Study On The Mechanism Of TDZD-8 Blocking GSK-3β To Relieve Pathological Pai

Objective: To investigate the analgesic effect of the Glycogen Synthase Kinase 3β(GSK-3β)specific inhibitor TDZD-8 in animal models of pathologic pain and its possible mechanism in order to provide potential therapeutic targets and research ideas for the treatment of pathologic pain.Methods:(1)The rat tibia was inoculated with MRMT-1 cells to establish an animal model of cancer-induced bone pain,and TDZD-8 was injected intrathecally.Paw withdrawal threshold(PWT)was used to evaluate pain behavior in rats.The destruction of tibia was observed by X-ray and H&E staining.H&E staining was used to observe the inflammatory infiltration of spinal dorsal horn.The expression changes of GSK-3β,Drp1,GFAP,Iba1,IL-1β and NLRP3 inflammasome related proteins in the spinal cord were detected by immuneofluorescence(IF)and Western blotting(WB).The morphological changes of spinal mitochondria were observed by transmission electron microscopy.The changes of mitochondrial related protein expression in spinal cord were analyzed by proteomics.(2)Neuropathic pain model was established by intraperitoneal injection of OXA;Intrathecal injection of TDZD-8 was used for drug administration;The changes of pain behavior were detected by PWT and spontaneous foot contraction reflex.The expression changes of GSK-3β,Iba1,IL-1β and NLRP3 inflammasome related proteins in spinal cord were detected by IF and WB.(3)The mouse knee joint was injected with CFA to establish inflammatory pain model;TDZD-8 was administrated intraperitoneally.Knee joint morphological changes were measured during the experiment.The changes of pain behavior were detected by PWT,spontaneous foot contraction reflex and rotary rod fatigue.The changes of GSK-3β,GFAP,IL-1β,Bcl-2,Bax,Cyto C and NLRP3 inflammatory bodies in spinal cord were detected by IF and WB.The level of antioxidant Mn-SOD in spinal cord was detected.Additionally,the protective effects of TDZD-8 on TNF-α or IL-1β-induced mitochondrial function damage were investigated in cultured glial cells.Results:(1)Three animal models of neuropathic pain,inflammatory pain and cancerous pain were successfully constructed,and TDZD-8 treatment had significant analgesic effect.(2)TDZD-8 treatment significantly inhibited GSK-3β activity in spinal cord.(3)TDZD-8 alleviated pathological pain by inhibiting inflammatory response that was associated with the activations of spinal cord glial cell and NLRP3 inflammasome.(4)TDZD-8 inhibits oxidative stress response,attenuates mitochondrial division,and reduces mitochondrial permeability to improve mitochondrial function and relieve pathological pain.(5)TDZD-8 inhibited inflammatory factors-induced mitochondrial dysfunction in cultured glial cells.Conclusion: TDZD-8 can effectively relieve pathologic pain by inhibiting the activity of GSK-3β,the mechanism of which may be related to the regulation of glia and NLRP3inflammasome-mediated inflammatory response and attenuation of mitochondrial dysfunction-associated oxidative stress.