P2X7 Receptor Mediated NLRP3 Inflammasomes Regulating Microglia M1/M2 Polarization In Bone Cancer Pain Model

Background:Bone cancer pain(BCP)is the most severe and intractable type of cancer pain,which can seriously affect patient’s quality of life and shortens the survival time of patients.To date,the avaliable cancer pain treatment strategies follows the international three ladder analgesic regimen.Opioid analgesic is most commonly used in advanced cancer,but with great individual differences and poor long-term analgesic effect.The main reason is the complexity of the pathogenesis of bone cancer pain.Therefore,it is an urgent problem to solve the pathogenesis of BCP and seek effective and targeted prevention and treatment methods.Microglia play an important role in the central sensitization of chronic pain.Recent studies have shown that activated microglia could release a series of neurotoxic substances to stimulate neurons and form neuronal sensitization.Microglia have strong plasticity and induce two polarized phenotypes,pro-inflammatory(M1)phenotype and anti-inflammatory(M2)phenotype in response to stimuli.Multiple proinflammatory cytokines secreted by M1 microglia activate neighboring neurons resulting in central sensitization,while M2 microglia subsequently relieve pain by having secreting antiinflammatory cytokines that suppress inflammatory respones and mediate restoration of immune homeostasis.Studies have shown that promoting microglial polarization to M2 in the occurrence of multiple chronic pain,including BCP,can effectively provide pain relief,but the specific targets are not clearly defined.The P2X7 receptor(P2X7R)is a non-selective ATP gated ion channel predominantely present in microglia in the spinal cord.Studies have demonstated that P2X7R was involved in the regulation of microglial M1/M2 phenotype transformation in an ischemic pain model.P2X7R activation causes K+outflow and Ca2+ influx,where K+outflow activates the NLRP3 inflammasomes,and Ca2+ influx can activate related signaling pathways such as MAPK to promote the nuclear displacement of NF-κB to further promote the transcription of the NLRP3 inflammasomes.Recently,it has been shown that overexpressed NLRP3 inflammasomes in models of central nervous system can promote microglia polarization to type M1,which in turn exacerbates the inflammatory response.However,it has not been reported whether P2X7R promotes the M1/M2 phenotype transformation by regulating the NLRP3 inflammasomes and then participates in the central sensitization of BCP.To investigate whether suppression of P2X7R/NLRP3 signaling can alleviate BCP by modulating M1 to M2 polarization,we used the breast cancer induced bone pain female rat models and the co-stimulating B V2 microglial activation model to investigate whether suppression of P2X7R/NLRP3 signaling could alleviate BCP by modulating M1 to M2 polarization.Objective:Based on the BCP rat model by directly injecting Walker 256 breast cancer cells into the tibia to investigate whether suppression of P2X7R could alleviate BCP by modulating M1 to M2 polarization;LPS and BzATP co-stimulating BV2 microglial activation model to investigate whether P2X7R participates in the central sensitization by promoting spinal microglia to M1 type polarization.Methods:Part 1:P2X7 receptor regulates microglia to M1 phenotype involved in central sensitization of bone cancer painResearch methodsExperiment 1:Bone cancer pain model was established by injecting Walker-256 breast cancer cells into the tibia in female rats.32 healthy SPF Sprague-Dawley(SD)female rats were randomized into four groups(8 per group):Sham group,bone cancer pain 7 day group(BCP 7d),bone cancer pain 14 day group(BCP 14d),bone cancer pain 21 day group(BCP 21d).The test indexes include:(1)bone destruction and tumor cell infiltration by tibia X-ray on days 7,14 and 21 after surgery;(2)50%Paw withdrawal thresholds(50%PWTs)、number of spontaneous flinches(NSF)、limb use score was measured one day before and 4,7,11,14,17,21 days after modeling to assess the behavior of bone cancer pain in rats;(3)Western blot to determine protein levels of CD86,iNOs and M2 markers CD 163 and Arg-1 in microglia in spinal cord tissue;(4)Immunofluorescence staining technology to detect the localization of P2X7R in the spinal cord tissue.(5)P2X7R expression in spinal cord tissue was determined by immunofluorescence staining and Western blot.Experiment 2:Based on the BCP model,the intraperitoneal injection of P2X7R specific antagonist BBG to evaluate the role of P2X7R in regulating microglial M1/M2 phenotype transformation in bone cancer pain.Thirty-two healthy adult SPF female rats were randomized into four groups(8 of each group):Sham+NS group,Sham+BBG group,BCP+NS group,or BCP+BBG group.Rats in the Sham+BBG and BCP+BBG groups were intraperitoneally administered with BBG(50mg/kg)from day 7 to day 21 at 8 am,once daily.The detection indexes include:(1)One day before modeling and 4,7,11,14,17,21(days)after modeling,50%PWTs,NSF,and limb use score were measured to evaluate the effect of BBG on pain behavior in rats;(2)Western blot for protein levels of CD86,iNOs,TNF-α and IL-18,M1 markers CD163,Arg-1 and anti-inflammatory factors IL-4 and IL-10 to analyze the effects of BBG on spinal cord microglia polarization.Experiment 3:Establish of LPS and BzATP co-stimulate BV2 microglia activation model,and a P2X7RsiRNA intervention model to study whether P2X7R could regulate the polarization of BV2 microglia to M1 phenotype.Experimental groups:Control-NC siRNA group,Control-P2X7R siRNA group,LPS+BzATP+NC siRNA group,and LPS+BzATP+P2X7R siRNA group.The detection indexes include:(1)The protein levels of BV2 microglia M1 markers CD86,iNOs and M2 markers CD163,Arg-1 by Western blot.M1-related inflammatory factors TNF-α,IL-18 and M2-related antiinflammatory factors IL-4,IL-10 mRNA levels by RT-PCR.Which was used to analyze the effect of P2X7R on BV2 microglia polarization.Results:1.The rat bone cancer pain model was successfully established,and the BCP spinal cord dorsal horn microglia were polarized to M1 phenotype.(1)X-ray showed that bone destruction and tumor cell infiltration were observed in the BCP group on day 14 after Walker 256 cell injection.On the day 21,cancer cells completely infiltrated the bone marrow cavity by day and the tibia was severely damaged.The tibia of the Sham group rats did not show any tumor infiltration and bone destruction.(2)Animal behavioral hypersensitivities were evaluated from three perspectives.50%PWTs was significantly decreased on days 7,11,14,17,21 after surgery(p<0.05)compared to the Sham group.Besides,higher NSF level and hind limb use score in the BCP group,and significantly higher than the sham group on days 7,11,11,14,17,21(p<0.05).(3)Western blot results showed that the M1 markers CD86 and iNOs were significantly elevated on BCP14d,while the M2-markers CD 163 and Arg-1 were significantly suppressed.1.4 Immunofluorescence staining revealed that most P2X7R co-localized with marker Iba-1 positive cells of microglia in the dorsal horn of the ipsilateral spinal cord but not with astrocyte and neuron marker.1.5 Immunofluorescence staining and Western blot results showed that the levels of P2X7R in the dorsal horn of the spinal cord was upregulated over time in the BCP group when compared to the Sham group.2.The P2X7R-specific antagonist,BBG,could effectively alleviated BCP and promoted microglia polarized to M2 phenotype.(1)BBG treatment significantly elevated 50%PWTs on days 7,11,14,21(p<0.05);NSF level and limb use score in BBG group were significantly lower than in the BCP group(p<0.05).(2)Western blot results showed that BBG significantly suppressed the expression of the M1 markers CD86,iNOs and pro-inflammatory factors TNF-α,IL-18.Besides,M2 markers CD 163,Arg-1 and the anti-inflammatory factors IL-10 and IL-4 were significantly elevated in the BBG treatment group compared to the BCP group.3.P2X7R siRNA inhibited LPS and BzATP-induced microglia polarization towards to M1 phenotype and promoted microglia polarization towards to M2 phenotype in BV2 cell lines(1)Western blot showed that co-stimulation with LPS and BzATP significantly elevated the expression of M1 phenotypic markers iNOs and CD86.However,expression levels of M2 markers CD163 and Arg-1 were suppressed(p<0.05);Pretreatment with P2X7R siRNA reversed LPS and BzATP-induced changes in protein levels of CD86,iNOs,CD163 and Arg-1(p<0.05).(2)The RT-PCR assay showed that co-stimulation with LPS and BzATP significantly elevated the expression M1-associated proinflammatory factors TNF-α and IL-18,inhibition of M2-related anti-inflammatory factors IL-10 and IL-4 mRNA levels(p<0.05);Pretreatment with P2X7R siRNA could reversed LPS and BzATP-induced changes in mRNA levels of TNF-α,IL-18,IL-4 and IL-10(p<0.05).Conclusion:In cancer-induced bone pain model and the LPS and BzATP co-stimulated BV2 microglial activation model,P2X7R activation promotes microglia to type M1 polarization involved in the central sensitization of bone cancer pain.Part 2:P2X7 receptor regulates microglia to M1 polarization involved in central sensitization of bone cancer pain by activating NLRP3 inflammasomesObjective:Based on the BCP rat model and LPS and BzATP co-stimulating BV2 microglial activation model to investigate whether P2X7R receptor is involved in the central sensitization of bone cancer pain by activating NLRP3 inflammasomes and promoting spinal microglia to M1 type polarization.Methods:Experiment 1:Based on the BCP model,BBG was injected intraperitoneally to detect the expression of NLRP3 inflammasomes-associated protein in spinal cord tissue,and then to determine whether P2X7R can regulate the expression of NLRP3 inflammasomes in the BCP model.This experiment utilized the spinal cord tissue in Part 1,Experiment 2.The detection indexes include:Western blot for detecting the protein expression of NLRP3,ASC and Caspase-1,an important component of the NLRP3 inflammasomes in the spinal cord tissue.Experiment 2:Establish a BV2 microglial activation model of LPS and BzATP and a P2X7RsiRNA intervention model to study whether P2X7R can regulate the expression of NLRP3 inflammasomes in BV2 cells.Experimental groups:Control-NC siRNA group,Control-P2X7R siRNA group,LPS+BzATP+NC siRNA group,and LPS+BzATP+P2X7R siRNA group.The detection indexes include:Western blot to detect the protein expression of NLRP3,ASC and Caspase-1 in BV2 microglia.Experiment 3:Based on the BV2 microglial activation model,P2X7R regulated BV2 microglial polarization by activating NLRP3 inflammasomes by co-treatment of P2X7RsiRNA with the agonist NLRP3 inflammasin(Nigericin).Experimental groups:Control group,LPS+BzATP group,LPS+BzATP+siRNA group,LPS+BzATP+siRNA+N group(Nigericin:NLRP3-specific agonism).The detection indexes include:(1)Western blot detects the protein expression of P2X7R,NLRP3,ASC and Caspase-1 in BV2 microglia;(2)Protein levels of the M1 markers CD86,iNOs,and the M2 markers CD163,and Arg-1 in BV2 microglia were determined by Western blot;(3)The levels of M1 associated proinflammatory factors,TNF-α、IL-18 and M2 related antiinflammatory factors IL-4、IL-10mRNA were determined by RT-PCR.Results:1.The P2X7R-specific antagonist BBG significantly downregulated the expression of the NLRP3 inflammasomes in spinal cord tissues.The Western blot results showed that the expression of the NLRP3,ASC and Caspase-1 were significantly increased in the BCP group.However,the level of NLRP3,ASC,and Caspase-1 in the spinal cord tissue were significantly lower in the BBG treatment group than in the BCP group(p<0.05).2.In BV2 microglia,P2X7R regulates the expression of the NLRP3 inflammasomes.Western blot results showed that the expression levels of P2X7R,NLRP3,ASC and caspase-1 proteins were higher in BV2 cells treated with LPS+BzATP than the PBS group.Result also showed that P2X7R siRNA can significantly reduce the expression levels of the above proteins in BV2 cells treated with LPS and BzATP.3.P2X7R regulates the transformation of BV2 microglia to the M1 type by activating the downstream NLRP3 inflammasomes.(1)Western blot results showed that the NLRP3 inflammasomes-specific agonist Nigericin can effectively reverse the expression of NLRP3,ASC and Caspase-1 protein levels in BV2 cells treated with LPS and BzATP compared to P2X7R siRNA(p<0.05).(2)Western blot results showed that Nigericin could reverse the expression of CD86,iNOs,CD163 and Arg-1 protein levels in BV2 cells treated with LPS and BzATP compared to P2X7siRNA(p<0.05).(3)The RT-PCR test showed that Nigericin reversed the P2X7RsiRNA-elicited changes in TNF-α,IL-18,IL-4,and IL-10 in LPS and BzATP exposed BV2 cells(p<0.05).Conclusion:In this study,a BCP rat model was established to confirm that the P2X7R of spinal microglia participates in the central sensitization of BCP by activating the NLRP3 inflammasomes and promoting the M1-type polarization of spinal microglia.Also we used LPS and BzATP co-stimulating BV2 microglial activation model to further verified that P2X7R receptor promotes spinal microglia to M1 type polarization by activating NLRP3 inflammasomes.In conclusion,P2X7R participate in the central sensitization of bone cancer pain by activating the NLRP3 inflammasomes and promoting the M1-type polarization of spinal microglia.Therefore,P2X7R/NLRP3 is likely to be an effective target for the prevention and treatment of bone cancer pain.