Regulation Of Neuroinflammation In Dorsal Root Ganglia By Gut Microbia Contributes To Morphine Tolerance

Chronic pain is a major public health problem.About 30% of the population live with this disorder.Opioids are still the gold standard for its pharmacological treatment in the clinical setting.Morphine,as a classic opioid,is widely used in the clinical treatment of acute and chronic pain.However,long-term use of morphine is often limited by the development of analgesic tolerance.Opioid tolerance is characterized by a progressive lack of response to opioids that can be overcome by escalating doses to achieve equivalent pain relief.Therefore,morphine tolerance limits its clinical application,which is a tough problem in treatment of chronic painIntensive research into the neurobiological mechanisms of morphine tolerance in the past decades.It is documented that repeated and long-term exposure to morphine causes opioid receptor–mediated adaptive changes within the nervous system,including desensitization,internalization,downregulation,and phosphorylation of opioid receptors or heterodimerization with other receptors.Moreover,chronic opioid exposure leads to activation of anti-opioid systems release from primary afferents and spinal dorsal horn glutamate receptor activation;descending spinal facilitation;glial cell activation and cytokine release.However,morphine tolerance is still ineffectively managed by current drugs.It is also documented that MOR expressed by nociceptors represents a critical and susceptible element driving analgesic tolerance and OIH,but the mechanism is still undefined.The dysregulation of microbiota-gut-brain axis plays a key role in many kinds of neurological diseases,but its role in morphine tolerance is still unclear.we proposed a hypothesis that dysregulation of microbiota-gut-brain axis plays a key role in morphine tolerance in mice.Part one: gut microbiome mediated morphine tolerance in mice1.Specific Aims:(1)To investigate whether gut microbiota involved in morphine tolerance(2)To analyze gut microbial composition in morphine tolerance mice(3)To clarify the mechanism underlying morphine-altered gut microbiota community and enhanced intestinal permeability and its association with morphine tolerance.2.Methods:(1)Adult male C57 mice were used in the present studies.We examined the general condition,locomotion,basal pain threshold,motor ability of mice following bacterial depletion with oral gavage of an antibiotic cocktail(ABX).(2)Faecal microbiota transplantation was performed from morphine tolerance mice to normal mice.Chronic tolerance was also tested with tail-flick assay following repetitive treatment of morphine(10 mg/kg,s.c.)twice a day for 7 days,and the analgesic effect was measured 30 min after each injection in male mice.(3)Tolerance model was established by repetitive morphine injection(10 mg/kg,s.c.),which was given twice a day for 7 days.fecal contents were collected later and analyzed for microbial composition by Next generation sequencing.(4).Serum FITC concentrations were measured following oral administration of FITC-dextran.saline.To determine the effects of chronic morphine on bacterial translocation,mesenteric lymph node(MLN),liver,and spleen tissue samples were collected and cultured on blood agar plates(BD Biosciences)for 24–48 hours and colony forming units(CFUs)quantified.3.Results:(1).The efficacy of ABX treatment was evidenced by gross morphological changes indicative of bacterial clearance;(2).Reduction of gut bacteria prevented antinociceptive tolerance to chronic morphine,but there were no significant difference between both groups concerning the general condition,locomotion,basal pain threshold,development of morphine dependence,motor ability of mice;(3).Faecal microbiota transplantation accelerated morphine tolerance.(4)16s r RNA sequencing showed that chronic morphine treatment significantly altered the gut microbial composition and induces preferential expansion of gram-positive communities(5).H&E staining demonstrateed mucosal damage with chronic morphine exposure also increased gut permeability and bacterial translocation,which were abated by propranolol treatment.4.Summary:Gut microbiome participated in morphine tolerance,destruction of colonic mucosa,bacterial translocation and increasing gut permeability.Part two: effects of gut microbiota on regulation of neuroinflammation in Dorsal Root Ganglia induced by morphine1.Specific Aims:(1)To investigate whether gut microbiota involved neuroinflammation induced by morphine.(2)To investigate whether gut microbiome mediated D R G neuroinflammation induced by morphine via macrophage.(3)To elucidate the mechanisms underlying the hyperexcitability of dorsal root ganglia neurons induced by long-term morphine treatment.2.Methods:(1)Using immunofluorescence and RT-PCR to investigate expression of macrophage maker F4/80,inflammatory cytokines and chemokine in DRG of moephine tolerance mice.(2)Macrophage depletion by injection of Clodronate Liposomes and then access the influence of morphine antinociceptive tolerance.(3)Whole-cell patch clamp recordings were performed in vitro on single DRG neurons isolated.3.Results:(1)Chronic morphine significantly increased IL-1? concentration in serum.Gut bacteria depletion prevented chronic morphine-induced IL-1? expression.(2)The result of immunofluorescence and RT-PCR showed that macrophage maker F4/80 expression was increased in DRG.Gut bacteria depletion prevented chronic morphine-induced F4/80 expression.(3),The result of RT-PCR showed that macrophage maker F4/80,inflammatory cytokines and chemokine increased in DRG.Gut bacteria depletion prevented chronic morphine-induced F4/80,inflammatory cytokines and chemokine expression.(4)Macrophage depletion in vivo prevented morphine antinociceptive tolerance.(5)Chronic morphine induced increase in electrical excitability of DRG neurons,which can be reduced by macrophage depletion.4.Summary:Gut microbiota mediated DRG neuroinflammation induced by morphine via macrophage.Part three: the mechanisms of neuroinflammation induced by morphine via gut microbiota and TLR21.Specific Aims:(1)To investigate whether TLR2 involved morphine tolerance.(2)To elucidate the mechanisms of TLR2 mediated morphine tolerance2.Methods:(1)To identify TLRS involved the development of morphine tolerance through RT-PCR?Western Blot(2)To evaluate the development of moephine tolerance in TLR2 KO mice and TLR4 KO mice.(3)Tolerance model of TLR2 KO mice was established as WT mice.Fecal contents were collected later and analyzed for microbial composition by Next generation sequencing.(4)Using immunofluorescence and RT-PCR to investigate expression of macrophage maker F4/80,inflammatory cytokines and chemokine in DRG of TLR2 KO moephine tolerance mice3.Results:(1)The result of RT-PCR and Western Blot showed that TLR2 expression was increased in morphine tolerance model.(2)Depression TLR2,but not TLR4,prevented antinociceptive tolerance to chronic morphine.(3).Intrathecal injection of TLR2 agonist PGN accelerated antinociceptive tolerance to chronic morphine.Intrathecal injection of TLR2 neutralizing antibody prevented morphine tolerance.(4)16s r DNA content exhibited a trend towards reduced species richness for the morphine treated TLR2 KO mice.(5).The expression of F4/80,inflammatory cytokines and chemokine did not significant increase in TLR2 mice treated with morphine4.Summary:TLR2 mediated morphine tolerance through influence of gut microbiota and activation of macrophage.Conclusions:1.Morphine altered gut microbiota community and enhanced intestinal permeability,which was associated with morphine tolerance.2.Macrophage and following neuroinflammation,induced by long-term morphine treatment via gut microbiota,contributed to the hyperexcitability of dorsal root ganglia neurons.3.TLR2 mediated morphine tolerance through influence of gut microbiota and activation of macrophage.