| Objective:Pain is an uncomfortable emotional experience.According to the duration of pain,it can be considered as acute pain and chronic pain.If a variety of diseases and drugs,as well as other factors,can damage the nervous system(including peripheral and central nervous systems)and result in dysfunctions,we call them neuropathic pain.Neuropathic pain is known as a chronic pain which usually manifests a series of syndromes for the patients.The diversity of the neuropathic pain is shown by the various natures of pain and the changes of pain thresholds and the complexity can be shown by sensory disturbances as well as movement disorders.Both of them are due to the complexity of the pathogenesis of neuropathic pain.The current research cannot fully explain the pathogenesis,so there are less effective treatment strategies and methods.On the one hand,patients have to suffer much pain in body;on the other hand,they also suffer great mental torture,which will greatly attenuate their work efficiency and quality of life.Furthermore,the complexity and difficulty to treat the neuropathic pain bring huge mental and economic burden to the patients as well as their families,which may add tremendous pressure to the society,especially the health resources and the human resources,and may increase the burden of nursing personnel,forming a vicious spiral and leading to more mental pain and body pain.Since the first discovery of the AQP family in 1988,more and more scientists have paid great attention to its importance as a mediator and controller on water molecules.Neuroedema is one of the most important manifestations of nerve injury,and it influences the process of nerve restoring its own function.In recent years,the research on AQP4 has become the focus,especially in the field of neuroscience.As research progressed,it was gradually found that AQP4 also had a large number of functions beyond water balance.It may change the structure of astrocytes and the connection between astrocytes,and may regulate its functions,such as the migration of astrocytes.In addition,AQP4 also acts on the spontaneous or subsequent increment of astrocytes and the process of scar formation after injury.Some studies show that AQP4 also plays a role in the complex neuroendocrine system.For a long time,we have found that in the central nervous system,there are more glial cells than neurons,but we know less functions of them compare to neurons.They do not perform the function of communicating,but play secondary roles such as nutritional support.The most important functions are performed by neurons.So we’ve always took for granted that only neurons are responsible for pain,but many studies have shown that previous theories cannot explain all pains,which made many scientists to research deeper into the field of pain.They found that glial cells do not act as a spectator,but played a very important role.For example,glial cells can up-regulate or down-regulate the function of neurons in a variety of ways.Among glial cells,astrocytes and microglia are more representative.According to the current research,the activated glial cells play an important role in the whole process of neuropathic pain.Without them,the whole process cannot go smoothly.That is to say,if we can understand how the cells related to pathological pain(including activated astrocytes and microglia)work,it will be an important finding for the study of pathological pain.The activation of glial cells is a very complex process,the surface of astrocytes exists a large number of ion channels and receptors,these receptors may be activated by interleukin-1 which is released by some damaged neurons;some may be activated by degraded products of damaged neurons.These are the important processes after astrocytes activated by stimulation,and some receptors can alter excitation threshold of astrocyte activation,indirectly involved in the process.Many ion channels which can change the concentration of various ions inside and outside the cell,resulting in the response to various neurotransmitters.These series of ion channels and receptors are activated,are not a simple separate arrangement,but need a complete information system,which can act as a bridge to connect all the related factors,signal transduction pathway have this ability precisely.Mitogen activated protein kinase(mitogen-activated protein,kinases,MAPKs)is particularly important for information transmission in cell,different members of the MAPKs family,such as extracellular regulated kinase(extracellular signal-regulated kinase,ERK),c-Jun N-terminal kinase(c-Jun N-terminal,kinase,JNK)and p38 signaling pathway,can change the varies extracellular stimulation into the corresponding intracellular forms and cause further reaction,such as development and differentiation.Previous studies have shown that activation of MAPKs plays a crucial role in the occurrence,development and continuous complex process of neural structure,and this activation is mainly concentrated in neurons in posterior horn of spinal cord.In recent years,studies and reports on AQP4 mainly concentrate on the brain,but there are less studies on spinal cord and spinal ganglia;there are studies on the effects of AQP4 and MAPK signal pathway in the activation of glial cells and inflammatory factors expression,but the correlation between AQP4 and phosphorylation of MAPK signaling pathway in peripheral nerve injury is not clear,much less of the ERK,JNK,p38 signal pathway research.This paper use the model of chronic constriction injury of the sciatic nerve,apply animal behavior,immunofluorescence and other methods,from feeling,movement,expression,inflammatory factors and other aspects,made a systematic study of the relationship between AQP4 and the phosphorylation of MAPK signaling pathway after peripheral nerve injury,and provide a theoretical basis for clinical treatment on peripheral nerve injury.Methods: animal behavior: application of heat pain stimulation and mechanical pain mensuration detection the recovery of nerve function after giving TGN-020,a AQP4 inhibitor.Vivo experiment: According to the literature,make chronic constriction injury(CCI)model of Sprague Dawley mouse sciatic nerve by the classical method.The intervention method:giving TGN-020 in abdomen,because DMSO is the dissolving,the same concentration of DMSO is used as the control group.The tissue was the L4,L5 spinal ganglion and spinal cord of the corresponding segment of the sciatic nerve.Application of Western blot method,on the 1st,4th,7th,14 th,21st day after nerve injury,detect activation degree,expression quantity and the trend of p-ERK,p-JNK,p-p38 and GFAP which are the activation product of MAPK signaling pathway after AQP4 stimulation;detect activation and expression of p-ERK,which is the activation products of the MAPK signaling pathway after AQP4 stimulation in glial cell and neun by immunology chemical fluorescence double staining method;detect the expression of relevant inflammatory factors such as TNF,IL-6 and IL-1 p using ELISA method.Statistical analysis: the data analysis was carried out by Graph Pad Prism5 software,and all the data were expressed by mean number+-standard deviation.If there is a comparison between multiple groups,the single factor variance analysis is applied.If two groups are compared,Bonferroni test is applied.If P<0.05,the difference is statistically significant.Results:As the immunofluorescence staining showed that the number of co-location of p-ERK and Neu N positive cells increased significantly after injury,and significantly decreased after TGN-020 treatment,revealing that TGN-020 could alleviate the negative reaction of neurons after nerve injury.For the mechanism of pathological pain,the results of ELISA showed levels of IL-6 and IL-1 beta,TNF alpha were increased after injury,indicating that inhibition of AQP4 can reduce the expression of inflammatory cytokines induced by injury;Western blot showed the expression of GFAP protein was significantly increased,and ERK,JNK,p38 pathway were activated;at the same time,fluorescent staining was also found GFAP protein,the marker of astrocytes in spinal cord and satellite glial cells in dorsal root ganglion,was strongly positive with the cell body became hypertrophy.TGN-020 can inhibit the expression of AQP4,weaken the activation of glial cells induced by nerve injury,and affect the activation of ERK,JNK and p38 signaling pathways.We also found that the role of ERK was the most obvious in each signal pathway,and on the basis of this,immunofluorescence double staining showed that the number of cells expressing p-ERK and GFAP at the same time increased obviously after injury,and could be significantly reduced by TGN-020.The results of animal behavior test showed that TGN-020 alleviated the abnormal pain sensitivity resulting from nerve injury,which made the nerve function easier to recover.Conclusion:1、Inhibition of AQP4 attenuated the neuropathic pain induced by the sciatic nerve injury.2、Inhibition of AQP4 reduced the expression of inflammatory factors,astrocyte activation and MAPKs signaling pathway in dorsal root ganglion and spinal dorsal horn,thereby improving neurological function.3、Inhibition of AQP4 decreased the satellite glial cells activation via inhibition of ERK pathway in dorsal root ganglion and spinal dorsal horn,so as to attenuate neuropathic pain.4、Inhibition of AQP4 reduced neuropathic pain according to inhibiting the activation of the ERK pathway in the dorsal root ganglion nerve and the posterior horn of the spinal cord. |
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