The Rhythmic Characteristics Of Rheumatoid Arthritis Inflammation And Regulatory Roles Based On Clock Genes

Background:Rheumatoid Arthritis(RA)is an autoimmune disease primarily characterized by erosive joint inflammation.Patients often present with symmetrical pain,swelling,and stiffness in multiple joints of the limbs,with symptoms that are typically exacerbated in the morning.The primary pathological feature of RA is synovitis,in which various inflammatory cytokines participate in the inflammatory response in the synovium and play crucial roles in disease progression.In joints affected by RA,pro-inflammatory factors,such as tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β),can activate multiple inflammatory cells,including fibroblast-like synoviocytes(FLSs),leading to the occurrence of inflammation.Activated FLSs,in turn,produce a large amount of inflammatory mediators,contributing to the persistence and exacerbation of inflammation.Simultaneously,the pathological process of RA triggers the RANKL/RANK-mediated bone-destruction signaling pathway,activating nuclear factor-κB(NF-κB)and stimulating effector cells,especially RA-FLS,to secrete pro-inflammatory cytokines including IL-6 and TNF-α.This process promotes the differentiation and maturation of osteoclasts,exacerbates bone erosion,and leads to bone destruction.In contrast,osteoprotegerin(OPG),as a decoy receptor for RANKL,competitively binds to RANKL,inhibiting the binding of RANKL to its receptor RANK.This blockage prevents the activation of the RANKL/RANK signaling pathway,thereby halting the formation of osteoclasts,bone resorption,and delaying bone destruction.The balance of the RANKL/OPG system is crucial for bone destruction in RA.Biological clock genes play significant roles in the pathology of RA,with complex and close regulatory relationships between clock genes and various inflammatory cytokines and signaling pathways in RA.This involves a series of crucial clock gene regulatory elements,including CLOCK,BMAL-1,Period(PER-1,PER-2,and PER-3),and cryptochrome(CRY-1 and CRY-2).The effects of inflammatory processes on the clock gene regulatory are manifold.Researches have demonstrated that TNF-α and IL-1β can directly affect the expression of core clock genes,including CLOCK and BMAL-1,interfering with their normal circadian rhythm changes.Simultaneously,they affect the stability and function of PER and CRY proteins,disrupting the negative regulatory loop of the biological clock and leading to rhythmic disturbances.The regulatory relationship between clock genes and inflammation is both bidirectional and interactive.Inflammation can interfere with the normal rhythm of the biological clock,and the dysregulation of this clock may exacerbate inflammatory responses.Therefore,a comprehensive understanding of the interactions between pro-inflammatory cytokines,bone destruction factors,osteoprotegerin,and clock genes in the disease process of RA is crucial to reveal the pathological mechanisms of RA and gain a deeper understanding of its clinical symptoms.This understanding is essential for the development of novel therapeutic strategies.Methotrexate(MTX)and Tripterygium Glycoside(TG)are widely used in clinical practice as first-line drugs for RA treatment.Further researches on the mechanisms of action of MTX and TG,particularly their roles in regulating inflammation,bone destruction,and circadian rhythm,could provide more options and directions for personalized treatment.Therefore,this study is based on clock genes and aims to investigate the rhythmic characteristics of inflammatory cytokines,bone-resorption factors,and bone protective agents in the pathology of RA,as well as the regulatory roles of clock genes in RA inflammation,and initially reveal the effects of MTX and TG on bone destruction in RA and their effects on circadian rhythms,at three levels:(i)the genomic profile of RA patients;(ii)animal experiments(in vivo);and(iii)cell experiments(in vitro),by analyzing wholegenome RNA-seq data from RA patients,establishing collagen-induced arthritis(CIA)models,and utilizing IL-6/s IL-6R-induced rheumatoid arthritis fibroblast-like synoviocyte(RA-FLS)models,to provide a scientific basis and theoretical foundation for systematically understanding the chronobiological mechanisms of RA and optimizing treatment strategies.Part Ⅰ: Genome-wide RNA-seq feature mapping and functional analysis of RA patients Objective: This study aimed to analyze the whole-genome RNA-seq dataset of patients with RA using bioinformatic methods to hierarchically investigate patterns of gene expression under different pathological conditions of RA and to further explore the expression patterns of circadian clock-related genes in RA.Methods: Data were screened in NCBI’s Gene Expression Omnibus(GEO)database with the keyword "Rheumatoid Arthritis" and “Homo” in strict accordance with precise and comprehensive criteria.Among the 376 sets of candidate datasets,GSE141529 and GSE235508 were analyzed.Bioinformatics tools,including differential analysis,principal coordinate analysis,and pathway enrichment analysis were used to confirm the characteristic expression profiles of RA-,RA-active-,and MTX-treated patients,identify the major enrichment pathways,and analyze the pattern of changes in bioclock genes.Results: Significant differences were found in gene expression between patients with RA and healthy controls,and the differential genes were mainly enriched in the pathways of "circadian control”," immune inflammation " and " cell cycle”.Patients with RA and healthy individuals were grouped significantly on the basis of differential gene expression.Biological clock genes exhibited different dynamic patterns of change in different pathological states and treatment processes of RA,especially PER-2 and CRY-1,which showed statistically significant differences.Conclusion: This part of the study revealed that signaling pathways,including rhythm regulation,immuno-inflammatory and cell cycle are the main pathways enriched by differential genes under different pathological states in RA patients.These results confirmed that biological clock genes have different expression patterns in different pathological stages of RA.Part Ⅱ: Circadian patterns of pro-inflammatory cytokines,bone-resorption factors,and bone protective agents,and the regulatory role of clock genes in CIA rat models Objective: This study explored the circadian patterns of pro-inflammatory cytokines,boneresorption factors,and bone-protective agents as well as the regulatory role of clock genes by constructing collagen-induced arthritis(CIA)rat models(in vivo).Methods: This study investigated changes in the arthritis index,paw volume,and histopathological alterations in the joints of CIA rats at different zeitgeber time(ZT)(ZT2,ZT6,ZT10,ZT14,ZT18,and ZT22).The expression of pro-inflammatory cytokines(TNF-α,IL-6,IL-17,IL-1β,and MIF),bone resorption factors(DKK-1 and RANKL),and the bone protective agent,osteoprotegerin(OPG),in the serum of CIA rats was detected by ELISA at different ZT.Protein expression of the pro-inflammatory factor MIF,bone-resorption factors,and OPG in joint tissues of CIA rats was detected by western blot at different ZT.The expressions of clock genes CLOCK,BMAL-1,PER-2,and CRY-1 in joint tissues of CIA rats were measured using RT-q PCR at different ZT.Circadian rhythm analysis was conducted using the Circa Compare package in R and the fitted cosine curves were visualized graphically.Results: Compared with the control group,CIA rats in the model group exhibited a significantly increased arthritis index and paw volume.Hematoxylin and eosin(HE)staining revealed cartilage damage,synovial tissue hyperplasia,and inflammatory cell infiltration.The histopathological score of the joint tissue was significantly higher than that in the control group.Compared to the control group,the expression of pro-inflammatory cytokines and bone-resorption factors was significantly increased,and the expression of OPG was significantly decreased in the CIA model group.The expression of TNF-α,IL-6,IL-17,IL-1β,MIF,DKK-1,RANKL increased significantly,while the expression of OPG decreased significantly during the dark period(ZT14,ZT18,and ZT22).Western blot analysis of joint tissues demonstrated that the expression of RANKL was significantly increased in the CIA model group,displaying a diurnal rhythm of decreasing during the light period and increasing during the dark period.The expression of OPG in the CIA model group significantly decreased at ZT2 and ZT22,exhibiting a diurnal rhythm of that increasing during the light period and decreasing during the dark period.The expression of DKK-1 in the CIA model group significantly increased at ZT2,ZT6,ZT10,ZT18,and ZT22,showing a diurnal rhythm of decreasing during the light period and increasing during the dark period.In the CIA model group,MIF expression was significantly increased at ZT2,ZT6,ZT10,ZT18,and ZT22,displaying a clear diurnal rhythm of decreasing during the light period and increasing during the dark period.RT-q PCR results of the expression CLOCK and BMAL-1 in joint tissues of CIA rats was higher than that in the control group,with a characteristic decrease during the light period and an increase during the dark period.The expression levels of PER-2 and CRY-1 were lower in the CIA model group than the control group,showing an increase during the light period and a decrease during the dark period.Diurnal changes in CLOCK and BMAL-1 were opposite to those in PER-2 and CRY-1.Further analysis of circadian rhythms demonstrated that CLOCK and BMAL-1 exhibited diurnal patterns in terms of their m RNA expression levels in the control group,whereas no diurnal patterns were observed in the CIA model group.The m RNA expression levels of CRY-1 in both model and control groups showed diurnal patterns,with significantly lower amplitudes in the CIA model group.Conclusion: The results of this part of the study indicated that expression levels of proinflammatory cytokines(TNF-α,IL-6,IL-17,IL-1β,and MIF)and bone-resorption factors(DKK-1 and RANKL)in CIA rats significantly increased at night,while expression level of OPG decreased at night,suggesting that the inflammatory response is exacerbated during the nocturnal period.The protein expression of RANKL,DKK-1,and MIF in joint tissues of CIA rats was significantly increased,whereas OPG protein expression was decreased.RANKL,OPG,DKK-1,and MIF exhibited diurnal rhythms,with diurnal changes in expression of RANKL,DKK-1,and MIF opposite to those of OPG.Dynamic changes in clock genes(CLOCK,BMAL-1,PER-2,and CRY-1)in the control and CIA groups also suggested that suggested that there may be a close regulatory relationship between RA inflammation and clock genes.Part Ⅲ: Circadian patterns of RANKL/OPG in RA-FLS and the regulatory role of clock genes in inflammationObjective: This section of the study investigated the circadian patterns of RANKL/OPG and the regulatory role of clock genes in inflammation through the establishment of IL-6/s IL-6R-induced rheumatoid arthritis fibroblast-like synoviocyte(RA-FLS)models,and explored the effects of MTX and TG on the RANKL/OPG ratio and circadian rhythms.Methods: This study collected synovial tissue from patients with RA,cultured synovial cells,and identified them by microscopic observation and flow cytometry.RT-q PCR and western blot were used to investigate the effects of different concentrations and incubation times of IL-6/s IL-6R on RANKL expression in RA-FLS.Western blot was used to detect the expression levels of RANKL and clock genes(CLOCK,BMAL-1,PER-2,and CRY-1)in IL-6/s IL-6R-induced RA-FLS.Circadian rhythm analysis and visualization of the fitting cosine curves were performed using the Circa Compare package in R.CRISPR technology was used to knockout clock genes and explore their regulatory roles in inflammatory factors.The CCK-8 assay was used to assess the effects of MTX and TG on the viability of RA-FLS.Additionally,RT-q PCR was performed to determine the effects of MTX and TG on the expression of RANKL and OPG in RA-FLS,and circadian rhythm analysis was conducted using the R language Circa Compare package.Results: Synovial cells were observed under the microscope and exhibited a spindle-shaped morphology when passaged to the fifth generation.Flow cytometry analysis revealed that these cells were RA-FLS,positive for cadherin-11 but negative for CD14 and HLA-DR.The expressions of RANKL m RNA and protein in IL-6/s IL-6R-induced RA-FLS were the highest when the concentration of IL-6/s IL-6R was 50 ng/m L and the incubation time was48 h.Western blot analysis of RANKL and clock gene expression in IL-6/s IL-6R-induced RAFLS showed opposing trends with a 24-hour periodicity for RANKL,CLOCK,BMAL-1,and PER-2,CRY-1 proteins.Circadian rhythm analysis using the R language Circa Compare package revealed significant 24-hour circadian rhythms in all groups.After knocking out CLOCK and BMAL-1,inflammatory cytokines IL-1β,IL-17,and TNF-α were significantly reduced,while knocking out PER-2 and CRY-1 resulted in significant elevations of these inflammatory cytokines.RT-q PCR analysis of the effects of MTX and TG on expression levels of RANKL and OPG m RNA in IL-6/s IL-6R-induced RA-FLS revealed that untreated RA-FLS exhibited distinct circadian rhythms in RANKL and OPG m RNA expression,with opposing rhythmic trends.Both MTX and TG significantly inhibited RANKL m RNA expression,with MTX exhibiting the more pronounced effect.Both MTX and TG promoted OPG m RNA expression,with MTX exhibiting significantly stronger stimulatory effects than TG.However,MTX and TG exerted no significant effects on RANKL and OPG rhythms.CCK-8 assays demonstrated that both MTX and TG significantly inhibited the proliferation of IL-6/s IL-6R-induced RA-FLS cells.Conclusion: The expression of RANKL and clock genes protein in IL-6/s IL-6R-induced RA-FLS exhibited significant circadian rhythms.Specifically,the patterns of expression of RANKL,CLOCK,and BMAL-1 were in contrast with those of PER-2 and CRY-1,suggesting that RA-FLS are regulated by circadian rhythms mediated by clock genes.Furthermore,clock gene knockout experiments demonstrated that clock genes play a regulatory role in the expression of inflammatory factors in RA.RANKL and OPG m RNA expression in RA-FLS displayed distinct circadian rhythms,with opposing rhythmic trends.MTX and TG inhibited RA-FLS proliferation.Additionally,both MTX and TG suppressed RANKL m RNA expression and promoted OPG m RNA expression in RA-FLS.Notably,MTX exhibited stronger inhibitory effects on RANKL and a more significant promotional effect on OPG than TG.However,MTX and TG had no significant effects on RANKL and OPG rhythms.