Rheumatoid arthritis is a common autoimmune disease, however, available regimes exert little influence on it's long-term prognosis. The aim of the current study is to investigate potential effects of 1,7-dihydroxyl-3,4-dimethoxyl-xanthone (XAN) in HFLS-RA cells and describe the underlying mechanisms of induction of NF-κB activity. Viability of cells was measured by MTT assay. Flow cytometry was employed to assess the pro-apoptotic effects. Modulation on NF-κB signaling was investigated by RT-qPCR, Western-blot and immunofluorescence methods. It was found that XAN induced proliferation inhibition and apoptosis of HFLS-RA cells in the concentration-dependent manner, which were strengthened by pyrrolidinedithiocarbamic acid but antagonized by IKK16. NF-κB signaling was abrogated shortly after the treatment of XAN via various means including mRNA expression, phosphorylation and nuclear translocation, which leaded to up-regulation of p38 and down-regulation of X-linked inhibitor of apoptosis protein. Simultaneous suppressions on p-IKKβ, p-IκB and p-p65 suggested the regulation on NF-κB was IKKβ mediated. Meanwhile, XAN promoted the expression of IKKα, which has a possible connection to pro-apoptotic effects suggested by the up-regulated cleaved PARP. These findings indicated IKKβ/NF-κB mediates the proliferation of HFLS-RA cells inhibited by XAN, and divergent regulations on IKKs could provide synergic effects on the cells' proliferation.
Background
The bark of
Securidaca inappendiculata
Hassk. is traditionally used for treating inflammatory diseases and bone fractures in China. We have previously validated the xanthone-enriched fraction (XRF) of
S. inappendiculata
with anti-rheumatic potentials, but mechanism underlying the joints protective effects is still largely unknown.
Materials and Methods
The male rats with collagen-induced arthritis (CIA) were treated with XRF. The therapeutic efficacy of XRF was evaluated by arthritis score changes, morphological observation of paws, histological examinations and serological analyses. Protein expression in tissues and cells was investigated by either immunohistochemical or immunoblotting methods, while levels of mRNA expression were investigated by RT-qPCR. Metabolites in serum were detected by LC-MS approach. The joints homogenates were used for analyzing possible targeted genes by genome microarray analyses.
Results
Treatment with XRF and methotrexate (MTX) led to significant decrease in arthritis scores, and alleviated deformation of paws in CIA rats. In addition, XRF and MTX reduced circulating TNF-α, IL-1β and IL-17α in the serum and down-regulated TLR4/NF-κB and JNK pathways in joints of CIA rats. Compared to MTX, XRF-loading microemulsion significantly protected joints, which was accompanied by dramatic decrease in MMP3. Differential genes-based KEGG enrichment and metabolomics analysis suggested that XRF reduced fatty acids biosynthesis by regulating PPAR-γ signaling.
S inappendiculata
-derived 1,7-dihydroxy-3,4-dimethoxyxanthone (XAN) up-regulated PPAR-γ expression in macrophages, but suppressed it in pre-adipocytes in vitro, which was synchronized with SIRT1 changes. Adiponectin production and SCD-1 expression in pre-adipocytes were also decreased. Aside the direct inhibition on MMP3 expression in synovioblast, the presence of XAN in macrophages-pre-adipocytes co-culture system further reinforced this effect.
Conclusion
This study revealed the joint protective advantages of the bioactive fraction from
S. inappendiculata
in CIA rats over MTX, and demonstrated that
S. inappendiculata
-derived xanthones suppressed the erosive nature of synovioblast acquired under inflammatory circumstances by regulating PPAR-γ signaling-controlled metabolism-immunity feedback.
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