The Analgesic Effects Of RBmK-AGAP In Rat Bone Cancer Pain Model Via Inhibiting The Activation Of NF-?B Pathway In Spinal Astrocytes

Background and purpose: Cancer pain is one of the most common and unbearable symptoms of cancer patients,which seriously impacts the quality of life of patients.At present,the management of patients with cancer pain is mainly based on the guidelines of the World Health Organization's(WHO)“analgesic ladder”,but 20%-40% patients still have no pain relief.And bone cancer pain is a disease which is the most difficult to treat and control.rBmK-AGAP is a polypeptide monomer purified from scorpion venom,which had been expressed in Escherichia coli using a recombinant method.It has anti-tumor and analgesic effects.But until now there are no research about the rBmK-AGAP's effect to cancer pain.In addition,some previous studies showed that the activation of glial cells and NF-?B signaling pathway are involved in the development and maintenance of bone cancer pain.In this study we aim to explore the analgesic effect of rBmK-AGAP through a pro-clinical bone cancer pain model and whether the mechanism is related to inhibit the activation of glial cells and NF-?B signaling pathway in spinal cord.Methods: 1.Rats were randomly divided into the following four groups: sham operation group(Sham+Veh),sham operation with drug administration group(Sham+AGAP),bone cancer pain group(BCP+Veh),bone cancer pain with drug administration group(BCP+AGAP).According to the previously described method,we did the surgery and successfully established the rat tibia bone cancer pain model.To quantitatively evaluate the rats' pain behaviors,we measured the rats' mechanical threshold and spontaneous limb use score before surgery and on the 3rd,5th,7th,9th,11 th and 13 th day after the surgery.rBmK-AGAP(0.05 mg/kg)was administered intraperitoneally when rats appeared pain behavior.Rat body weight was also measured to reflect the health status of the rat.2.Radiographic images of the left tibia were taken after behavioral testing at day 13,using a digital camera exposure to an X-ray source.It can reflect the degree of bone destruction and demonstrate the success of the rat tibia bone cancer pain model.3.The expression levels of GFAP,Iba-1,P-P65,P65,I?B? and P-IKK? in spinal cord were detected by Western blot and immunofluorescence experiments,as well as fluorescence intensity and phenotypic changes in glial cells to determine the activation of glial cells and NF-?B signaling pathway in spinal cord.Specific cell types activated by the NF-?B signaling pathway were confirmed by immunofluorescence double staining experiments.4.The expression levels of inflammatory cytokine IL-6 and IL-1? were detected by ELISA.5.We culture the primary astrocytes from cerebral cortexes of neonatal rats(24h).we use the western blot and doublestaining method to demonstrate that weather rBmK-AGAP can inhibit the activation of NF-?B signaling pathway in primary astrocytes after the TNF? treatment in vitro.Results: 1.In the rats inoculated with Walker256 cancer cells,the mechanical allodynia and spontaneous limb use score showed a significantly lower at the 7th day post the surgery compared with the Sham+Veh rats and Sham+AGAP rats.Compared with the BCP+Veh group,the rBmK-AGAP group can significantly increase the 50%PWT,with the best analgesic effect at 30 minutes to 1 hour after the intraperitoneal injection.rBmK-AGAP was administered daily by intraperitoneal injection from 7to 13 day after the inoculation.And the result is rBmK-AGAP can attenuate the rats bone cancer pain behavior and does not have the effect to the normal baseline.There was no significant difference in body weight between the four groups.2.X-ray radiographic images of rats' tibia taken at 13 th day which is the end point of the study,and the results showed that bone destruction caused by Walker256 cancer cells compared with normal rats.rBmK-AGAP have no significant effect to the degree of bone destruction through 3-point scale method.3.The result of western blot and immunofluorescence experiments showed that compared with normal rats,the expression level of GFAP and Iba-1 in BCP+Veh group rats,as well as the fluorescence intensity and the number of astrocytes and microglia is increased.rBmK-AGAP can decrease the expression level of GFAP in spinal cord,and inhibit the phenotypic changes of astrocytes.4.rBmK-AGAP can inhibit the expression of P-P65 and P-IKK? in spinal cord,inhibit the NF-?B signaling pathway.Then we demonstrated the specific cell type in spinal cord by double staining experiment.rBmK-AGAP can inhibit the NF-?B signaling pathway in astrocytes and neurons.5.It was confirmed by ELISA that rBmK-AGAP can inhibit the expression levels of inflammatory factors IL-6 and IL-1?.6.We culture the primary astrocytes and demonstrated that rBmK-AGAP can inhibit NF-?B signaling pathway in vitro.Conclusion: This study confirmed that rBmK-AGAP can attenuate the bone cancer pain behavior by rat tibia bone cancer pain model.The mechanism is related to inhibiting the activation of NF-?B signaling pathway and the activation of astrocytes in spinal cord.In addition,we still demonstrated that rBmK-AGAP can inhibit the NF-?B signaling pathway of primary astrocytes in vitro.