Study On The Effect And Regulatory Mechanism Of Protein 4.1R On LPS-induced Sepsis

BackgroundSepsis is a common complication form severe burns,trauma and major surgical operations,and it has become one of the main causes of mortality in critically ill patients.Clinically,sepsis caused by bacterial infections accountes for more than 95%,and the release of endotoxin lipopolysaccharide from bacterial infections is responsible for the occurrence of sepsis.The monocyte/macrophage system is the first line against the invasion of LPS,and Toll like receptor 4 located at the macrophage surface can recognise LPS,which can activate the intracellular signal transduction in macrophage,and then macrophage produces large amounts of inflammatory cytokines,such as TNF-α,IL-1,IL-6 and IL-12.Those cytokines can be transport to the distal target organ or target cells through blood circulation or paracrine and exert biological effects to cause systemic inflammatory response,and lead to the occurrence of sepsis ultimately.Considering the critical role of macrophages in the development of sepsis induced by LPS,it is of great significance to find and identify the new regulatory molecules of inflammatory response in macrophages.Protein 4.1R is a member of the protein 4.1 family,which also includes protein4.1B,protein 4.1N,protein 4.1G.The members of protein 4.1 family have four conserved domains:FERM domain,FERM-adjacent regulated domain,spectrin actin binding domain and the C-terminal domain.Protein 4.1R is a membrane skeleton protein,which founctions as an adapter protein to link various transmembrane proteins to cytoskeletal protein and plays a role in maintaining normal cell morphology,mediating cell movement and adhesion and regulating cell growth and differentiation.In recent years,protein 4.1R has received much attention because of its important regulatory role in immune cells.In CD4~+T cells,protein 4.1R can suppress the cell proliferation and differentiation of CD4~+T cell through binding linker for activation of T cells(LAT)and inhibiting the phosphorylation of LAT by ZAP-70.Recent report has showed that moesin also containing the FERM domain can participate in the TLR4/NFκB signaling pathway in macrophages,these researches suggest that protein 4.1R may play a role in inflammatory response of macrophage induced by LPS and may participate in the occurrence and development of sepsis.ObjectiveFor establishment of a mouse sepsis model,Wide type(4.1R+/+)and 4.1R gene knockout(4.1R-/-)C57BL/6 mice were intraperitoneally inoculated with LPS,which was used to study the effect of protein 4.1R on sepsis.Macrophages induced by the bone marrow cells of 4.1R+/+and 4.1R-/-mice were used to investigate the effect of protein 4.1R on inflammatory response of macrophage induced by LPS.So then,this study was aimed to establish a fundamental research for further elucidating of the pathogenesis of sepsis,and to provide a new idea for the treatment of sepsis.Method1.4.1R+/+and 4.1R-/-C57BL/6 mice were intraperitoneally inoculated with LPS and the survival time of mice were monitored.2.ELISA was used to detect the secretion of proinflammatory cytokines TNF-α,IL-6,IL-1βand chemokine MCP-1 in the serum of 4.1R+/+and 4.1R-/-sepsis mice.3.The liver tissues of mice with 4.1R+/+and 4.1R-/-sepsis mice were sectioned and observed by H&E staining,and the immunohistochemical changes of macrophage were investigated.4.The fluorescence quantitative PCR was used to detect the expression of pro-inflammatory cytokines TNF-α,IL-6,IL-1βand chemokine MCP-1 in the liver tissues of 4.1R+/+and 4.1R-/-sepsis mice.5.The bone marrow derived macrophages(BMDM)were induced by the bone marrow cells of 4.1R+/+and 4.1R-/-mice in vitro,and the purity of 4.1R+/+BMDM and 4.1R-/-BMDM cells was detected by flow cytometry.6.The expression of 4.1R in 4.1R+/+BMDM and 4.1R-/-BMDM cells was identified by PCR technology and Western blotting,and the exon map of 4.1R gene in BMDM cells was plotted.7.The fluorescence quantitative PCR and ELISA was used to detect the expression and the secretion of proinflammatory cytokines TNF-α,IL-6,IL-12 and chemokine MCP-1 in 4.1R+/+BMDM and 4.1R-/-BMDM cells stimulated by LPS.8.Western blottin was used to detect the activation of MAPKs signaling pathway in 4.1R+/+BMDM and 4.1R-/-BMDM cells stimulated by LPS.Results1.The 4.1R-/-mice had an earlier onset and a higher mortality rate in the sepsis model.2.The secretion of pro-inflammatory cytokines TNF-α,IL-6,IL-1βand chemokine MCP-1 in the serum of 4.1R-/-sepsis mice was significantly higher than that of4.1R+/+sepsis mice.3.The edema and injury of liver tissue in 4.1R-/-sepsis are more serious than4.1R+/+sepsis mice,and the number of macrophages infiltrated in the liver tissue of4.1R-/-sepsis mice is more than 4.1R+/+sepsis mice.4.The expression of pro-inflammatory cytokines TNF-α,IL-6,IL-1βand chemokine MCP-1 in the liver tissues of 4.1R-/-sepsis mice were significantly higher than that in 4.1R+/+sepsis mice.5.The purity of 4.1R+/+BMDM and 4.1R-/-BMDM cells was 94.7%and 94.0%respectively,which suggested that 4.1R+/+BMDM and 4.1R-/-BMDM cells could be used for subsequent experiments.6.The absent expression of 4.1R in 4.1R-/-BMDM cells in gene level and protein level was identified with PCR technology and Western blotting respectively,and 4.1R gene in BMDM cells lacked exon 14,exon 15,exon 16.7.The result of fluorescence quantitative PCR and ELISA showed that the expression levels and the secretion of proinflammatory cytokines TNF-α,IL-6,IL-12and chemokine MCP-1 in 4.1R-/-BMDM cells stimulated by LPS were significantly higher than that of 4.1R+/+BMDM cells.8.The result of western blotting showed that the expression of p-JNK protein and p-ERK protein in 4.1R-/-BMDM cells stimulated by LPS was significantly higher than that of 4.1R+/+BMDM.ConclusionProtein 4.1R can inhibit the production of pro-inflammatory cytokines in macrophages stimulated by LPS and plays an important negative regulatory role in LPS-mediated sepsis.