Study On The Effect And Mechanism Of Necroptosis In Neuropathic Pain Induced By Paclitaxel

Objective: To observe the role of cell necroptosis in paclitaxel-induced neuropathic pain(PIPN),and further study its related mechanism,providing novel directions and theoretical basis for revealing the pathogenesis and prevention of PIPN.Methods:We established rat PIPN models by intraperitoneal injection of paclitaxel intermittently(2mg/kg,4 times in total with a cumulative dose of 8mg/kg).The pain behavioral assessments were evaluated by mechanical withdrawal threshold(MWT)test and thermal withdrawal latency(TWL)test.Dorsal root ganglion(DRG)and spinal dorsal horn cell necrosis were observed by in vivo propidium iodide(PI)staining and transmission electron microscopy.The expression changes of two proteins related to necroptosis,namely receptor interacting protein kinase 3(RIP3)and mixed family kinase domain-like protein(MLKL),in DRG and spinal cord were detected by Western blot.The spatial expression changes of RIP3 and MLKL in DRG,spinal neurons and glial cells and the M1/M2 type polarization of spinal microglia were observed by double immunofluorescence staining.The levels of inflammatory cytokines such as TNF-α,IL-1β,IL-4,IL-10 in DRG and spinal cord were detected by ELISA.The role of cells necroptosis in DRG and spinal cord in PIPN was observed by using of Nec-1,a specific inhibitor of necroptosis.The effect of macrophages on neuronal necroptosis in DRG of PIPN rats was observed by using of clodronate liposomes,a scavenger of macrophage.The effect of M1/M2 type polarization of microglia on cells necroptosis of spinal dorsal horn in PIPN rats was observed by administration of microglia activation inhibitor minocycline.Results: 1.The quartic paclitaxel administration in rats induced robust hyperalgesia and allodynia with significant neuronal necroptosis in DRG.Paclitaxel treatment increased the expression levels of RIP3 and MLKL which were mainly localized in DRG neurons.Paclitaxel promoted the activation of satellite glial cells and the release of pro-inflammatory factors such as TNF-αand IL-1β.All the changes mentioned above were inhibited by Nec-1treatment,suggesting that DRG neuron necroptosis was related to PIPNs.The administration of clodronate liposomes significantly reversed the macrophage infiltration in DRG,decreased the levels of pro-inflammatory cytokines TNF-α and IL-1β in DRG,reduced the expression levels of RIP3 and MLKL,and inhibited the number of cells necroptosis,and alleviated the abnormal pain behavior induced by paclitaxel.2.Paclitaxel treatment induced severe cells necroptosis in spinal dorsal horn,increased the expressions of RIP3 and MLKL locating in neurons,astrocytes and microglia.Paclitaxel also promoted the releases of pro-inflammatory cytokines TNF-α and IL-1β.All above mentioned changes were inhibited by Nec-1 treatment,suggesting that cells necroptosis in spinal cord was related to the development of PIPN.In addition,paclitaxel induced the M1 type polarization of microglia in spinal dorsal horn,but had no effect on M2 type polarization and anti-inflammatory cytokines IL-4 and IL-10.Minocycline significantly inhibited the M1 type polarization of microglia,decreased the numbers of necroptosis in spinal dorsal horn,and alleviated abnormal pain behavior induced by paclitaxel.Conclusions:1.Paclitaxel promoted macrophage infiltration in DRG and release of pro-inflammatory factors,triggering RIP3/ MLKL-dependent necroptosis of neurons,which was related to the development PIPN.2.Paclitaxel participated in PIPN by promoting the M1 type polari zation of microglia in spinal dorsal horn and releasing pro-inflammatory factors,triggering RIP3/ MLKL-dependent neuronal necroptosis.