Akt-AQP4 Signaling Mediates Neuropathic Pain Induced By Spinal Nerve Ligation In Rats By Activating Astrocytes

Neuropathic Pain(NP)is pain caused by diseases or nerve damage of the somatosensory nervous system.There are many mechanisms for neuropathic pain,including physical and chemical factors such as injury,age,exposure to specific toxins,and infection.Akt signal activation is involved in Bone cancer pain(BCP)in mice and sciatic nerve chronic constriction injury rats pathological pain control.Aquaporin 4(AQP4),as a special channel protein,is mainly expressed in astrocytes and has been reported to be beneficial to the pathogenesis of central nerve injury including neuropathic pain(NP).Here,we aim to verify the role of AQP4 and Akt pathway in neuropathic pain induced by spinal nerve ligation(SNL)and the central sensitization mechanism involved,especially the activation mechanism of astrocytes.To explore whether the Akt pathway is involved in the regulation of AQP4 expression.Objective:To investigate whether Akt-AQP4 signal axis regulates the occurrence and development of neuropathic pain after spinal nerve ligation in rats by activating astrocytes.Methods:1.Rat SNL model was made and the expression and distribution of p-Akt,AQP4 and GFAP were detected by immunofluorescence histochemical staining or western blot.2.Effects of intrathecal injection of different concentration gradients of AQP4 and Akt inhibitors TGN020 and MK-2206 on pain maintenance period of rats in SNL model.3.The effects of AQP4 inhibitor TGN020 on the mRNA expression of the neuronal excitatory marker c-FOS,the activation markers of astrocytes and microglia(GFAP and Iba-1),the pro-inflammatory factor(TNF-α,IL-1β)and the marker of astrocyte derived pain regulator(Connexin 43)were detected by RT-PCR.4.Primary rat lactation astrocytes were cultured in vitro and activated by the proinflammatory factor TNF-α.After the addition of the Akt inhibitor MK-2206,the expressions of Akt,p-Akt,AQP4 and GFAP were detected by western blot to clarify the regulation of Akt on AQP4 and the effect of Akt on the activation of astrocytes.5.The expression of AQP4 in astrocytes of spinal dorsal horn was detected by immunofluorescence double staining.Western blot was used to detect the effect of intrathecal injection of TGN020 on the activation of astrocytes(GFAP).Regulation of AQP4 expression in L4-5 spinal cord segment after intrathecal injection of MK-2206 and its effect on astrocyte activation.Results:In vivo,AQP4 expression was up-regulated in L4-5 spinal cord segment after the establishment of SNL model,In the dorsal horn of the spinal cord,AQP4 is expressed in the dendrites of astrocytes.During the neuropathic pain maintenance period,a single intrathecal administration of AQP4 inhibitor TGN020 could temporarily reverse the neuropathic pain induced by the SNL model.The mRNA levels of neuron excitatory factor(c-FOS),astrocyte activating factor(GFAP),proinflammatory cytokine(TNF-,IL-1β)and astrocyte derived pain regulator(Connexin 43)decreased after continuous injection of TGN020.The mRNA level of microglial activating factor(Iba-1)did not change significantly after continuous injection of TGN020.In addition,SNL also induces increased Akt phosphorylation in L4-5 spinal cord segments.Single intrathecal administration of Akt inhibitor MK-2206 also significantly alleviated the nociceptive state during the maintenance of neuropathic pain.In vitro,TNF-α induced astrocyte activation was accompanied by increased AQP4 and p-Akt expression.Akt inhibitor MK-2206 can regulate the activation of astrocytes by reducing the expression of GFAP and down-regulate the expression of AQP4 in a dose-dependent manner.In the SNL rat model,continuous intrathecal inj ection of TGN020 could also inhibit the activation of astrocytes,while repeated intrathecal injection of MK-2206 could significantly down-regulate the expression of AQP4 and GFAP.Conclusion:We demonstrate that both the AQP4 and Akt signaling pathways are involved in the regulation of neuropathic pain maintenance.The Akt-AQP4 axis can regulate the central sensitization state caused by neuropathic pain,especially the activation state of astrocytes.Interfering with Akt-AQP4 signal axis is a potential effective method for the treatment of neuropathic pain.