Mechanism Of NLRC3 Regulation On Microglia Mediated Inflammatory Response Of After Spinal Cord Injury

Background:Spinal cord injury belongs to central nervous system(CNS)injury diseases,serious secondary injury is one of the main causes of poor recovery of motor function.Targeting on regulation of the occurrence and development of inflammation and promoting the functional recovery after spinal cord injury is of great challenging problem that need to overcome.NLRC3,a member of the NOD-like receptor family,is highly expressed in the cytoplasm of T lymphocytes as an innate immune receptor which inhibits inflammatory response,energy metabolism and other cell life activities through its negative regulatory effects.As CNS innate immune cells,microglia play an important role in balancing inflammation in CNS,however,whether NLRC3 is expressed and plays a role in microglia remains unclear.How NLRC3 regulates inflammatory response after spinal cord injury and its specific molecular mechanisms have not been reported.Further exploration of these questions will help to better understand the role of NLRC3 in the nervous system and the role of microglia in balancing inflammation,which has important theoretical significance and potential value of clinical application for enhancing restoration of spinal cord injury.Objectives:To clarify the expression level of negative regulatory immune receptor NLRC3 in the wild type(WT)mouse brain and its response to inflammatory stimulation.To explore the role of NLRC3 in locomotor function recovery,neuroinflammation and neuronal damage after spinal cord injury,through NLRC3 knockout(KO)and cell co-culture.Methods:Part I,the NLRC3 m RNA expression levels in the main organs of C57BL/6J mice were observed and compared by RT-q PCR.Western blotting and immunofluorescence staining were used to compare NLRC3 expression levels in primary cultured neurons,astrocytes,microglia and oligodendrocytes under physiological conditions.The correlation between NLRC3 expression level and inflammatory activation of microglia stimulated by lipopolysaccharide(LPS)was analyzed.After intraperitoneal injection of LPS in adult WT and KO mice,the changes in overall behavioral activity of the mice were observed,and the morphological changes of microglia in the cerebral cortex were analyzed by immunofluorescence staining and sholl analysis.Part II,the motor function,depression,anxiety and other emotions of WT and KO mice under physiological state were observed through basic behavioral experiments rota-rod,open field and tail suspension.Spinal cord(crushed)injury models were performed on two genotypes of mice at T8-T9segments.Catwalk gait analysis and BMS score were used to evaluate the motor function recovery of the mice.The spinal cord tissue was taken from the injured site 7 days after the operation.The inflammatory reaction and cell infiltration were detected by Western blotting,immunofluorescence staining and flow cytometry.Part III,the primary cultured microglia were treated with LPS and co-cultured with CD3~+T cells derived from the spleens of WT and KO mice.After co-culture,the total protein of microglia was extracted and Western blotting was used to detect the inflammatory response of microglia.Primary neurons from the cerebral cortex of mice were treated with conditioned medium and neurobasal medium mixed accordingly,and Tuj1 immunofluorescence staining was used to detect neuronal damage.Results:Part I,(1)The expression of NLRC3 in the organs showed that the highest level was spleen,the brain expressed relatively low level of NLRC3 at the physiological condition.(2)The basic expression of NLRC3 in microglia cells was high.NLRC3 expression in microglia was significantly increased after LPS stimulation.After intraperitoneal injection of LPS,the activation level of microglia in the brain of KO mice was higher.(3)The activity of NLRC3 KO mice decreased significantly after LPS stimulation,compared with WT mice.Part II,(1)Under physiological conditions,there was no significant difference in motor function and emotional performance between WT and KO mice.After spinal cord injury surgery,the recovery of motor function in KO mice was significantly lower than that in WT mice.(2)The expression levels of i NOS,CCL2 and TNF-αwere not significantly different between WT mice and KO mice in the sham operation group,and the NF-κB signaling pathway was not significantly activated.After 7days of SCI,the NF-κB inflammatory signaling pathway was significantly activated in KO mice,compared with WT mice.The levels of i NOS,CCL2 and TNF-αwere significantly increased.(3)After spinal cord injury,KO mice showed more Iba-1~+and CD3~+T cells infiltration than WT mice.Flow cytometry further showed that there were more infiltration of CD11b~+CD45~+cells with higher activation level in KO mice in comparison of WT mice.Further typing of CD3~+T cells revealed more infiltration of CD3~+CD4~+and CD3~+CD4~-T cells with higher activation level overall.Part III,(1)After co-culture of LPS treated microglia with KO mouse CD3~+T cells,the levels of inflammatory signaling pathway proteins such as NF-κB p65,p-IKB-αand proinflammatory cytokine proteins in microglia were significantly increased.(2)The neurons were cultured with conditioned medium of LPS-treated microglia cells and KO mice co-culture,and the immunofluorescence staining showed that the neuronal processes were seriously damaged and the PI+neuron number was increased.Conclusions:NLRC3,as an inflammatory receptor,is expressed at a basic level in the mouse brain,especially in microglia,and can respond to inflammatory stimuli.NLRC3 has the effect of inhibiting inflammatory response.After spinal cord injury,the deficiency of NLRC3 caused a large number of inflammatory cell infiltration,resulting in a more intensive inflammatory response,and the motor function recovery of NLRC3 KO mice was poorer than that of WT mice.The co-culture of inflammatory microglia and CD3~+T cells in physiological state with NLRC3 deficiency caused positive feedback of inflammatory response,which aggravated the damage to cultured neurons in vitro.The data from this study suggest that NLRC3 play a protective role in spinal cord injury by inhibiting T cell chemotaxis to the injured area and alleviating microglia-mediated inflammatory response,thus promoting tissue protection and nerve function recovery in spinal cord injury.Therefore,NLRC3 has the potential to be a new target for spinal cord injury treatment.