The Role And Mechanism Of AMPK Regulating Spinal Cord Inflammatory Response And Autophagy Signaling In Bone Cancer Pain

Ob Jective: Cancer is one of the diseases with the highest incidence in the world.Cancer cells can destroy the structure of bone tissue,cause sensitization of the central nervous system and produce severe pain.The aim of this study was to explore the role and mechanism of AMPK in cancer-induced bone pain（CIBP）,to further analyze the analgesic effect of AMPK activator in CIBP,and to provide research strategies and potential targets for the treatment of cancer pain.Methods: A CIBP model was established by in Jecting MRMT-1 breast cancer cells into the bone marrow cavity of the tibia of male SD rats.Behavioral（mechanical pain）examination,imaging（X-ray）and morphological（HE staining）analysis were used to evaluate the success of the model.After the CIBP model was established successfully,the AMPK activator AICAR was in Jected intrathecally,and the threshold of 50% retraction was detected after 0 h,3h and 6h of administration.After behavioral tests,animals were sacrificed,and spinal cord tissues were collected for fixed sections and protein extraction,and tissue morphology and protein expression were further analyzed.The inflammatory infiltration of the spinal cord was analyzed by HE staining;the activation status of glial cells was determined by immunofluorescence;the AMPK level in the spinal cord was validated by proteomic analysis;the analgesic effect of AICAR was determined by behavioral test;the mitochondrial damage was observed by transmission electron microscopy;the expression levels of mitochondrial-related proteins,NLRP3 inflammatory signal components and autophagy-related proteins were analyzed by immunofluorescence and immunoblot.Cell-level analysis of the protective effect of AICAR on inflammation-induced mitochondrial damage.Western blot was used to detect protein levels in each group,mitochondrial membrane potential at cell level was analyzed by mitochondrial membrane potential kit,and intracellular reactive oxygen species level was detected by reactive oxygen species detection kit.Results:（1）The CIBP rat model was successfully established: the mechanical pain threshold of CIBP rats was reduced,the bone tissue was damaged by X-ray,and the bone trabecular loss by HE staining;（2）the spinal cord inflammation of CIBP rats was increased with the activation of glial cells;（3）the effect of changes in AMPK/Drp1 pathway activity on mitochondria in CIBP rats was decreased;（4）Increased NLRP3 inflammasome-mediated inflammatory signaling in CIBP rats: increased expression of NLRP3,Activated-caspase-1,and IL-1β;（5）Increased autophagy signaling in CIBP rats: increased expression of Parin,ASC,PINK1,LC3 B,p62,TOM20,and Cytochromoe c;intrathecal in Jection of AMPK-specific activator AICAR increases mechanical pain threshold,inhibits glial activation,decreases Drp1 mitotic activity,and downregulates spinal cord inflammation and autophagy signaling in CIBP rats.Conclusion: AMPK-specific agonist AICAR can reduce Drp1-mediated mitochondrial damage and dysfunction,and further down-regulate inflammatory and autophagy signals,thus alleviating the algesic behavior and exerting analgesic effect in CIBP rats.