The Underlying Mechanism Of The Role Of NLRP3 Inflammasome In Morphine Withdrawal

Objective:Morphine is a powerful analgesic and is widely used clinically.Morphine withdrawal is an adverse reaction caused by the sudden withdrawal of the drug after long-term dependence on morphine.The mechanism of morphine withdrawal is complex and diverse,and there are many reactions in the body related to it.Microglia are immune cells in the central nervous system,and NLRP3(NLR family pyrin domain containing 3)inflammasome is the key molecule to produce inflammatory factors.Microglia are involved in the pharmacological action of morphine,so the purpose of this article is to elucidate the role of NLRP3 inflammasome in morphine withdrawal.The molecular mechanism of NLRP3 inflammasome mediating morphine withdrawal was explored through in vivo and in vitro morphine withdrawal model and by behavioral testing,pharmacological intervention and molecular biology analysis.Thus,our study may provide a new therapeutic target for clinical relief of morphine withdrawal symptoms.Methods: 1.BV2 microglia were incubated with morphine for 24 hours,and then q-PCR experiments were performed to detect the changes in the m RNA level of NLRP3 inflammasome-associated factors.BV2 microglia was treated with morphine withdrawal,and incubated with the NLRP3 inhibitor MCC950(1 ?M),and then Western blot experiments were performed to detect the changes in the protein expression levels of NLRP3 inflammasome-associated factors.2.Adult male SD rats were randomly divided into four groups,including the control group,the morphine dependence group,the morphine withdrawal group,and the morphine withdrawal + inhibitors group.Morphine withdrawal rat model was developed.The morphine withdrawal group was injected with NLRP3 inhibitor ?-hydroxybutyrate(?-HB)and MCC950,and the changes in the rats' withdrawal behavior were detected.3.The spinal dorsal horn tissues of the first three groups of rats were taken for q-PCR experiments to determine whether morphine withdrawal affected the m RNA level of inflammasome-associated factors.4.The spinal dorsal horn tissues of the above four groups of rats were taken for Western blot(WB)experiments to determine whether morphine withdrawal affected the protein expression of NLRP3 inflammasome-associated factors.5.Wild-type(WT)and NLRP3 knockout(KO)mice were employed to establish morphine withdrawal model,and the behavioral changes of morphine withdrawal were observed to determine whether NLRP3 inflammasome is involved in morphine withdrawal.6.TLR2 KO mice were employed to establish morphine withdrawal model,and the behavioral changes of morphine withdrawal were observed to determine whether TLR2 is involved in morphine withdrawal.Results: 1.The results showed that the m RNA level of inflammasome-associated factors were not affected by chronic morphine treatment on BV2 microglia cell line.After morphine withdrawal,the protein expression levels of pro-Caspase-1 and NLRP3 were up-regulated.This up-regulation was inhibited by NLRP3 inhibitors,indicating that the NLRP3 inflammasome of microglia was activated by morphine withdrawal.2.Morphine withdrawal symptoms in rats were significantly reduced by the administration of NLRP3 inhibitors ?-hydroxybutyrate and MCC950.3.The results of q-PCR analysis showed that the m RNA levels of NLRP3 inflammasome-associated factors were not affected by morphine withdrawal,and indicating that the regulation of their m RNA levels was not involved in morphine withdrawal.4.The results of WB showed tha that after morphine withdrawal,the protein expression levels of pro-Caspase-1 and IL-1? were up-regulated,and this up-regulation was inhibited by NLRP3 inhibitors,indicating that morphine withdrawal was involved in the regulation of NLRP3 inflammasome-associated factors.5.The results of behavioral showed that the morphine withdrawal symptoms of NLRP3 KO mice were significantly reduced,which further proved that the NLRP3 inflammasome is involved in morphine withdrawal.6.The results of behavioral showed that the morphine withdrawal symptoms of NLRP3 KO mice were significantly reduced,indicating that TLR2 was involved in morphine withdrawal.Conclusions: 1.Activation of NLRP3 inflammasome in microglia is induced by morphine withdrawal.2.Spinal NLRP3 inflammasome is involved in the pathogenesis of morphine withdrawal.3.TLR2 is involved in the pathogenesis of morphine withdrawal.