The Mechanism Of Activated Autophagy In Alleviating Cancer-induced Bone Pain

BackgroundCancer-induced bone pain(CIBP)is one of the most common chronic pain.Since the microenvironment of bone tissue was suitable for tumor cell,about 2/3 of end-stage cancer patients have been demonstrated to have bone metastasis,which leads to CIBP.CIBP is often characterized by mechanical hyperalgesia and allodynia.At present,nonsteroidal anti-inflammatory drug and opioid analgesics are used to treat CIBP.However,the therapeutic effects are limited because of the side effects,such as constipation,nausea,excessive sedation,vomiting,respiratory depression,addiction and so on.Therefore,it's urgent to study the mechanisms involving in CIBP and discover new therapeutic targets.As defined by programmed cell death type II,autophagy is a process of relying on lysosomes to degrade metabolic wastes for recycling.A growing body of evidence has proved that the disorders of autophagy involve in tumors,immune diseases,cardiovascular diseases,neurodegenerative diseases and so on.Mounting studies suggest that autophagy plays a pivotal role in the mechanisms of tumor anti-radiotherapy,chemotherapy and targeted therapy.When damaged,proteins and organelles are degraded into small molecules by autophagy,maintaining the stability of the intracellular environment.Recent studies confirm that autophagy contributes to the occurrence and development of neuropathic pain.Rapamycin,an autophagy activator,significantly relieve nerve injury-induced mechanical and thermal hyperalgesia.However,whether autophagy participates in the pathogenesis of CIBP has not been reported.Inflammasomes,as a macromolecular protein complex,participate in the occurrence and development of inflammation and play an important role in the immune system.Under stress,inflammasomes are overactivated and cause a series of inflammatory reactions,resulting in tissue damage and tumor.A lot of studies show that inflammasomes are closely related to many diseases,such as arthritis,gastric cancer,metabolic diseases and allergic reaction.NLRP3 inflammasomes,as the most popular subtypes,are mainly distributed in monocyte macrophages,lymphocytes and microglia.Mounting evidence has demonstrated that many factors can activate both of inflammasomes and autophagy,including excessive release of reactive oxygen species,tissue damage and ischemia.Since autophagy regulates the activation and function of inflammasomes through different mechanisms,we assume that autophagy regulate CIBP through NLRP3 inflammasome signaling pathway.This study intended to explore the roles of autophagy and NLRP3 inflammasome in CIBP rat model.The autophagy agonist and MCC950 inhibitor were used to study the specific mechanisms of autophagy in the development of CIBP.Methods and Results1.The activated autophagy involves in cancer-induced bone pain MethodsExperiment 1: In this study,we randomly divided female Sprague-Dawley rats into three groups: na(?)ve group,sham group and cancer-induced bone pain(CIBP)group.Mechanical paw withdrawal threshold(MPWT)was measured before tumor cell implantation(TCI)and at day 3,7,14 and 21 after model establishment.Changes of autophagy-related protein were measured by Western blot and immunofluorescence,including Beclin-1,LC-3II and P62.Experiment 2: We randomly divided female Sprague-Dawley rats four groups: sham + vehicle group,CIBP + vehicle group,CIBP + Rap group and CIBP + 3-MA + Rap group.To demonstrate the role of autophagy in CIBP model,a single dose of rapamycin(1,5,10 mg/kg)was intraperitoneally injected at day 14.Then,rapamycin was continuously injected once daily from day 14 to day 18 to observe the cumulative analgesic effect on CIBP.To investigate whether autophagy inhibitor could reverse the effect of rapamycin,3-Methyladenine(3-MA)was administrated intraperitoneally 30 minutes before rapamycin.Western blot and immunofluorescence were utilized to detect the expression and distribution of spinal Beclin-1,LC-3II and P62.ResultsCompared with na(?)ve and sham group rats,the MPWT of CIBP group markedly decreased from day 7 to day 21.The injection of rapamycin(5 and 10 mg/kg,i.p)significantly improved the MPWT,while the dose of 1mg/kg couldn't improve obviously mechanical allodynia.The MPWT in 5 and 10mg/kg groups increased significantly,beginning at 0.5h,peaking at 2h,and lasting for 4h.Continuous administration of rapamycin markedly attenuated CIBP-related behaviors.However,the treatment with autophagy inhibitor 3-MA inhibited the analgesic effect of rapamycin.Western blot and immunofluorescence data showed that spinal Beclin-1,LC-3II and P62 were all activated.Moreover,the expression levels of Beclin-1 and LC-3II were further upregulated after the injection of rapamycin,while the expression of P62 was downregulated by treatment with rapamycin.However,the effects of rapamycin on Beclin-1,LC-3II and P62 were reversed by 3-MA.2.Inhibition of NLRP3 inflammasome signaling pathway relieves cancer-induced bone painMethodsExperiment 1: We randomly divided female Sprague-Dawley rats into three groups: na(?)ve group,sham group and CIBP group.The MPWT was respectively measured prior to and day 3,7,14 and 21 after TCI.Western blot and immunofluorescence were utilized to explore the expression and distribution of spinal NLRP3,ASC,Caspase-1 and IL-1?.Experiment 2: We randomly divided female Sprague-Dawley rats into four groups: sham + vehicle group,sham + MCC950 group,CIBP + vehicle group and CIBP + MCC950 group.To determine the role of NLRP3 inflammasome involving in CIBP rats,MCC950(1,5,10 mg/kg),a selective inhibitor of NLRP3,was intraperitoneally injected at day 14.Then,MCC950 was continuously administrated once daily from day 14 to day 18 to investigate the cumulative analgesic effect.Western blot and immunofluorescence were utilized to detect the expression and distribution of spinal NLRP3,ASC,Caspase-1 and IL-1?.ResultsCIBP-related behaviors were tested after MCC950 injection.The treatment with MCC950(5,10 mg/kg,i.p)significantly attenuated MPWT of CIBP group,while the dose of 1 mg/kg had no obvious effect.Repetitive injection of MCC950 at the dose of 5 mg/kg and 10 mg/kg from day 14 to day 18 significantly alleviated mechanical allodynia after TCI.However,the dose of 1 mg/kg had no effect on MPWT of CIBP rats.After TCI,the protein expression of spinal NLRP3,ASC,Caspase-1 and IL-1? were all increased.However,chronic treatment with MCC950 significantly suppressed the protein expression of NLRP3 inflammasome.3.Activation of autophagy attenuates cancer-induced bone pain by inhibiting the NLRP3 inflammasome signaling pathwayMethodsFemale Sprague-Dawley rats were randomly divided into four groups: sham + vehicle group,CIBP + vehicle group,CIBP + Rap group and CIBP + 3-MA + Rap group.To confirm the relationship between autophagy and NLRP3 inflammasome,Western blot and immunofluorescence were used to evaluate the expression and distribution of spinal NLRP3,ASC,Caspase-1 and IL-1?.ResultsThe expression levels of NLRP3,ASC,Caspase-1 and IL-1? were downregulated by chronic injection of rapamycin,while the effects were abolished by 3-MA.Moreover,the immunofluorescence data were consistent with Western blot.4.Statistical analysisAll data were presented as mean ± SEM and analyzed by the Graph Pad Prism.One-way analysis of variance(ANOVA)was used to analyze western blot data,followed by Bonferroni post hoc test.Two-way Repeated Measures ANOVA was used to analyze the behavioral data,followed by Bonferroni post hoc test.Statistical significance was indicated by p<0.05.Conclusions1.Rapamycin,the agonist of autophagy,could obviously relieve CIBP.2.NLRP3 inflammasome signaling pathway mediated the occurrence and development of CIBP.3.Rapamycin attenuated CIBP by inhibiting NLRP3 signaling pathway.SignificanceIn this study,autophagy was demonstrated to involve in the pathogenesis of CIBP,which could be considered as a novel target for alleviating CIBP.MCC950,a selective inhibitor of NLRP3,significantly attenuated CIBP through downregulating the NLRP3 inflammasome signaling pathway.Rapamycin could modulate the expression level of NLRP3 inflammasome signaling pathway.However,a further study was needed to confirm whether NLRP3 inflammasomes could regulate the level of autophagy in CIBP.