Securidaca inappendiculata-Derived Xanthones Protected Joints from Degradation in Male Rats with Collagen-Induced Arthritis by Regulating PPAR-γ Signaling.

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-1ß 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.

Metabolic Enzyme Triosephosphate Isomerase 1 and Nicotinamide Phosphoribosyltransferase, Two Independent Inflammatory Indicators in Rheumatoid Arthritis: Evidences From Collagen-Induced Arthritis and Clinical Samples.

Metabolic intervention is a novel anti-rheumatic approach. The glycolytic regulator NAMPT has been identified as a therapeutic target of rheumatoid arthritis (RA), while other metabolic regulators coordinating NAMPT to perpetuate inflammation are yet to be investigated. We continuously monitored and validated expression changes of Nampt and inflammatory indicators in peripheral while blood cells from rats with collagen-induced arthritis (CIA). Gene transcriptional profiles of Nampt+ and Nampt++ samples from identical CIA rats were compared by RNA-sequencing. Observed gene expression changes were validated in another batch of CIA rats, and typical metabolic regulators with persistent changes during inflammatory courses were further investigated in human subjects. According to expression differences of identified genes, RA patients were assigned into different subsets. Clinical manifestation and cytokine profiles among them were compared afterwards. Nampt overexpression typically occurred in CIA rats during early stages, when iNos and Il-1ß started to be up-regulated. Among differentially expressed genes between Nampt+ and Nampt++ CIA rat samples, changes of Tpi1, the only glycolytic enzyme identified were sustained in the aftermath of acute inflammation. Similar to NAMPT, TPI1 expression in RA patients was higher than general population, which was synchronized with increase in RFn as well as inflammatory monocytes-related cytokines like Eotaxin. Meanwhile, RANTES levels were relatively low when NAMPT and TPI1 were overexpressed. Reciprocal interactions between TPI1 and HIF-1α were observed. HIF-1α promoted TPI1 expression, while TPI1 co-localized with HIF-1α in nucleus of inflammatory monocytes. In short, although NAMPT and TPI1 dominate different stages of CIA, they similarly provoke monocyte-mediated inflammation.

Activation of Cholinergic Anti-Inflammatory Pathway in Peripheral Immune Cells Involved in Therapeutic Actions of α-Mangostin on Collagen-Induced Arthritis in Rats.

BACKGROUND: Studies have shown that α-mangostin (MG) could exert anti-rheumatic effects in vivo by restoring immunity homeostasis, and have indicated that activation of the choline anti-inflammatory pathway (CAP) may contribute to this immunomodulatory property. The current study was designed to further investigate the effects of MG on the CAP in peripheral immune cells and clarify its relevance to the potential anti-rheumatic actions. METHODS: The catalytic activity of acetylcholinesterase (AChE) and expression of α7-nicotinic cholinergic receptor (α7nAChR) in peripheral blood mononuclear cells (PBMCs) from rats with collagen-induced arthritis (CIA) or human volunteers were evaluated after MG treatment. Consequent influences on the immune environment were assessed by flow cytometry and ELISA analyses. Indirect effects on joints resulting from these immune changes were studied in a co-culture system comprised of fibroblast-like synoviocytes (FLSs) and PBMCs. RESULTS: MG promoted α7nAChR expression in PBMCs both in vivo and in vitro, and inhibited the enzymatic activity of AChE simultaneously. Activation of the CAP was accompanied by a significant decrease in Th17 cells (CD4+IL-17A+), while no obvious changes concerning the distribution of other T-cell subsets were noticed upon MG treatment. Meanwhile, MG decreased the secretion of TNF-α and IL-1ß under inflammatory conditions. PBMCs from MG-treated CIA rats lost the potential to stimulate NF-κB activation and pro-inflammatory cytokine production of FLSs in the co-culture system. CONCLUSION: Overall, the evidence suggested that MG can improve the peripheral immune milieu in CIA rats by suppressing Th17-cell differentiation through CAP activation, and achieve remission of inflammation mediated by FLSs.

1,7-Dihydroxy-3,4-Dimethoxyxanthone Inhibits Lipopolysaccharide-Induced Inflammation in RAW264.7 Macrophages by Suppressing TLR4/NF-κB Signaling Cascades.

Securidaca inappendiculata Hassk. is a traditional Chinese anti-rheumatic herbal medicine native to southern China. In this study, we identified a possible TLR4 inhibitor from this plant. General effects of its xanthone-rich fraction (XRF) on inflammation in vitro were investigated by immunoblotting experiments performed on lipopolysaccharides (LPS)-treated RAW264.7 cells, and the possible ligand of TLR4 within was screened out by analyzing chemical composition differences of the XRF containing cell culture medium under different inflammatory circumstances. The interaction between ligand and TLR4 was validated by cellular thermal shift assay (CETSA) and molecular docking simulation, and TLR4/NF-κB pathway status was investigated by immunoprecipitation, ELISA, immunofluorescence, dual-luciferase reporter, and immunoblotting experiments. Treatment with XRF resulted in significant decrease in p-p65 and p-JNK, and the signal accounting for 1,7-dihydroxy-3,4-dimethoxyxanthone (XAN) at 12.5 min with mass of 289.29 was greatly decreased in XRF containing medium after LPS stimulus because of enhanced interaction with increased TLR4. CETSA and molecular docking simulation demonstrated that XAN could bind to TLR4 directly on a smooth region adjacent to its contact interface with MD-2. XAN treatment inhibited the dimerization of TLR4 and transcriptional activity of NF-κB in HEK293T cells and decreased p65 accumulation in nucleus and pro-inflammatory cytokines production in RAW264.7 cells receiving LPS treatment. Overall evidences suggest that XAN could be a selective TLR4 inhibitor with potent anti-inflammatory effects. Also, it indicated that xanthone derivatives could have promising clinical application in many immune-mediated inflammations by acting as TLR4 inhibitors.

Selective regulation of IKKß/NF-κB pathway involved in proliferation inhibition of HFLS-RA cells induced by 1,7-dihydroxyl-3,4-dimethoxylxanthone.

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-IKKß, p-IκB and p-p65 suggested the regulation on NF-κB was IKKß 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 IKKß/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.