Resveratrol-induced SIRT1 activation inhibits glycolysis-fueled angiogenesis under rheumatoid arthritis conditions independent of HIF-1α.

OBJECTIVE: This study investigated the impacts of SIRT1 activation on rheumatoid arthritis (RA)-related angiogenesis. METHODS: HUVECs were cultured by different human serum. Intracellular metabolites were quantified by UPLC-MS. Next, HUVECs and rat vascular epithelial cells under different inflammatory conditions were treated by a SIRT1 agonist resveratrol (RSV). Cytokines and biochemical indicators were detected by corresponding kits. Protein and mRNA expression levels were assessed by immunoblotting and PCR methods, respectively. Angiogenesis capabilities were evaluated by migration, wound-healing and tube-formation experiments. To down-regulate certain signals, gene-specific siRNA were applied. RESULTS: Metabolomics study revealed the accelerated glycolysis in RA serum-treated HUVECs. It led to ATP accumulation, but did not affect GTP levels. RSV inhibited pro-angiogenesis cytokines production and glycolysis in both the cells, and impaired the angiogenesis potentials. These effects were mimicked by an energy metabolism interrupter bikini in lipopolysaccharide (LPS)-primed HUVECs, largely independent of HIF-1α. Both RSV and bikinin can inhibit the activation of the GTP-dependent pathway Rho/ROCK and reduce VEGF production. Abrogation of RhoA signaling reinforced HIF-1α silencing-brought changes in LPS-stimulated HUVECs, and overshadowed the anti-angiogenesis potentials of RSV. CONCLUSION: Glycolysis provides additional energy to sustain Rho/ROCK activation in RA subjects, which promotes VEGF-driven angiogenesis and can be inhibited by SIRT1 activation.

Xanthones as α-glucosidase inhibitors from the antihyperglycemic extract of Securidaca inappendiculata.

CONTEXT: Securidaca inappendiculata Hassk. (SI) is used to cure fractures and rheumatoid arthritis in China. Also, it is a potential antidiabetes drug; however, there are no reports on this. OBJECTIVE: The study was designed to evaluate the antihyperglycemic activities of fractions and compounds from SI, and attempt to explore the mechanism. MATERIALS AND METHODS: Antihyperglycemic activities were evaluated by the suppression on serum glucose levels in vivo and α-glucosidase inhibition assays in vitro. Fractions were given to mice by gastric intubation for 8 d. The high, medium, and low doses of fractions were equal to 10, 5, and 2.5 g/kg of the herb [SID (dichloromethane fraction) and SIE (ethyl acetate fraction) were doubled]. The serum glucose was monitored at 1 and 12 h after feeding. The silica gel and LH-20 chromatography were used to isolate active compounds. Structure-activity relationship analysis was based on IC50s and structures. RESULTS: The IC50s of SID, SIE, SIA (acetone fraction), SIM (methanol fraction), and acarbose were 712, 446, 1123, 1418, and 735 µg/mL. The postprandial and fasting serum glucose levels of SID, SIE, SIA, and SIM (high dose) were 5.5, 5.9, 6.2, 6.3 and 3.7, 3.5, 4.0, 5.0 mmol/L, while those of vehicle control were 7.5 and 5.6 mmol/L. Eleven xanthones isolated all exhibited inhibitory activities, mainly in a non-competitive reversible manner. The IC50s varied from 3.2 to 77.3 µg/mL. Structure-activity relationship analysis exhibited free hydroxyls contributed the most importance to the activity. CONCLUSION: The results indicated that xanthones from SI were powerful agents for antidiabetes.

Consumption of Saturated Fatty Acids-Rich Lard Benefits Recovery of Experimental Arthritis by Activating PPAR-γ.

SCOPE: This study investigates the impacts of lard and related fatty acids intake on rheumatoid arthritis (RA) animal models. METHOD AND RESULTS: Collagen-induced arthritis (CIA) and adjuvant-induced arthritis (AIA) are induced in SD rats and C57 BL/6 mice respectively, which are fed by lard-rich diet (LRD) for 42 days with intake restriction or not. AIA SD rats are treated by representative fatty acids for 30 days. Body weight, arthritis score, and metabolic profile are periodically recorded. Monocyte distribution, cytokine/metabolites levels, gene expression, and tissue damages are investigated by flow cytometry, ELISA, colorimetry, PCR, and histological methods. After being treated by fatty acids in vitro, THP-1 monocytes and the corresponding medium are collected for ELISA, PCR, immunoblotting, and reporter gene assays. Irrespective of intake amounts, LRD decreases inflammatory cytokines and inhibits glycolysis in all rheumatic rodents. Furthermore, it alters monocyte distribution and promotes PPAR-γ expression in AIA mice. Overall evidences show that both saturated (SF) and unsaturated fatty acids (USF) from lard can attenuate inflammation by activating PPAR-γ. Silencing PPAR-γ abrogates their anti-inflammatory effects in vitro. Besides, SF can stimulate TLR4/NF-κB pathway. CONCLUSION: Lard consumption is beneficial for active inflammatory arthritis recovery. Even SF can activate PPAR-γ and consequently attenuate inflammation.

α-Mangostin Inhibited M1 Polarization of Macrophages/Monocytes in Antigen-Induced Arthritis Mice by Up-Regulating Silent Information Regulator 1 and Peroxisome Proliferators-Activated Receptor γ Simultaneously.

Background: α-Mangostin (MG) showed the potentials in alleviating experimental arthritis, inhibiting inflammatory polarization of macrophages/monocytes, and regulating peroxisome proliferators-activated receptor γ (PPAR-γ) and silent information regulator 1 (SIRT1) signals. The aim of this study was to analyze the correlations among the above-mentioned properties. Methods: Antigen-induced arthritis (AIA) was established in mouse, which was treated with MG in combination with SIRT1/PPAR-γ inhibitors to clarify the role of the two signals in the anti-arthritic actions. Pathological changes were systematically investigated. Phenotypes of cells were investigated by flow cytometry. Expression and co-localization of SIRT1 and PPAR-γ proteins in joint tissues were observed by the immunofluorescence method. Finally, clinical implications from the synchronous up-regulation of SIRT1 and PPAR-γ were validated by experiments in vitro. Results: SIRT1 and PPAR-γ inhibitors (nicotinamide and T0070097) reduced the therapeutic effects of MG on AIA mice, and abrogated MG-induced up-regulation of SIRT1/PPAR-γ and inhibition of M1 polarization in macrophages/monocytes. MG has a good binding affinity to PPAR-γ, and MG promoted the co-expression of SIRT1 and PPAR-γ in joints. Synchronously activating SIRT1 and PPAR-γ was revealed to be necessary by MG to repress inflammatory responses in THP-1 monocytes. Conclusion: MG binds PPAR-γ and excites this signaling to initiate ligand-dependent anti-inflammatory activity. Due to certain unspecified signal transduction crosstalk mechanism, it then promoted SIRT1 expression and further limited inflammatory polarization of macrophages/monocytes in AIA mice.

AMPK/SIRT1 Deficiency Drives Adjuvant-Induced Arthritis in Rats by Promoting Glycolysis-Mediated Monocytes Inflammatory Polarization.

Background: Exact roles of many metabolic regulators in rheumatoid arthritis (RA) are to be clarified. This study aimed to further characterize the impacts of silent information regulator 1 (SIRT1) status changes on this disease. Methods: Fluctuation pattern of SIRT1 expression in adjuvant-induced arthritis (AIA) rats was monitored using periodically collected white blood cells. Another bath of AIA rats were treated by SIRT1 agonist resveratrol. Blood from these rats was used to separate monocytes and plasma, which were subjected to polymerase chain reaction (PCR), enzyme linked immunosorbent assay (ELISA), and biochemical analyses. Clinical implication of SIRT1 activation was verified by treating AIA rat monocytes with SIRT1 agonist and overexpression vector in vitro. Results: SIRT1 deficiency occurred in AIA rats, which was accompanied with down-regulation of interleukin 10 (IL-10) and arginase-1 (ARG-1). Resveratrol eased oxidative stress and increased IL-10 production in vivo. Results of ELISA analysis demonstrated that resveratrol attenuated AIA severity in rats. Furthermore, it restored the altered levels of triglyceride, lactate and pyruvate in blood. Resveratrol promoted IL-10 production, and suppressed glycolysis of AIA monocytes cultured in vitro. SIRT1 overexpression similarly reshaped differentiation profile of AIA monocytes, evidenced by changes in metabolism indicators, IL-10 production and AMP-activated protein kinase (AMPK) pathway status. Although overexpressing SIRT1 in normal cells did not affect glycolysis significantly, it attenuated AMPK antagonist-caused abnormality. Conclusion: SIRT1 deficiency is implicated in AIA-related immune abnormality and metabolism alteration. Activating this signaling with resveratrol would impair the inflammatory polarization of monocytes, and consequently ease the severity of RA.

Polyphenols from Securidaca inappendiculata alleviated acute lung injury in rats by inhibiting oxidative stress sensitive pathways.

Objective: Securidaca inappendiculata is a medicinal plant frequently used in the treatment of inflammatory diseases in south China. In this study, we aimed to explore its bioactive constituent which contributes to the anti-inflammatory activity. Methods: Polyphenol-enriched and polyphenol-deprived fractions (PRF and PDF, respectively) were separated from the ethanolic extract by HPD300 macroporous resin-based method, and their anti-inflammatory activities were investigated on a lipopolysaccharide (LPS)-induced acute lung injury (ALI) model in rats. The possible mechanism of action in alleviating acute inflammation was studied using RAW264.7 cells. Results: Both Folin-Ciocalteu and 1H nuclear magnetic resonance (NMR) analyses showed that polyphenolic content in PRF was approximately 10 times higher than that of PDF, and this observation reflected in their antioxidative capacities. PRF but not PDF significantly decreased the level of malondialdehyde, suppressed the expression of nicotinamide phosphoribosyltransferase (NAMPT) protein, and improved the severity of ALI in rats. PRF at 10 µg/mL effectively downregulated the expression of proteins NAMPT, HMGB1, TLR4, and p-p65, and scavenged the intracellular reactive oxygen species (ROS) in LPS-primed RAW264.7 cells. N-acetyl-L-cysteine exhibited similar inhibitory effects on ROS production and NAMPT-mediated TLR4/NF-κB activation in vitro, whereas nicotinamide mononucleotide antagonized all the changes induced by PRF during cotreatments. Conclusion: As an antioxidant, PRF exhibited potent anti-inflammatory activity under both in vivo and in vitro conditions by downregulating NAMPT and TLR4/NF-κB. Accordingly, polyphenols were identified as important bioactive constituents in S. inappendiculata targeting oxidative stress-sensitive pro-inflammatory pathways.

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.

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.

Bioactive fractions from Securidaca inappendiculata alleviated collagen-induced arthritis in rats by regulating metabolism-related signaling.

Securidaca inappendiculata is a xanthone rich medicinal plant that has been used in the treatment of inflammation and autoimmune diseases like rheumatoid arthritis (RA) for centuries; however, the material base and mechanism of action responsible for its anti-arthritis effect still remains elusive. The objective of this study is to evaluate the therapeutic effects of xanthone-enriched extract of the plant against collagen-induced arthritis (CIA) in rats and explore the underlying mechanisms. The xanthone-deprived fraction (XDF) and xanthone-rich fraction (XRF) were obtained by using a resin adsorption coupled with acid-base treatment method, and their chemical composition difference was characterized by UPLC-MS/MS analysis. Effects of the two on CIA were analyzed using radiographic, histological, and immunohistochemical analyses. The results indicated that XRF alleviated joint structures destructions with the higher efficacy than XDF, and decreased levels of TNF-α, IL-6, and anti-cyclic citrullinated peptide antibody in CIA rats significantly. Furthermore, XRF inhibited nicotinamide phosphoribosyl transferase (NAMPT) mediated fat biosynthesis and utilization indicated by clinical evidences and metabonomics analysis, which thereby disrupted energy-metabolism feedback. In addition, Toll-like Receptor 4 and High Mobility Group Protein 1 expressions were downregulated in XRF-treated CIA rats. Collective evidences suggest NAMPT could be an ideal target for RA treatments and reveal a novel antirheumatic mechanism of S. inappendiculata by regulating NAMPT controlled fat metabolism.

Fatty oil from Securidaca inappendiculata exerted therapeutic effects on adjuvant-induced arthritis in mice via suppression on fibroblast-like synoviocyte.

Securidaca inappendiculata Hassk. (SI) is a medicinal plant used to treat rheumatoid arthritis (RA) in South China. A substantial amount of fatty oil was isolated from SI (SIF), however little knowledge about its chemical composition and medicinal potentials was obtained. In this study, we analyzed its chemical composition with methyl esterification based GC-MS method, and investigated the therapeutic potentials on adjuvant-induced arthritis (AA) in mice. MTT and western-blot methods were employed to investigate its effects on proliferation rate and protein expressions in MH7A cells, respectively. It was revealed SIF was mainly comprised of saturated and monosaturated fatty acids, and the two predominant compounds were palmitic acid (36.89%) and oleic acid (31.12%). Treatment with SIF at 100 mg/kg resulted in significant alleviation of AA severity in mice, together with reduced synovial hyperplasia and inflammatory infiltration in joints, and decreased levels of sialic acid, malondialdehyde and alkaline phosphatase in serum. Results from immunohistochemical assays hinted the protective effects of SIF on joints were associated to the inhibition on production of some pathological factors in synovium, including IL-1ß, TNF-α and MMP-9. SIF inhibited the proliferation of MH7A cells in a concentration dependent manner, and abrogated phosphorylation of p65 in vitro. These evidences collectively suggested SIF could suppress the pathological functions of fibroblast-like synoviocyte, and protect joints from destruction under AA conditions.

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.