The Effect And Mechanism Of Autophagy In Astrocytes During Neuropathic Pain

BackgroundNeuropathic pain is a common type of chronic pain,which can hardly treated in clinical.Recent studies suggest that glial cells play an important role in the development of neuropathic pain.Astrocytes are the most abundant glial cells in the central nervous system(CNS),and reponsible for support,separation and nutrition of neurons.Astrocytes are also involved in the formation of the blood-brain barrier and synapses in central nervous system,which play a key role in maintaining homeostasis and protecting neurons from oxidative stress.Astrocytes are activated after peripheral nerve injury and play an important role in maintianing neuropathic pain.On one hand,activated astrocytes release various cytokines,such as CCL7,MMP2,which promote microglia activation and pain sensitization;on the other hand,because of the damage of blood brain barrier,many non neural cells,including T cells,B cells and macrophage cells intrude into CNS,which aggravate neuro-inflammation and pain sensitization.Autophagy is an evolutionarily conserved biological process and plays an important role in physiological and pathological condition.Autophagy is closely related with immune inflammation and oxidative stress.Studies show that autophagy is involved in antigen presentation and immune inflammation.Autophagy also could inhibit inflammatory reaction by removing damaged protein and organella.Autophagy is involved in differentiation and activation of astrocytes and impaired autophagy in astrocytes mediates the formation of various diseases.Inflammatory stimulation changes mitochondrial dynamic in astrocyte,and impaired autophagy affects mitochondrial function recovery.Whether autophagy in astrocytes is involved in the ocurrence and development of neuropathic pain and the related mechanisms remain to be proved.ObjectiveTo study the role and mechanism of autophagy in astrocytes in the development of neuropathic pain.MethodsL5 spinal nerve ligation(SNL)was operated to induced neuropathic pain in C57 mice,and the mechanical allodynia and thermal hyperalgesia was measured using the von Frey fiber needle and Hargreaves radiation calorimeter.The level of neuro-inflammation and ROS was measured through ELISA assay and immunofluorescence of 8-HOG,respectively.Western blot and immunofluorescence single or double staining were used to detect the changes of LC3-? and P62 in spinal cord after SNL.Autophagy inhibition test was used to test autophagy flux.Intrathecal injection of autophagy inhibitor(3-mA)or autophagy inducer(rapamycin)at the early stage and late stage of neuropathic pain to detect the effect of autophagy on the level of neuro-inflammation and ROS.Through combination the cell experiment and in vivo experience to further explore effect and related molecular mechanism of astrocyte autophagy on neuro-inflammation and ROS.Results1)The thresholds of mechanical allodynia and thermal hyperalgesia decreased significally in mouse after SNL.What's more,the expression level of IL-1?,TNF-?,CCL7,MMP2 and ROS were increased markedly in spinal and the ROS indicator 8-HOG was mainly colocalized with the neuronal marker NeuN.2)The western blot and immunofluorescence results showed that LC3-?in spinal cord were significantly increased,reaching peak on day 3 after SNL and returning to normal levels on day 14 after SNL.However,the p62 level continuously increased after SNL,which maintains at least 28 days.Our electron microscopy results showed that the number of autophagosomes increased significantly in SNL3 d spinal cord tissue,while the number of lysosome was not.On day 14 after SNL,the number of autophagosomes in spinal cord was rarely,while lysosomes increased.Autophagy inhibition test showed autophagy flux impaired in SNL14 d spinal cord tissue.LC3-? was mainly expressed in neurons on day 3 after SNL,and p62 was mainly expressed in the astrocytes,which indicated that impaired autophagic flux was time specific and cell specific in spinal cord.3)Intrathecal injection of autophagy inhibitor(3-MA)aggravated neuropathic pain,and intrathecal injection of autophagy inducer(Rapamycin)alleviated neuropathic pain both in the early stage or late stage,however,inhibition or activation of autophagy at different stages of neuropathic pain had different effect on neuro-inflammation and ROS.In the early stage of neuropathic pain,activation of autophagy reduced IL-1?,TNF-? in spinal cord,but increased CCL7 and MMP2 level;inhibition of autophagy increased IL-1?,TNF-? level,but had no effect on CCL7,MMP2 levels.In the late stages of neuropathic pain,inhibition of autophagy increased levels of neuro-inflammation and ROS,however,activation of autophagy but had no effect on the level of ROS level,although decreased the neuro-inflammation level.4)We found that NRF2 is at least partly involved in the regulation of autophagy on ROS,because at late stage of neuropathic pain,rapamycin treatment significantly reduced NRF2 levels in spinal cord.Our cell tests also showed that autophagy activation reduced NRF2 level in primary astrocytes.Activation of NRF2 by inhibiting Keap1 significantly decreased ROS level and increase the threshold of neuropathic pain in rapamycin treatment group,but had no effect on the pain threshold of DMSO group.5)Cell experiments showed that inhibition of autophagy decreased levels of CCL7,MMP2 at the early satge of IL-1?,TNF-? stimulation,but increased the levels of CCL7,MMP2 at the late satge of IL-1?,TNF-? stimulation.JNK/NF-?B pathway,but not p38/ERK pathway mediates the increase of CCL7,MMP2 induced by autophagy inhibition at the late satge of IL-1?,TNF-? stimulation,because inhibition of autophagy increased the phosphorylation of JNK and NF-?B but not ERK and p38.What's more,the inhibition of JNK,NF-?B rather than ERK,p38 could significantly reverse the increase of CCL7 and MMP2 mediated by autophagy inhibition.Conclusion1)Impaired autophagy flux in spinal cord was time specific and cell-type specific after SNL.2)Inhibition or activation of autophagy aggravated or allevated neuropathic pain,respectively,both at the early and late stage.3)Inhibition or activation of autophagy at different stages of neuropathic pain had different effect on neuro-inflammation and ROS.4)NRF2 pathway in astrocytes was at least partly involved in the regulation of autophagy on ROS.5)In astrocytes,it was JNK/NF-?B pathway,but not p38/ERK pathway mediated the increased level of CCL7,MMP2 induced by autophagy inhibition at the late satge of IL-1?,TNF-? stimulation.