| Background:Rheumatoid arthritis(RA)is a chronic autoimmune and inflammatory disease mainly characterized by symmetrical multiarticular lesions,and its pathogenesis is not completely clear.The main pathological basis is synovial inflammation and pannus formation.The hyperplastic rheumatoid fibroblast-like synoviocytes(RA-FLSs)form pannus and have abnormal function of migration,invasion and secretion.This abnormal function is a crucial part of the pathophysiological behavior of fibroblast-like synoviocytes(RA-FLSs)and is the key to joint destruction.The role of oxidative stress in RA has gradually attracted the attention of researchers.Furthermore,the chronic inflammation in RA is closely related to redox imbalance,and this redox imbalance may be one of the causes of RA.Advanced oxidized protein products(AOPPs)is a novel biomarker of oxidative stress,which is not only a product of oxidative stress,but also has pro-inflammatory effects.Our previous study has confirmed that the level of AOPPs is increased in RA patients,and it is closely related to the disease activity of RA.Based on the above theories and results,we focused on the effect of specific effects of oxidative stress substances represented by AOPPs on the RA-FLSs,and analyzed the role of oxidative stress in RA.MethodsRA-FLSs was treated with different concentrations of AOPP-HSA(50-200?g/ml).In the first part,transwell chamber was used to observe the migration and invasion ability of RA-FLSs induced by the AOPPs.In the second part,the secretion and mRNA expression levels of TNF-?,IL-6,MMP-3 and MMP-13 were detected by ELISA and Q-PCR under the induction of AOPPs.In the third part,the activation of NF-?B in RA-FLSs was detected by Western blot and immunofluorescence when adding the AOPPs.ResultsPart ?:The migration and invasion ability of RA-FLSs induced by AOPPs1.Influence of AOPPs on RA-FLSs migration abilityCompared with the control group and the unmodified HSA group,RA-FLSs treated with AOPP-HSA(100?g/ml)significantly enhanced the cell migration ability after 48h.(Number of migrated cells:control group,34.67±4.51;HSA group,32.67±2.06;AOPPs group,71.67±2.52).In the blocking experiment,anti-RAGE antibodies could partially inhibit AOPPS-induced migration.(Number of migrated cells:AOPPs+anti-RAGE antibody group,48.01±2.65).2.Influence of AOPPs on RA-FLSs invasion abilityCompared with the control group and the unmodified HSA group,RA-FLSs treated with AOPP-HSA(100?g/ml)significantly enhanced the cell invasion ability after 48h.(Number of invaded cells:control group,22.33±4.16;HSA group,27.67±5.03;AOPPs group,80‘67±3.06).In the blocking experiment,anti-RAGE antibodies could partially inhibit AOPPs-induced invasion.(Number of invaded cells:AOPPs± anti-RAGE antibody group,44.67±5.03).Part ?:The secretion and mRNA expression of TNF-?,IL-6,MMP-3 and MMP-13 in RA-FLSs induced by AOPPs1.Determination of the concentrations of cytokines TNF?,IL-6,MMP-3 and MMP-13 in cell supernatantAfter incubation with 0,50,100,200?g/ml AOPP-HSA or 100?g/ml unmodified HSA for 48 h,the contents of inflammatory cytokines IL-6,TNF?,MMP-3 and MMP-13 in cell supernatant were up-regulated in a dose-dependent manner(P<0.05).Although the expressions of IL-6,TNF?,MMP-3 and MMP-13 in 200?g/ml AOPPs group were slightly lower than those in 100?g/ml AOPPs group,they were still significantly higher than those in the control group.There was no significant difference in the secretion of IL-6,TNF?,MMP-3 and MMP-13 in the unmodified HSA compared with the normal control group(P>0.05).RA-FLSs was co-incubated with 100?g/ml AOPP or 100?g/ml unmodified HSA after 0,6,12,24,48 h.With the extension of time,the contents of inflammatory cytokines IL-6,TNF?,MMP-3 and MMP-13 in the supernatant were upregulated in a time-dependent manner(P<0.05).There was no significant difference in the secretion of IL-6,TNF?,MMP-3 and MMP-13 in the unmodified HSA compared with the normal control group(P>0.05).In the blocking experiment,the anti-RAGE antibody could significantly inhibit the secretion of IL-6,TNF?,MMP-3 and MMP-13 induced by AOPPs.2.Expression of mRNA levels of cytokines IL-6,TNF?,MMP-3 and MMP-13 in RA-FLSsAfter incubation with 0,50,100,200?g/mL AOPP-HSA or 100?g/mL unmodified HSA for 48 h,the mRNA expressions of IL-6,TNF?,MMP-3 and MMP-13 increased in a dose-dependent manner with the increase of stimulus dose(P<0.05).Although the mRNA expressions of IL-6,TNF?,MMP-3 and MMP-13 in 200?g/ml AOPPs group were slightly lower than those in 100?g/ml AOPPs group,they were still significantly higher than those in the control group.There was no significant difference in the expression of mRNA levels of IL-6,TNF?,MMP-3 and MMP-13 between the unmodified HSA and the control group(P>0.05).RA-FLSs were co-incubated with 100?g/ml AOPP-HSA or 100?g/mL unmodified HSA after 0,6,12,24 and 48 h.The mRNA expression of IL-6,TNF?,MMP-3 and MMP-13 were upregulated with time prolonging in a time-dependent manner(P<0.05).Compared with the normal control group,there was no significant difference in the expression of mRNA levels of IL-6,TNF?,MMP-3 and MMP-13 in the unmodified HSA(P>0.05).In the blocking experiment,the anti-RAGE antibody could significantly inhibit the mRNA expression of IL-6,TNF?,MMP-3 and MMP-13 induced by AOPPs.Part 3:Activation of NF-?B in RA-FLSs by AOPPs1.Detection of the changes of NF-?B by Western blot in RA-FLSsAfter incubation with 50,100,200?g/ml AOPP-HSA or 100?g/mL unmodified HSA for 3 h,in RA-FLSs increased the phosphorylation of NF-?B p65 and decreased the protein of I?B? in a dose-dependent manner(P<0.05).The largest effect of AOPPs on the RA-FLSs was at 100?g/ml.There was no significant difference in the reduction of p65 phosphorylation and I?B? in unmodified HSA compared with control group(P>0.05).After incubation with 100?g/ml AOPP-HSA or 100?g/ml unmodified HSA for 0,1,3 and 6h,respectively,the phosphorylation of NF-?B p65 increased with the prolonging of stimulation time,and the protein of I?B? decreased further with the prolonging of stimulation time in a time-dependent manner(P<0.05).The maximum effect was achieved at 3 hours.There was no significant difference in the reduction of p65 phosphorylation and I?B? in unmodified HSA compared with control group(P>0.05).In the blocking experiment,the anti-RAGE antibody could effectively inhibit the change of NF-?B induced by AOPPs.2.Detection of nuclear transfer of NF-?B p65 subunit by immunofluorescence assayRA-FLSs was co-incubated with 100?g/ml AOPP-HSA or 100?g/ml unmodified HSA for 3h,respectively.Immunofluorescence staining showed p65 entering the nucleus.However,p65 remained in the cytoplasm in the control group and the unmodified HSA group.In the blocking experiment,the anti-RAGE antibody could significantly inhibit the transfer of NF-?B p65 to the nucleus induced by AOPPs.Conclusions1.AOPPs can increase the migration ability of RA-FLSs in a time-dependent and dose-dependent manner.2.AOPPs can increase the invasion ability of RA-FLSs in a time-dependent and dose-dependent manner.3.AOPPs can up-regulate the expression of IL-6 and TNFa in RA-FLSs in a time-dependent and dose-dependent manner.4.AOPPs can up-regulate the expression of MMP-3 and MMP-13 in RA-FLSs in a time-dependent and dose-dependent manner.5.AOPPs can activate the NF-?B signaling pathway in RA-FLSs in a time-dependent and dose-dependent manner.6.AOPPs induced the changes in the migration,invasion and secretion of RA-FLSs by activating the RAGE receptor.Blocking RAGE receptor can significantly inhibit the above changes.7.Anti-RAGE antibody can significantly block NF-?B signaling pathway activated by AOPPs.8.Unmodified HSA had no significant effect on RA-FLSs,which confirmed that the oxidative modification of protein may influence the biological effect of RA-FLSs.9.This study confirmed that AOPPs may activate NF-?B signaling pathway through binding with RAGE receptor,and induce the change of migration,invasion and secretion function in RA-FLSs. |
PDF Full Text Request