The Role Of NOD2 On MSU Crystal-induced Gout Inflammation

Backgroung Gouty arthritis is an acute inflammatory joint disease caused by monosodium urate(MSU),and is the most common inflammatory arthritis in man.Current studies have shown that MSU act as dangerous-associated molecular patterns(DAMPs)which can be recognized by pattern-recognition receptors(PRRs)in the cell membrane or cytoplasm,and then activate TLR4/NF-κB,NLRP3 pathways.Nucleotide-binding oligomerization domaincontaining protein(NOD2),as a typical PRRs,can be recognized as pathogen associated molecular patterns(PAMPs)and DAMPs in pathogens.After binding to the ligand,it mediates the release of inflammatory factors by activating NOD2/NF-k B and mitogenactivated protein kinase(MAPK)pathways.More and more studies have shown that NOD2 is related to chronic inflammatory diseases,such as Crohn’s disease,Blau syndrome and early-onset sarcoidosis.However,whether NOD2 is involved in the gout inflammatory response has not been reported.PurposeTo explore the molecular mechanism of NOD2 in the pathogenesis of MSU-induced gouty inflammation.Methods1.Twelve-week old C57BL/6 mice were used for in vivo experiments,mouse air pouch model were created,and NOD2 protein was knocked down and overexpressed by injecting si RNA and lentivirus into the air pouch.MSU was injected into the air pouch and the mice were sacrificed 8 hours after stimulation,and the lavage fluid and cells in the air pouch were collected.Count the white blood cells of the lavage fluid in the air pouch,collect the white blood cells in the lavage fluid to detect the relative expression of the specific markers(i NOS,CD86)on the surface of M1 macrophages by Q-PCR,and use the ELISA kit to detect the content of interleukin 1 beta(IL-1β)and monocyte chemoattractant protein-1(MCP-1)inflammation in the lavage fluid.The air pouch synovium was collected,fixed with paraformaldehyde,and F4/80 and NF-κB phosphorylation level in synovial tissue were detected by immunohistochemistry.HE staining detects the infiltration of inflammatory cells in the synovial tissue of air pouch.2.Transfecting si RNA and lentivirus to knock down and overexpress NOD2 protein respectively.Under the stimulation of MSU,ELISA was used to detect the expression of inflammatory factors IL-1β and MCP-1 in the supernatant of THP-1 cells.Western blotting analysis the phosphorylation level of NF-κB protein in THP-1 cells and the activation of caspase-1.Experiments in vitro were conducted to explore the involvement of NOD2 in the inflammatory response induced by MSU,and to study the inflammatory mechanism of NOD2 in the pathogenesis of gout.Results1.(1)Through the mouse air pouch model,it was found that in the NOD2 knockdown experiment,the white blood cell count and the expression level of specific markers(i NOS,CD86)on the surface of M1 macrophages in the air pouch lavage fluid of the MSU-NOD2-si RNA group.And the expression levels of inflammatory factors IL-1β and MCP-1 were significantly lower than those in the MSU-NC group.The immunohistochemical results of mouse air pouch synovial tissue showed that the phosphorylation levels of NF-κB and F4/80 in the synovial tissue of the MSU-NOD2-si RNA group were significantly lower than those in the MSU-NC group.The results of HE staining showed that the infiltration of inflammatory cells in the synovial tissue of the MSU-NOD2-si RNA group was significantly less than that of the MSU-NC group.(2)In the NOD2 overexpression experiment,the white blood cell count,the expression level of specific markers on the surface of M1 macrophages and the expression level of the inflammatory factor IL-1β and MCP-1 in the air pouch lavage fluid of MSU-NOD2-LV mice were significantly higher than MSU-NC group.The immunohistochemical images of mouse synovial tissue showed that the F4/80 and NF-κB phosphorylation levels of synovial tissue in the MSU-NOD2-LV group were significantly higher than those in the MSU-NC group.HE staining showed that the infiltration of inflammatory cells in the synovial tissue of mice in the MSU-NOD2-LV group was significantly higher than that in the MSU-NC group.2.(1)By knocking down the NOD2 protein in THP-1 cells,we found that after MSU stimulation,the levels of NF-κB protein phosphorylation and caspase-1 activation of THP-1 cells in the MSU-NOD2-si RNA group were significantly lower than those of MSU-NC group.(2)THP-1 cells were transfected with lentivirus to overexpress NOD2 protein.We found that after MSU stimulation,the levels of NF-κB protein phosphorylation and caspase-1 activation of THP-1 cells in the MSU-NOD2-LV group were significantly lower than MSU-NC group.ConclusionIn this study,through the experiments in vitro and in vivo,we found that NOD2 participates in the gout inflammatory response by regulating the expression of inflammatory factors stimulated by MSU.Further studies have shown that NOD2 affects the function of macrophages by regulating the phosphorylation level of NF-κB protein and the activity of caspase-1.This project preliminarily explores the regulatory role of NOD2 in the inflammatory process induced by MSU,supplements the inflammatory regulation mechanism of gout,and provides a new direction for further research and treatment of gout.