| BackgroundThree different activation pathways can initiate the complement system,including the lectin(MBL pathway),the classical and the alternative pathway,classical pathway.The complement membrane attack complex C5b-9 is the final product of complement system activation.Many studies have found that the complement membrane attack complex C5b-9 can activate monocytes,macrophages,endothelial cells,platelets,etc.,and these cells can induce inflammation by releasing inflammatory factors(such as IL-1β,IL-18,etc.)and cytokines.The inflammatory factors(IL-1β,IL-18)play an important role in the formation of macrophage foam cell.Studies have shown that the secretion of IL-1βand IL-18 is mainly regulated by NLRP3 inflammasome.Thus,we suspect that the complement membrane attack complex C5b-9 may promote the formation of THP-1-derived macrophage foam cell by activating the NLRP3 inflammasome.ObjectiveTo investigate the effect of the complement membrane attack complex C5b-9 on the formation of THP-1-derived macrophage foam cell and explore its possible mechanism.MethodsTHP-1-derived monocytes are cultured and induced into THP-1-derived macrophages by phorbolesters.And then THP-1-derived macrophages were treated with 0ug / ml,25 ug / ml,50 ug / ml,100 ug / ml Ox-LDL for 24 h and at 0 hours,12 hours,24 hours,48 hours,RT-PCR was used to detect the expression of the complement C9,NLRP3,caspase-1,IL-1β,IL-18.ELISA was used to detect the concentrations of C5b-9,IL-1β,and IL-18 secreted by THP-1-derived macrophages.The cholesterol content in THP-1-derived macrophages was detected by biochemical method,and oil red O staining was used to observe the lipid droplets in THP-1-derived macrophages.Furthermore,50 nmol and100nmol fluorescent control siRNA were used to transfect THP-1-derived macrophages for 24 hours and 48 hours,respectively,according to the optimal concentration and time of fluorescent control siRNA transfection,complement C9 siRNA S1,S2 and S3 with different sequences were used to transfect THP-1-derived macrophages.The mRNA expression of the NLRP3,caspase-1,IL-1β,and IL-18 of THP-1-derived macrophages were respectively detected by RT-PCR.The secretion of the complementmembrane attack complex C5b-9,IL-1β and IL-18 of THP-1-derived macrophages were respectively detected by ELISA.The content of cholesterol and lipid in THP-1-derived macrophages were evaluated by biochemical method and oil red O staining.Results1.THP-1-derived monocytes treated with 100nmol/l phorbolester for24 hours can be differentiated into THP-1-derived macrophage.2.THP-1-derived macrophages were treated with 0μg/ml,25μg/ml,50μg/ml,and 100μg/ml of ox-LDL for 24 hours.Compared with the incubation with THP-1-derived macrophages at 0μg/ml,ox-LDL incubated at 25μg/ml-50μg/ml increased the mRNA expression of the complement C9,NLRP3,caspase-1,IL-1β,and IL-18 of THP-1-derived macrophage in a concentration-dependent manner(P<0.05),and at which50ug/ml ox-LDL incubated with THP-1-derived macrophages for 24 hours,the mRNA expression reached a peak.Moreover,THP-1-derived macrophages were treated with 50 μg/ml ox-LDL for 0-48 hours.Compared with the incubation with THP-1-derived macrophages for 0hour,ox-LDL incubated for 0-24 hours increased the mRNA expression of the complement C9,NLRP3,caspase-1,IL-1β,and IL-18 of THP-1-derived macrophage in a time-dependent manner(P<0.05),and when 50ug/ml ox-LDL incubated with THP-1-derived macrophages for24 hours,the mRNA expression of the complement C9,NLRP3,caspase-1,IL-1β,and IL-18 were the highest.(P <0.05).3.THP-1 macrophages were treated with 0-100 μg/ml ox-LDL for 24 hours.Compared with the incubation with THP-1-derived macrophages at 0μg/ml,ox-LDL incubated at 25μg/ml-50μg/ml gradually increased the concentrations of the complement membrane attack complex C5b-9,IL-1β and IL-18(P<0.05),and the concentrations reached a peak at 50μg/ml ox-LDL.THP-1-derived macrophages were treated with 50 μg/ml ox-LDL for 0-48 hours.Compared with the incubation with THP-1-derived macrophages for 0 hour,ox-LDL incubated for 0-24 hours increased the concentration of the complement membrane attack complex C5b-9,IL-1β,and IL-18 of THP-1-derived macrophage in a time-dependent manner,and when 50ug/ml ox-LDL incubated with THP-1-derived macrophages for 24 hours,the concentration of the complement membrane attack complex C5b-9,IL-1β,and IL-18 were the highest(P<0.05).4.0μg/ml,25μg/ml,50μg/ml,100μg/ml of ox-LDL treated THP-1macrophages for 24 hours.Compared with the incubation with THP-1-derived macrophages at 0μg/ml,ox-LDL at 25μg/ml-50μg/ml gradually increased the contents of TC,FC,CE,CE/TC(%)and lipid droplets of macrophage(P<0.05),and the contents reached a peak at 50μg/ml ox-LDL.THP-1-derived macrophages were treated with 50 μg/ml ox-LDL for 0-48 hours.Compared with the incubation withTHP-1-derived macrophages for 0 hour,ox-LDL incubated for 0-24 hours increased the contents of TC,FC,CE,CE/TC(%)and lipid droplets of macrophage in a time-dependent manner,and when 50ug/ml ox-LDL incubated with THP-1-derived macrophages for 24 hours,the contents of TC,FC,CE,CE/TC(%)and lipid droplets of macrophage were the highest(P<0.05).5.Transfection with 50 nmol and 100 nmol fluorescent control siRNA for 48 hours,the transfection efficiency was the highest.Besides,RT-PCR detection revealed that the efficiency of complement C9 siRNA S2 was the best.6.Compared with the control,complement C9 siRNA transfected THP-1-derived macrophages,the mRNA expressions of the NLRP3,caspase-1,IL-1β,and IL-18 were all reduced.The secretion of the complement membrane attack complex C5b-9,IL-1β,and IL-18 were all reduced.Samely,the formation of lipid droplets and macrophage foam cell were decreased(P<0.05).complement membrane attack complex C5b-9,NLRP3,caspase-1,IL-1β,and IL-18ConclusionsThe complement membrane attack complex C5b-9 promotes the release of L-1β and IL-18,as well as the formation of lipid droplets and macrophage foam cell by activing NLRP3 inflammasome. |
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