CXCL12/CXCR4 Axis Contributes To Morphine Tolerance Through Regulation Of Spinal Cord Autophagy In A Model Of Cancer Pain

Background and purposeCancer pain seriously affects the life quality of patients.Morphine is widely used to control moderate to severe cancer pain in the world,although long-time administration will cause morphine tolerance and greatly reduce its analgesic effect.The specific mechanisms of morphine tolerance are quite complicated.How to effectively inhibit morphine tolerance has become a concern.CXC-motif chemokine ligand 12(CXCL12)and CXC-motif chemokine receptor 4(CXCR4)are widely distributed in human tissues and organs,binding to mediate a variety of biological functions.Although increasing studies in recent years have shown CXCL12 contributes antinociceptive tolerance of normal murine to morphine,the role and mechanism of CXCL12 in cancer-pain rats of morphine tolerance have not been reported yet.Autophagy is a cellular process of degrading damaged organelles and proteins and recycling,which plays an important role in maintaining cellular homeostasis.Several studies have indicated the involvement of autophagy in cancer pain and morphine tolerance.In this study,we aim to establish a model of cancer pain by inoculating Walker 256 tumor cells to the plantar of rats,and to investigate the role of CXCL12 in morphine tolerance and whether the role is mediated by the regulation of autophagy in spinal cord.MethodsAdult male Wistar rats,weighing 160 g～180 g,were inoculated with 100μl Walker 256 tumor cells(1×10~6 cells)on the right plantar to establish the cancer pain model for 3 days after successful intrathecal catheterization.4 groups of rats(n=8)were randomly divided 5 days after tumor cells inoculation(TCI).Separate groups of rats received drugs once a day for 7 days.Saline group:saline 20μl(i.t.).Saline+morphine group:saline 20μl(i.t.)+morphine 10 mg/kg(i.p.);CXCL12+morphine group:CXCL12 100 ng(i.t.)+morphine 10 mg/kg(i.p.);AMD3100+morphine group:AMD3100 20μg(i.t.)+morphine 10 mg/kg(i.p.).Morphine was administered 30minutes after intrathecal injection in the latter 3 groups.Mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were tested on ipsilateral paw of rats at several timepoints(before catheterization,before TCI,5 days after TCI,and30 minutes after the last administration on each day)to evaluate antinociception of morphine.After 7-day behavior tests,the rats were decapitated for harvesting spinal cord.Western blot experiments were performed to detect the expression of microtubule-associated protein 1 light chain 3(LC3),Beclin-1 and p62.ResultsMWT and TWL of the right hind paw of rats in separate groups were significantly decreased 5 days after TCI(P<0.05),which indicated a cancer pain model.Morphine treatment resulted in the development of tolerance on day 6 in the saline+morphine group.Pre-treatment of CXCL12 i.t.accelerated morphine tolerance.Conversely,AMD3100 administered prior to morphine attenuated antinociception tolerance.Western blot analysis showed that morphine increased the expression of LC3-II/I,Beclin-1 and p62(P<0.05),which indicated an activation of autophagy but inhibition of autophagy influx.Co-administration of CXCL12 and morphine alleviated increases in LC3-II/I and Beclin-1 induced by morphine,but aggravated p62 expression(P<0.01).Instead,AMD3100 treatment decreased the elevation of p62 induced by morphine(P<0.05).ConclusionsThe CXCL12/CXCR4 signaling contributes to the development of tolerance to morphine in a model of cancer pain.Autophagy in spinal cord is involved in the role of CXCL12/CXCR4 axis in tolerance of cancer-pain model to morphine.