Experimental Study On Stimulation Of Macrophage TLR2-NADPH-NLRP3 Inflammatory System With COM

THE FIRST PART: THE ROLE OF TLR2/NADPH OXIDASE PATHWAY IN COM CRYSTAL-MACROPHAGES REACTION SYSTEMObjective To observe the expression of TLRs receptor in macrophages stimulated by COM crystals.To investigate the role of TLRs-NADPH signaling pathway in the crystal-macrophages reaction system.Methods The relative m RNA expression of TLR2 m RNA,TLR4 m RNA,p91 phoxm RNA and p47 phoxm RNA were detected by Real-time PCR,which were induced by 500 ug / ml of COM.The protein expression of TLR2 were detected by flow cytometry.The protein expression of p91 phox and p47 phox were detected by Western-blot.The expression of ROS in the cells was observed by fluorescence microscopy.Thereafter,macrophages were pretreated with apocynin.The expression of ROS in the cells was observed by fluorescence microscopy.FSL-1,a TLR2 receptor inhibitors,was pre-treated with macrophages,and Western blotting was applied to detect protein level of p47 phox.RESULTS The expression levels of TLR2 m RNA expression were significantly increased after stimulation with COM crystals(P <0.01).There was no significant change in TLR4 m RNA expression(P>0.05).The expression levels of p91 phox and p47 phox protein and m RNA expression were significantly increased after stimulation with COM crystals(P <0.01).The intracellular ROS levels of macrophages were meaningfully increased by the method of using DCFH-DA(P<0.01).Cells were pre-incubated with a NADPH oxidase inhibitors(apocynin),followed by treatment with COM crystal(500ug/ml).The blockade of NADPH oxidase by apocynin resulted in decreased the intracellular ROS levels of macrophages(P<0.01).Cells were pre-incubated with a TLR2 receptor inhibitors(FSL-1),followed by treatment with COM crystal(500ug/ml).According to the western blotting analysis of macrophages cell lysates,the blockade of TLR2 receptor by FSL-1resulted in decreased the protein levels of p47 phox in macrophages(P<0.05).CONCLUSION Macrophages may be recognize COM crystals or matrix bound to the crystas by TLR2 receptors.The activation of NADPH oxidase produces a large amount of ROS.TLR2/NADPH oxidase pathway may play an important role in the formation of calcium oxalate stones.THE SECOND PART :COM INDUCES NLRP3 INFLAMMASOME ACTIVATION IN MACROPHAGE VIA NADPH OXIDASE DEPENDENT ROS PRODUCTIONObjective To clear the role of the NLRP3 inflammasome activation in the formation of calcium oxalate kidney stone,and to explore the activation mechanism of the NLRP3 inflammasome.To investigate the involvement of NADPHoxidase derived reactive oxygen species(ROS)in the inflammatory response of crystal-macrophages.To evaluate the effect of atorvastatin on NLRP3 inflammasome in macrophages stimulated by COM crystals.Methods The relative m RNA expression of NLRP3 m RNA and Caspase-1 m RNA were detected by Real-time PCR,which were induced by 500 ug / ml of COM.The protein expression of NLRP3 and Caspase-1(P20)were detected by Western-blot.The expression of IL-1β and IL-18 in the supernatant was measured by enzyme-linked immunosorbent assay(ELISA).Macrophages were pretreated with apocynin,FSL-1 or atorvastatin,and then were incubated for another 24 h with 500ug/ml COM crystal.The protein expression of NLRP3 and Caspase-1(P20)were detected by Western-blot.RESULTS Exposure of macrophages to COM crystal(500ug/ml)significantly increased the protein expression level of NLRP3,Caspase-1(P20),and the m RNA level of NLRP3,Caspase-1(*P<0.01).The crystal of COM significantly stimulated macrophages to release IL-1β and IL-18(*P<0.05).The protein expression level of NLRP3,Caspase-1(P20)was down-regulated in macrophages incubated with apocynin,FSL-1 or atorvastatin.CONCLUSION The NLRP3 inflammasome activation engeages in the formation of the stone.NADPH oxidase/ROS is involved in COM crystal induced NLRP3 inflammasome activation.Atorvastatin reduces the activation of NLRP3 inflammasome.THE THIRD PART: ROLE OF NLRP3/ HMGB1 PATHWAY IN CRYSTAL-MACROPHAGE INFLAMMATORY RESPONSEObjective To observe the expression of HMGB1 in cell supernatants.To investigate the role of NLRP3-HMGB1 signaling pathway in the inflammatory response of crystal-macrophages.Methods The relative m RNA expression of HMGB1 m RNA were detected by Real-time PCR,which were induced by 500 ug / ml of COM.The protein expression of HMGB1 were detected by Western-blot.Macrophages were transfected by NLRP3-Si RNA.After COM crystal stimulation,the protein expression of HMGB1 were detected by Western-blot.RESULTS The crystal of COM significantly stimulated macrophages to release HMGB1(*P<0.05).The silencing of the NLRP3 gene obviously reduced the protein expression level of HMGB1(*P<0.01).CONCLUSION The crystal of COM crystals might promote the release of HMGB1.NLRP3-HMGB1 inflammatory signaling pathway plays an important role in the inflammatory response of crystal-macrophages.