Mechanism Research On Mas1/PKC Pathway Regulating Fascin1 Phosphorylation Mediated Microglial Phagocytosis After Spinal Cord Injury

Part1 Fascin1 can regulate the phagocytosis of myelin fragments in microglia and affect the recovery of spinal cord function after spinal cord injuryBackground: The main pathological feature of spinal cord injury is that the injury and rupture of spinal cord blood vessels lead to ischemic necrosis of local tissues,resulting in a large number of myelin fragments in the core and surrounding areas of the injury.Excessive accumulation of myelin fragments will seriously hinder nerve regeneration and functional recovery.After spinal cord injury,the removal process of myelin fragments is mainly completed by microglia and macrophages.However,microglia have a "faster" reaction speed and a "stronger" ability to clear myelin fragments than macrophages.When microglia play the phagocytic function,the membrane deformation involved in the depolymerization and polymerization of F-actin is an inevitable condition.Fascin1,as a powerful actin-binding protein,is highly expressed in microglia,and can provide mechanical force for membrane deformation by regulating F-actin.However,it is not known whether Fascin1 mediates the phagocytosis of myelin fragments by microglia after spinal cord injury.Methods: Build Cx3cr1^cre;Fascin1^fl/fl transgenic mice were used to establish a mouse spinal cord injury model,and tissue immunofluorescence staining was used to verify whether the model was successfully constructed.After that,Cx3cr1^cre after spinal cord injury was measured by tissue immunofluorescence staining;The survival of neurons in Fascin1^fl/fl transgenic mice and wild-type mice was detected,and the recovery of motor function was detected by using the motor function BMS score and footprint analysis.Immunofluorescence staining was used to evaluate the changes of phagocytosis of microglia after specific knockdown of Fascin1 in vivo and in vitro.Results: Compared with wild-type mice,the expression of Fascin1 in Cx3cr1+microglia decreased significantly on the 14 th day after spinal cord injury,indicating that Cx3cr1^cre;Fascin1^fl/fl mouse model was successfully constructed.The results of motor function BMS score and footprint analysis suggest that specifically knocking down Fascin1 in Cx3cr1+microglia will significantly damage the motor recovery after spinal cord injury in mice.In terms of the phagocytic function of microglia,the results of immunofluorescence staining in vivo and in vitro suggest that specifically knocking down Fascin1 in Cx3cr1+microglia will significantly damage its phagocytic ability.Conclusion: The specific knockdown of Fascin1 in microglia will damage the motor recovery after spinal cord injury in mice,and Fascin1 is an important functional protein that mediates the phagocytosis of microglia.However,its own and upstream regulation mechanism are still unclear and need further exploration and exploration.Part2 Mas1/PKC pathway regulates Fascin1 phosphorylation balance and mediates microglial phagocytosis after spinal cord injuryBackground: Fascin1 itself can be phosphorylated.In non-phosphorylation state,it can combine and stabilize two adjacent F-actin bundles to maintain the stability of organelle membrane;In phosphorylation state,F-actin beam will be depolymerized to prepare for dynamic changes of cell membrane.When Fascin1 functions,PKC can phosphorylate the 39 th serine residue（S39）in Fascin1 protein molecule after translocation activation,thus realizing negative regulation of Fascin1 protein function.The G protein-coupled receptor Mas1 may act as an upstream receptor to regulate the function of PKC.However,after spinal cord injury,it is not known whether Mas1/PKC axis leads to microglial phagocytosis disorder by mediating Fascin1 phosphorylation imbalance.Methods: The mouse spinal cord injury model was prepared by T10 clamp method,and the expression of p-Fascin1 and Fascin1 were detected after spinal cord injury.Using the mouse spinal cord injury model,primary mouse microglia and the transcriptome data of spinal cord injury microglia,we explored and verified the expression changes of Mas1 and PKC and the upstream and downstream regulatory relationship.The phagocytosis of myelin fragments by Cx3cr1+microglia after interference with Mas1/PKC axis function was detected.To verify in vivo the effect of Mas1 receptor agonist captopril on the rescuing of Fascin1 phosphorylation imbalance in microglia after spinal cord injury and on the survival of neurons and the recovery of spinal cord function in mice.Results: The phagocytic capacity of microglia was the strongest on the third day after injury,and the phosphorylation level of Fascin1 was also high,which was highly correlated.In vitro experiments further proved that the phagocytosis of microglia was impaired with the down-regulation of Fascin1 phosphorylation.By analyzing the transcriptome data of macrophages/microglia after spinal cord injury,it is speculated that Mas1/PKC γ It may play a major role in the phosphorylation of Fascin1 in microglia after spinal cord injury.Mas1/PKC by using specific agonists and inhibitorsγ The results suggest that Mas1 can be used as a PKC γ Its upstream receptor regulates the phosphorylation of Fascin1 and the phagocytosis of microglia.After that,intraperitoneal injection of captopril can enhance the ability of microglia to phagocytose myelin fragments,help protect the remaining neurons,and promote the recovery of motor function after spinal cord injury.Conclusion: In this part of the study,we found Mas1 and PKC by analyzing transcriptome data γ It may regulate the phosphorylation of Fascin1.At the same time,we also proved that captopril can save the phosphorylation of Fascin1 in microglia and enhance the phagocytosis of microglia by up-regulating Mas1 in vivo and in vitro,which provides a theoretical basis for further clinical transformation.