,constipation and withdrawal effects after acute and chronic fentanyl injection of morphine or pethidine.Afterwards,we used ELISA to detect the concentration of cAMP in different brain regions of mice in the Exon5+/+group and the Exon5-/-group after acute and chronic injection of fentanyl.It was found that the concentration of cAMP in the Exon5-/-group was significantly higher than that in the Exon5+/+ group within the PAG region after continuous injection of fentanyl for 7 days.Western blot was used to detect the expression and phosphorylation levels of ERK in the PAG region of mice in the Exon5+/+group and the Exon5-/-group after acute and chronic injection of fentanyl.It was found that the phosphorylation level of ERK in the Exon5-/-group was significantly higher than that in the Exon5+/++ group after continuous injection of fentanyl for 7 days.Immunofluorescence staining was used to detect the expression level and distribution of β-arrestin2 and MOR in the PAG region of Exon5+/+and Exon5-/-group mice after chronic injection of fentanyl.It was found that the amount of β-arrestin2 collected in the PAG region of Exon5-/-group mice was significantly more than that of Exon5+/+group mice.Finally,we cultured primary neurons in the PAG region of the Exon5+/+group and the Exon5-/-group.After 30 minutes of fentanyl intervention,significant MOR endocytosis was observed in the primary neurons of the Exon5-/-group,while MOR endocytosis was not observed in the primary neurons of the Exon5+/+group.Conclusion and SignificantBased on the above-mentioned behavior observation of mice with three genotypes,we cleared that MOR-1B variants could postpone the development of analgesia tolerance and constipation tolerance of fentanyl.Subsequent experiments in vitro showed that MOR-1B variants could inhibit cAMP-ERK signal transduction in the PAG region and prevent MOR endocytosis induced by beta-arrestin2 to delay the development of fentanyl tolerance,which may be related to the dimers between MOR-1B variants and beta-arrestin2.The importance of opioids in the treatment of pain is beyond question.Hundreds and thousands of opioid compounds for traditional MOR have been developed over the years,and eventually dozens of opioids have been made available to clinicians,but the vast majority of these opioids do not separate the analgesic effects from the opioid side effects.It is expected to be able to solve this problem in the future because of MOR variants unique pharmacology function.MOR-1B variants regulate the development of fentanyl tolerance specifically,and this prompts us to develop a new opioid without tolerance through transforming the molecular structure of fentanyl and MOR-1B variants as the design targets in the future.We can also improve the enrichment of this new opioid in the PAG region in order to achieve the best curative effect. |
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