Study On The Effects Of S100A4 In Neuropathic Pain Of Rats

Objective:In clinical practice,pain is accompanied by various diseases,which is a complex physical and mental experience.It includes the sensation and the reaction caused by nociceptive stimulus.Pain can be considered as a warning sign that the body has been harmed,thus causing a series of defensive protective responses.But sometimes the pain gets out of control.Some long-term and severe pain makes the body overwhelmed,such as the neuropathic pain.Neuropathic pain is triggered by pathological changes in the somatosensory nervous system.As this change alters the structure and function of this system,pain occurs spontaneously,and the response to harmful and harmless stimuli gets magnified pathologically.At present,in the research into mechanisms of neuropathic pain,some important ones have been revealed such as NMDA receptors,inflammatory mediators,the balance of spinal opioids and antagonistic systems,central sensitization,central deinhibition,and spinal glial cells activation.The S100 protein contains at least 25 members,which constitutes the largest EF-hand signaling proteome in the human body.They were initially classified as an abundant set of low molecular?10-12 k Da?acidic proteins that are highly enriched in neural tissue.In cells,S100 protein has a variety of targets.Currently known are various enzymes,receptors,cytoskeleton and transcription factors,etc.,which can participate in various aspects of proliferation,differentiation,apoptosis,Ca²⁺regulation,etc.,and can maintain cell homeostasis and participate in energy metabolism plays an important role in the intracellular processes of inflammation and tumor invasion.Extracellular S100 protein is capable of acting on a variety of cells,including monocytes,macrophages,neutrophils,lymphocytes and mast cells in the immune system,neurons,astrocytes,microglia and Schwann cells in the nervous system,myoblasts and chondrocytes in the articulation system,endothelial cells,vascular smooth muscle cells,epithelial cells and myocardial cells in the circulatory system,so they can regulate the immune response,inflammatory cell migration and trend chemical properties,affect tissue development and repair,and promote tumor cell invasion.S100A4 is one of the danger-associated molecular patterns.There are many related receptors for inflammation-related signal transduction,such as Toll-like receptor 4,the advanced glycation end product receptor?RAGE?of the MAPK pathway,and the interleukin?IL?-10 receptor of the STAT pathway.It can activate these receptors,causing the corresponding cells to release inflammatory factors such as IL-1?,IL-6,TNF-?.Vitro experiments have confirmed that extracellular oligomeric S100A4 is an effective factor for neurite growth and primary neuron survival.S100A4 maintains a high level of expression in nerve growth and injury sites,suggesting that the protein effects in neuronal plasticity.Extracellular S100A4 can protect neurons by up-regulating multiple antioxidant enzymes and neuroprotective genes via the IL-10R/JAK/STAT3 pathway.It may also indirectly activate Erb B receptor triggering signals to induce neuroprotection via JAK/STAT,ERK and Akt.The increased expression of S100A4 in astrocytes after spinal cord injury may indicate that this molecule plays a role in the response of astrocytes to injury.In the dorsal root ganglion,S100A4?+?cells seem to constitute a subpopulation of neurons,some of which co-express calcitonin gene-related peptide?CGRP?and Isolectin B4?IB4?.Peripheral nerve or dorsal root injury induces significant up-regulation of S100A4 expression in satellite cells in ganglia and Schwann cells at the site of the injury and distal stump.S100A4 effects a lot in inflammatory response and nerve injury,these two processes are closely related to the generation of neuropathic pain.We suspect that S100A4 plays a certain role in neuropathic pain.This study main focuses on the expression and function of S100A4 in neuropathic pain animal models,and explored its possible role in neuropathic pain.Methods:This study used a seven-week-old male SD rat to prepare a spinal nerve ligation model?SNL?for experiments.The research material is spinal cord and L5 dorsal root ganglia.Immunofluorescence staining is used to detect the expression position of S100A4in the model and is co-labeled with other related interacting molecules and cell markers to distinguish cell types.Western blot experiments are used to detect changes in S100A4content in the same part under different conditions.Intrathecal injection of neutralizing antibodies was used to interfere with animal models of pain,and behavioral changes were recorded,and molecular changes were compared.Co-immunoprecipitation was used to verify the direct interaction between S100A4 and other molecules in order to explore its possible mechanism.Results:In this study,frozen sections of dorsal root ganglia of rat SNL model were immunofluorescent histochemically stained.We found that at 3d,7d,and 14d after modeling,the fluorescence intensity of the cells expressing s100A4 was not significantly different from that of the control group.On the 7th day after modeling,the number of cells expressing S100A4 was significantly higher than that of the control group.Western blot results showed that S100A4 protein was more than that in the control group after modeling,and the difference was statistically significant.Immunofluorescence staining also found that S100A4 was relatively concentrated in the spinal dorsal horn region.The expression on the operative side?the dominant side of Iba1?was higher than that on the contralateral side.S100A4 at 7d and 14d after SNL had larger fluorescence area than the control group,and the difference in integrated optical density was statistically significant.Western blot experiments supported that the expression of S100A4 protein increased significantly at 7d,14d,21d,and 28d after SNL,and the difference was statistically significant.Immunofluorescence double-labeling staining showed that S100A4 expressing cells in the dorsal root ganglion in the spinal dorsal horn had some co-staining with cells expressing CGRP,IB4 and P2X3,and S100A4 was partially co-stained with GFAP,IB4,and Iba1 in the spinal dorsal horn.The mechanical pain threshold test results in rats showed that there was no change in PWT after surgery.Compared with the control group,the PWT values of the placebo group?SNL/salt?at 1d,3d,5d,and 7d were significantly reduced.At the same time,the PWT of the experimental group?SNL/antibody?was higher than the placebo group?SNL/salt?at 3d,5d,and 7d after surgery,and the difference was statistically significant.In rat SNL,intrathecal injection of S100A4 antibody can increase its mechanical paw withdrawal threshold at 3d,5d,and 7d after surgery.Western blot was used to determine the S100A4 content.The results suggests in DRG,S100A4 in the experimental group?SNL/antibody?and the placebo group?SNL/salt?was higher than the control group?CON?;the experimental group?SNL/antibody?S100A4 protein was significantly lower than the placebo group?SNL/salt?.In the spinal cord,S100A4 in the experimental group?SNL/antibody?and the placebo group?SNL/salt?was also higher than the control group?CON?;When compared with the placebo group?SNL/salt?,S100A4 was lower in the experimental group?SNL/antibody?.This result suggests that intrathecal injection of S100A4 antibody can make S100A4 less in the DRG and spinal cord of the rat SNL model.The results of co-immunoprecipitation?co-ip?experiments supports that S100A4 acts directly on RAGE.At the same time,immunofluorescence staining showed that S100A4 and RAGE had obvious co-staining in the gelatinous region of the spinal dorsal horn.All the results imply that S100A4 participates in the development of neuropathic pain by coworking with RAGE.Conclusion:S100A4 is increased in the DRG and spinal dorsal horn in the rat SNL model.Intrathecal injection of S100A4 neutralizing antibody can effectively relieve mechanical pain in rats.Thence,S100A4 partial impacts the occurrence of neuropathic pain through RAGE.