The Molecular Mechanisms Of ?/? Opioid Receptor Heteromers Activate Microglia To Mediate Morphine Tolerance

Objective:Spinal microglia M1 type activation and proinflammatory factor release may be related to morphine tolerance mechanisms.?/?opioid receptor heteromers?MDOR?are involved in the regulation of analgesia,and our preliminary experiments suggest that it is related to the morphine tolerance process.In this study,morphine was used to incubate microglial cells to explore the role of?/?opioid receptor heteropolymers in regulating microglial activation,P2X4R?P2X4 receptor,P2X4R?and proinflammatory factor IL-1?under morphine stimulation.Here,we further explored the role of?/?opioid receptor heteromeric interfering polypeptide(MOR^TM1-TAT)in the expression of M1 type activation markers CD86,P2X4R and hyperalgesia in morphine-resistant rat.Methods:The experiments are divided into cell experiments and animal experiments.For cell experiment,For cells are divided into three parts?n=6?.In the first part were the Control group,Morphine group,MDOR-agonist group?MA group?,Morphine+MDOR-interferor group?MI group?.The type of microglial activation was detected by cellular immunofluorescence;the expression of P2X4R in cells was detected by immunoblotting;the content of?/?opioid receptor heteromers was detected by co-immunoprecipitation.In the second part,cells were randomly divided into the control group?morphine group?MOR^TM1-TAT group?5-BDBD group?Morphine+MDOR-interferor group?MI group?and morphine+5-BDBD group.The IL-1?mRNA was detected by RT-PCR method and the release of proinflammatory cytokines IL-1?was detected by Elisa method.In the third part were the the control group?morphine group?5-BDBD group and morphine+5-BDBD group.Each group was added with a final concentration of 3?M ATP and incubated for 4h.The IL-1?protein in the medium was detected by Elisa method.For animal experiment,twenty-four healthy male rats were divided into three groups:Control group,Morphine group,Morphine+MOR^TM1-TAT group.The changes of PWT in rats were measured before administration,15min,30min,60min,90min and 120min after MOR^TM1-TAT administration.The expression of CD86 and P2X4R was detected by using Western blot in the three groups after MOR^TM1-TAT injection for 120 minutes.Results:The cell experiment:in the first part:compared with the Control group,the expression of CD86?P<0.05?and?/?opioid receptor heteromers?P<0.05?in the MA group and Morphine group were significantly increased,and there was no significant change in the expression of CD206?P>0.05?;compared with Morphine group,the CD86?P<0.05?and?/?opioid Receptor heteromer expression?P<0.05?in the MI group decreased and CD206expression increased significantly?P<0.05?.In the second part:Compared with the Control group,the Morphine group and MT group had significantly increased IL-1?mRNA and IL-1?protein?P<0.05?;Compared with Morphine group,the IL-1?mRNA?P<0.05?and IL-1??P<0.05?protein expression were reduced in the MI group?P<0.05?,and no significant differences were observed between Morphine and MT groups?P>0.05?.In the third part,Compared with Control group,the release of IL-1??P<0.05?was increased in Morphine group;Compared with Morphine group,the release of IL-1??P<0.05?was significantly decreased in MT group.The animal experiment:Compared with Control group,the PWT values of Morphine group was significantly decreased?P<0.05?.Compared with morphine group,the PWT values of morphine+MOR^TM1-TAT group was significantly increased?P<0.05?at 15,30,60,90 and 120 minutes after injection.This indicated that intrathecal injection of MOR^TM1-TAT could significantly relieve morphine tolerance.Compared with control group,the expression of CD86 and P2X4R in spinal cord of rats in morphine group was significantly increased?P<0.05?.Compared with morphine group,the expression of CD86 and P2X4R in morphine+MOR^TM1-TAT group was significantly reduced at 120minutes after MOR^TM1-TAT drug injection?P<0.05?.This indicated that interference drugs(MOR^TM1-TAT)plays an analgesic role through reducing the expression of P2X4R and the M1 type activation of microglia.ConclusionMorphine incubation can lead to an increase in?/?opioid receptor heteromers and promote M1 type microglial activation.Interfering with the formation of?/?opioid receptor heteromers can inhibit morphine-induced proinflammatory microglial activation and morphine tolerance response.Its mechanism may be that the decrease in?/?opioid receptor heteromers leads to a decrease in the expression of P2X4R in microglia,which in turn inhibits the proinflammatory activation of microglia and the release of the proinflammatory cytokines IL-1?.