Endoplasmic Reticulum Stress Contributes To The Development Of Morphine Tolerance And Hyperalgesia

Morphine,as a classic opioid,is widely used in the clinical treatment of acute and chronic pain,neuropathic pain and cancer pain.The long-term use of morphine produces tolerance,which limits the clinical application of drugs,becoming the problem of pain diagnosis and treatment.This issue has aroused wide attention of scholars,a large number of different levels of research,but the mechanism of morphine tolerance is not yet fully elucidated.The main clinical feature of morphine tolerance are the diminished analgesic effect,shortened duration,aggravated pain and hyperalgesia,and more dose needed to be given in clinical application to achieve the desired analgesic effect.However,high-dose morphine may aggravate adverse drug reactions.Morphine tolerance involves desensitization and internalization of opioid receptors as well as heteropolymerization with other receptors,inflammatory response,and down-regulation of glutamate transporters,other mechanisms are still under investigation.The study of morphine tolerance mechanism is of great significance to the safe medication in clinical pain treatment.In addition,it has been reported in the literature that the molecular chaperone binds to immunoglobulin protein(BIP / GRP78)on the endoplasmic reticulum,also known as BIP / GRP78,which plays an important role in the development of morphine tolerance,whereas BIP / GRP78 plays an important role in regulating endoplasmic reticulum stress(ER stress).Studies have shown that endoplasmic reticulum stress can induce the activation of the NLRP3 inflammasome and the increasing of LCN2(Lipocalin-2)protein levels,and astrocytes play an important role in the development of morphine tolerance.Therefore,our present study focused on the role of endoplasmic reticulum stress-induced activation of NLRP3 inflammasome and the increasing of LCN2 in astroglial cells in morphine analgesia,tolerance and morphine-induced hyperalgesia.Objective: To establish a rat model of morphine tolerance and investigate the occurrence and related protein expression of endoplasmic reticulum(ER)in the dorsal horn of L2-L6 spinal cord in rats,effects of IRE1-NLRP3 and PERK-LCN2 signaling pathway on morphine analgesia,tolerance and morphine-induced hyperalgesia.Methods:(1)Morphine,morphine plus 4-PBA and morphine plus TUDCA were intrathecally injected into the rats twice a day for 7 consecutive days,to observe the effects of endoplasmic reticulum stress inhibitors on morphine analgesia and tolerance.The Von Frey filament was used to determine the mechanical pain threshold.Western blot was used to determine whether morphine induces endoplasmic reticulum stress-related protein production,as well as NLRP3 inflammasome and LCN2 protein expression.The accumulation of unfolded protein in spinal dorsal horn of morphine tolerant rats was detected by immunofluorescence.(2)In vitro morphine treatment of C6 cell line,Western blot detection of endoplasmic reticulum stress-related protein expression,NLRP3 inflammasome and LCN2 expression.(3)Rats were intrathecally injected IRE1 endonuclease inhibitor STF-083010 and inflammatory inhibitor ?-hydroxybutyrate(?-HB),observing the effect on morphine analgesia and tolerance by tail flick test.The mechanical pain threshold was measured by Von Frey filaments.The activation of NLRP3 inflammasome was detected by western blot.(4)Rats were intrathecally injected with PERK inhibitor GSK2656157 and LCN2 interfering RNA respectively.The effects of morphine analgesia and tolerance were observed by tail flick test.Von Frey filament was used to determine the mechanical pain threshold.LCN2 protein expression was detected by western blot.(5)Diabetic rat models were established.Morphine,morphine plus 4-PBA and morphine plus TUDCA were injected intrathecally.The effect of endoplasmic reticulum stress inhibitor on the hyperalgesia was tested by formalin test.(6)SNI rat model was established.Morphine,morphine plus 4-PBA and morphine plus TUDCA were injected intrathecally,and the mechanical pain threshold was measured by Von Frey fiber.Results:(1)Intrathecal administration of endoplasmic reticulum stress inhibitors 4-PBA and TUDCA enhanced morphine analgesia and relieved morphine tolerance and hyperalgesia.In the morphine dose-response curve experiment,4-PBA and TUDCA can prevent rightward shift of morphine tolerance curve.4-PBA and TUDCA can reverse morphine tolerance after morphine tolerance.(2)The result of immunofluorescence showed the unfolded protein reaction in spinal cord of morphine tolerant rats.Western blot results showed that the expression of endoplasmic reticulum stress-related protein in spinal cord of morphine tolerant rats increased.(3)The results of Western blot in morphine tolerant rat spinal cord showed that NLRP3 inflammasome activation and LCN2 protein expression were increased,and IRE1 endonuclease inhibitor STF-083010 and inflammatory body inhibitor ?-hydroxybutyrate(?-HB),PERK inhibitor GSK2656157 and LCN2 interference RNA can relieve morphine tolerance and pain sensitivity.(4)Endoplasmic reticulum stress inhibitors 4-PBA and TUDCA can relieve the diabetic formalin-induced pain.(5)Endoplasmic reticulum stress inhibitors 4-PBA and TUDCA can alleviate the hyperalgesia in SNI model.Conclusion: The IRE1-NLRP3 inflammasome and PERK-LCN2 signaling pathways are involved in the formation and development of morphine tolerance.