FOXO3a Modulates The Inflammation And Oxidative Stress Via Regulating TGF-β And HO-1 In Ankylosing Spondylitis

ObjectiveAnkylosing spondylitis(AS)is a genetically predominant chronic autoimmune inflammatory disease.AS is clinically incurable,requires lifelong treatment,and has a high morbidity and disability rate in young adults,which has been considered as a major public health problem.Although the genetic basis of AS is known,the molecular mechanisms responsible for the development of AS remain poorly understood.Therefore,in-depth exploration of the molecular mechanisms that affect the pathogenesis and progression of AS has important clinical value and public health significance.FOXO3a(Forkhead box O3a)transcription factor could regulate the expression of various downstream target genes,thereby exerting different biological effects.Among them,oxidative stress has been confirmed to be involved in the occurrence and development of AS.FOXO3 a plays a pivotal role in many aspects of autoimmune diseases,but the specific molecular mechanism of FOXO3 a in AS has not yet been explored.Therefore,this study aimed to investigate whether FOXO3 a modulates inflammation and oxidative stress in AS.Methods1.Bioinformatics Research: Firstly,we applied bioinformatics analysis to predict the downstream target genes that have binding sites with FOXO3 a,took the intersection of multiple databases,combined with literature,and screened out the genes that not only have binding sites with FOXO3 a but also are related to the pathogenesis of AS.2.Population research: We employed a case-control study involving 50 AS patients and 50 healthy controls(1:1 individual matched with patients for age and gender).T cells were separated by magnetic beads in peripheral blood.Quantitative Real-time PCR(q RT-PCR)was applied to detect the transcription levels of FOXO3 a and downstream target genes.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory cytokines [Interleukin-1β(IL-1β),IL-8,IL-17 A,IL-23,tumor necrosis factor-α(TNF-α)] and oxidative stress factors [superoxide dismutase(SOD),catalase(CAT),total antioxidant capacity(T-AOC),malondialdehyde(MDA)].The expression levels of FOXO3 a and downstream target genes in T cells of AS group and control group were compared,and their correlation with inflammatory cytokines,oxidative stress factors and clinical characteristics of AS was evaluated,and then the downstream target genes of FOXO3 a in this study were determined.3.In vitro experiment: Gene overexpression and knockdown of FOXO3 a in Jurkat cells were conducted via lentivirus and small interfering RNA,respectively.q RT-PCR was used to detect FOXO3 a,transforming growth factor-β(TGF-β)and heme oxygenase-1(HO-1)transcription level.Western blotting(WB)was applied to detect the protein levels of FOXO3 a,TGF-β and HO-1.ELISA was implemented to determine the levels of inflammatory cytokines and oxidative stress factors.Finally,we applied chromatin immunoprecipitation(CHIP)and dual-luciferase reporter assay to verify whether FOXO3 a directly binds to the promoter regions of TGF-β and HO-1and to determine the direction of its regulation.Results1.Results of Bioinformatics Research: Bioinformatics analysis revealed that genes that both have binding sites for FOXO3 a and are related to the pathogenesis of AS include TGF-β,HO-1,vitamin D receptor(VDR),hypoxia-inducible factor-1α(HIF-1α),kelch-like ECH-associated protein 1(KEAP1)and nuclear factor erythroid2-related factor 2(NRF2).2.Results of Population research: A total of 50 AS patients and 50 healthy controls were included in the case-control study.The average age of AS patients and healthy individuals were 35.60 ± 11.19 and 35.80 ± 11.30 years,respectively.The proportion of males in both the case and control groups was 74.0%.There was no significant difference in age and gender between the two groups.Downregulated FOXO3 a expression was first confirmed in AS patients.Among the predicted downstream target genes,except NRF2,all genes differentially expressed between AS patients and controls.However,FOXO3 a expression was only positively correlated with TGF-β and HO-1 expression in AS patients,therefore they were selected as downstream targets for follow-up studies.IL-1β,IL-8,IL-17 A,IL-23 and TNF-αwere significantly higher expressed in AS compared to controls.The levels of SOD,CAT,and MDA in AS patients were significantly higher than those in controls,and the activity of T-AOC in AS patients was significantly lower than that in healthy controls.The correlation analysis of FOXO3 a,TGF-β and HO-1 with inflammatory cytokines and oxidative stress factors showed that FOXO3 a transcription level was significantly negatively correlated with IL-8 and IL-17 A,and observably positively correlated with T-AOC activity.The transcription level of TGF-β was markedly negatively associated with IL-8,IL-17 A and MDA,and positively related to T-AOC activity.The transcription level of HO-1 was signally correlated with IL-8,IL-17 A,TNF-α,SOD,CAT and MDA,and dramatically negatively related to T-AOC activity.The correlation analysis of FOXO3 a,TGF-β and HO-1 with the clinical characteristics of AS patients showed that FOXO3 a was memorably negatively correlated with the Bath Ankylosing Spondylitis Functional Index.TGF-β was observably negatively related to Bath Ankylosing Spondylitis Disease Activity Index.And there were significant positive correlations between HO-1 and erythrocyte sedimentation rate,C-reactive protein,and Ankylosing Spondylitis Disease Activity Score.3.Results of in vitro experiments: In vitro experiments indicated that the m RNA expression of TGF-β and HO-1 were significantly increased by lentivirusFOXO3a(Lv-FOXO3a)and decreased by si RNA-FOXO3a(Si-FOXO3a).As expected,the above results also verified at the protein level.Lv-FOXO3 a obviously reduced the levels of IL-8,IL-17 A,and IL-23 cytokines.Correspondingly,SiFOXO3 a markedly enhanced the levels of them.The activities of SOD,CAT,and TAOC were obviously elevated in Lv-FOXO3 a group.Nevertheless,the level of MDA was up-regulated slightly in Lv-FOXO3 a group without statistically significant.Similarly,Si-FOXO3 a obviously decreased the activity of SOD,CAT,and T-AOC,but no remarkable difference on MDA was observed.CHIP assay demonstrated that FOXO3 a could directly bind to the site from-590 to-597 bp before the TGF-βpromoter region and the site from-165 to-173 bp before the HO-1 promoter region.Dual-luciferase reporter assay clarified that FOXO3 a transcription factor could bind to the above sites in the promoter regions of TGF-β and HO-1 and activate the expression of corresponding genes.ConclusionTaken together,FOXO3 a expression was obviously decreased and was associated with inflammation and total antioxidant capacity in AS patients.FOXO3 a could directly regulate TGF-β and HO-1 to alleviate inflammation and oxidative stress.Therefore,the modulation of cellular inflammation and oxidative stress via FOXO3 a mediated TGF-β and HO-1 activation partially takes part in the occurrence and development of AS,which might provide clues to further understand the pathogenesis of AS.