| Objective: RA is a common autoimmune disease affecting almost 1% population worldwide.Although the existing conventional RA drugs can relieve symptoms,but they cannot curb the disease process.Meanwhile,their toxicity and side effects are obvious.Securidaca inappendiculata Hassk.is a traditional herbal anti-rheumatic medicine used in south China.We have confirmed that xanthone derivatives from it have promising potentials in treatment of RA.In this study,we further confirmed that these compounds contributed to the anti-inflammatory property of this plant directly by targeting TLR4,and improved the immune homeostasis by intervening into the immune-metabolism feedback in CIA rats.Methods: 1.To identify components in XRF accounting for the eased inflammation in RAW264.7 cells pre-treated by LPS,we systematically evaluated the status of typical inflammatory pathways using WB method.The bioactivity of compounds with high affinity with TLR4 was further validated by molecular docking simulation,cellular thermal shift assay,immunoprecipitation,ELISA,double luciferase reporter and immunofluorescence techniques.2.To decipher the implication of regulation on immunemetabolism feedback by XRF treatments,the CIA model was adopted in the in vivo study.The CIA were treated by either MTX(0.33 mg/kg)or XRF(in the form of XRF-SU or XRF-ME,50 mg/kg)by gavage for 28 days.The therapeutic efficacy was preliminarily assessed by arthritis scores,serological analysis,morphology observation and histological examination(stained by HE/SOFG).The possible therapeutic targets were screened out using the genome microarray strategy,and obtained results were further validated by immunohistochemical,WB and metabonomics analyses.Using the co-culture system comprised of macrophage and pre-adipocytes,we investigated the interplay between immune system and energy metabolism under different physiological/pathological conditions,and assessed the influence of a representative S.inappendiculata-derived compound on this feedback using QPCR,ELISA and WB methods.Results: 1.XRF stimulus inhibited the activation of TLR4/NF-κB in LPS-primed RAW264.7 cells.2.A representative component from S.inappendiculata XAN selectively binds to TLR4 on RAW264.7 cells,and consequently hinders the dimerization of TLR4.3.XAN reduced the secretion of TNF-α and IL-1β in LPS-stimulated RAW264.7 cells in vitro,avoided the accumulation of p-p65 in nucleus,and inhibited the transcriptional activity of NF-κB.4.XRF treatments reduced circulating TNF-α,IL-1β and IL-17α in serum,down-regulated TLR4/NF-κB and JNK pathways in joints,and impaired the M1 polarization of macrophages in CIA rats.Compared with MTX,XRF exhibited more potent effects in alleviating the joints structure degradation.5.The genome microarray analysis indicated that XRF promoted the expression of many extracellular matrix genes,typically collagens.Meanwhile,many genes involved in rheumatoid arthritis as well as PPAR pathway were greatly altered during XRF treatments.6.The LC-MS analysis-based metabonomics study revealed that the biosynthesis of fat acids was decreased upon XRF treatments,while the degradation of type II Collagen seems to be slowed down.7.XAN up-regulated PPAR-γ expression in macrophages,but suppressed it in pre-adipocytes in vitro,which was synchronized with opposite SIRT1 changes.Adiponectin production and SCD-1 expression in pre-adipocytes were decreased too.Furthermore,the inhibition of XRF on MMP3 expression in synovioblast was further reinforced by the macrophagespre-adipocytes co-culture system.Conclusion: 1.S.inappendiculata-derived xanthones are capable in easing macrophage-controlled inflammations by targeting TLR4,which consequently lead to improved synovitis and other local inflammatory symptoms.2.XAN and relative components have the potential in suppressing the erosive nature of synovioblast acquired under inflammatory circumstances by regulating PPAR-γ signaling controlled metabolism-immunity feedback. |
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