NAMPT/SIRT1 Expression Levels in White Blood Cells Differentiate the Different Rheumatoid Arthritis Subsets: An Inspiration from Traditional Chinese Medicine.

Background: Rheumatoid arthritis (RA) patients are prone to developing different metabolic complications. Traditional Chinese Medicine attributes this uncertainty to varied syndrome types. Methods and Results: We retrospectively analyzed some serological indicators of active RA patients and healthy individuals. Randomly selected RA patients were divided into three groups according to NAMPT and SIRT1 expression levels in white blood cells (WBCs). Their disease severity and metabolic status were compared. Representative blood samples were subjected to a UPLC-MS/MS-based metabolomics analysis. Different human WBCs were treated with oleic acid and palmitic acid in vitro. The results indicated that blood glucose and lipid levels were decreased in RA patients, but their decrease was not in accordance with disease severity. Nutrients in the patients highly expressing SIRT1 were well preserved, with the lowest levels of RF and ß-CTX and the highest levels of adiponectin and resistin. Most of them exhibited cold symptoms. When SIRT1 deficiency was obvious, lipid depletion became evident, irrespective of expression levels of NAMPT. Simultaneous high-expression of SIRT1 and NAMPT coincided with the increase in production of lactic acid and the prevalence of hot symptoms. Despite the low levels of IL-6, joint injuries were severe. The corresponding WBCs were especially sensitive to fatty acids anti-inflammatory treatments. The levels of CCL27, CCL11, CCL5, AKP, CRP and ESR were similar among all the groups. Conclusion: NAMPT overexpression is a risk factor for joint injuries and nutrient depletion in RA. Supplementation with lipids would exert beneficial effects on these RA patients. Its aftermath would cause even severe inflammation. Contrarily, SIRT1 up-regulation restrains inflammation and lipid depletion.

Glycolysis aggravates methotrexate toxicity by fueling RFC1-controlled intestinal absorption in rheumatic rats.

Methotrexate (MTX) is a first line anti-rheumatic drug. This study was designed to investigate the impact of rheumatoid arthritis (RA) conditions on its oral absorption, and clarify the relevance with changes of MTX absorption-related transporters in rheumatic models. MTX was orally administered to healthy, collagen-induced arthritis (CIA), and adjuvant-induced arthritis (AIA) rats. MTX plasma concentrations were determined by a validated liquid chromatography-mass spectrometry method. We found that intestinal MTX absorption was significantly increased in CIA/AIA rats versus healthy controls. This finding was supported by small intestine-based MTX uptake assay in vitro. Meanwhile, intestinal expression of both reduced folate carrier 1 (RCF1) and proton-coupled folate transporter (PCFT) remained unchanged. The everted intestinal sac assay confirms RFC1 is the key transporter accounting for intestinal MTX absorption, as its antagonist salicylazosulfapyridine showed potent capacity in reducing MTX uptake. No correlation between RA-related cytokines and RCF1 expression was observed in clinical samples. We further revealed that when cultured with AIA rat or RA patient serum, lactate and adenosine triphosphate (ATP) production as well as MTX uptake in MDCKII cells were significantly increased, and this increase was completely abrogated by ATP production-related metabolic inhibitors. Thanks to its inhibitory effects on MTX bioavailability, the glycolysis inhibitor shikonin diminished MTX-induced injuries of kidney and liver in AIA rats. These data demonstrate that glycolysis-driven high energy metabolism increases MTX absorption in rheumatic subjects, leading to the exacerbated toxicity. These findings will have important implications in optimizing MTX regimens for RA treatment with better efficacy and lower toxicity.

The Effect of α7nAChR Signaling on T Cells and Macrophages and Their Clinical Implication in the Treatment of Rheumatic Diseases.

Rheumatoid arthritis (RA) is one of the most common autoimmune disease and until now, the etiology and pathogenesis of RA is not fully understood, although dysregulation of immune cells is one of the leading cause of RA-related pathological changes. Based on current understanding, the priority of anti-rheumatic treatments is to restore immune homeostasis. There are several anti-rheumatic drugs with immunomodulatory effects available nowadays, but most of them have obvious safety or efficacy shortcomings. Therefore, the development of novel anti-rheumatic drugs is still in urgently needed. Cholinergic anti-inflammatory pathway (CAP) has been identified as an important aspect of the so-called neuro-immune regulation feedback, and the interaction between acetylcholine and alpha 7 nicotinic acetylcholine receptor (α7nAChR) serves as the foundation for this signaling. Consistent to its immunomodulatory functions, α7nAChR is extensively expressed by immune cells. Accordingly, CAP activation greatly affects the differentiation and function of α7nAChR-expressing immune cells. As a result, targeting α7nAChR will bring profound therapeutic impacts on the treatment of inflammatory diseases like RA. RA is widely recognized as a CD4+ T cells-driven disease. As a major component of innate immunity, macrophages also significantly contribute to RA-related immune abnormalities. Theoretically, manipulation of CAP in immune cells is a feasible way to treat RA. In this review, we summarized the roles of different T cells and macrophages subsets in the occurrence and progression of RA, and highlighted the immune consequences of CAP activation in these cells under RA circumstances. The in-depth discussion is supposed to inspire the development of novel cell-specific CAP-targeting anti-rheumatic regimens.

Qing-Luo-Yin Alleviated Experimental Arthritis in Rats by Disrupting Immune Feedback Between Inflammatory T Cells and Monocytes: Key Evidences from Its Effects on Immune Cell Phenotypes.

BACKGROUND: Qing-Luo-Yin (QLY) is an anti-rheumatic herbal formula. Despite the well-investigated therapeutic efficacy of QLY, its immune regulatory properties are largely unknown. CD4+ T cells and monocytes are two key parameters in rheumatoid arthritis (RA). This study investigated the changes in these cells in QLY-treated RA animal models. MATERIALS AND METHODS: RA models were induced in male SD rats and were orally treated with QLY. Dynamic metabolic changes in collagen-induced arthritis (CIA) rats were monitored by 1H NMR approach. The immunity profiles of CIA and adjuvant-induced arthritis (AIA) rats were evaluated using immunohistochemical, PCR, ELISA, cytokine chip, flow cytometry, and immunofluorescence experiments. The bioactive components in QLY were identified by bioinformatic-guided LC-MS analyses. The compounds with high abundance in QLY decoction and easily absorbed were taken as key anti-rheumatic components and used to treat blood-derived immune cells using in vitro experiments. RESULTS: The results indicated that QLY decreased Th17 cells frequency and T cells-released IL-6, IL-17 and GM-CSF in CIA rats, which was attributed to the impaired lymphocyte maturation and altered differentiation. QLY inhibited lactic acid production and inflammatory polarization in the monocytes during the peak period of AIA and CIA. AIA monocytes elicited significant increase in Th17 cells counts, IL-6 and IL-1ß secretion in co-cultured splenocytes, which was abrogated by QLY. QLY-containing serum suppressed the phosphorylation of JNK and p65 in AIA lymphocyte-stimulated normal monocytes and consequently inhibited iNOS and IL-1ß expression as well as IL-6 and IL-1ß production. Matrine, sinomenine and sophocarpine were identified as major bioactive compounds in QLY. These identified compounds effectively inhibited the development of inflammatory T cells using concentrations detected in QLY-treated rats. At higher concentrations (20-fold increase), the chemical stimuli significantly suppressed the production of IL-1ß in AIA monocytes by inhibiting JNK and p65 pathways. CONCLUSION: By targeting inflammatory T cells and monocytes as well as disrupting their interplay, QLY improved immune environment in RA models especially during the active stages of disease.

α-Mangostin Alleviated HIF-1α-Mediated Angiogenesis in Rats With Adjuvant-Induced Arthritis by Suppressing Aerobic Glycolysis.

A previously validated anti-rheumatic compound α-mangostin (MAN) shows significant metabolism regulatory effects. The current study aimed to clarify whether this property contributed to its inhibition on synovial angiogenesis. Male wistar rats with adjuvant-induced arthritis (AIA) were orally treated by MAN for 32 days. Afterwards, biochemical parameters and cytokines in plasma were determined by corresponding kits, and glycometabolism-related metabolites were further accurately quantified by LC-MS method. Anti-angiogenic effects of MAN were preliminarily assessed by joints based-immunohistochemical examination and matrigel plug assay. Obtained results were then validated by experiments in vitro. AIA-caused increase in circulating transforming growth factor beta, interleukin 6, hypoxia inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in blood and local HIF-1α/VEGF expression in joints was abrogated by MAN treatment, and pannus formation within matrigel plugs implanted in AIA rats was inhibited too. Scratch and transwell assays revealed the inhibitory effects of MAN on human umbilical vein endothelial cells (HUVECs) migration. Furthermore, MAN inhibited tubule formation capability of HUVECs and growth potential of rat arterial ring-derived endothelial cells in vitro. Meanwhile, MAN eased oxidative stress, and altered glucose metabolism in vivo. Glycolysis-related metabolites including glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglyceric acid and phosphoenolpyruvic acid in AIA rats were decreased by MAN, while the impaired pyruvate-synthesizing capability of lactate dehydrogenase (LDH) was recovered. Consistently, MAN restored lipopolysaccharide-elicited changes on levels of glucose and LDH in HUVECs culture system, and exerted similar effects with LDH inhibitor stiripentol on glycometabolism and VEGF production as well as tubule formation capability of HUVECs. These evidences show that MAN treatment inhibited aerobic glycolysis in AIA rats, which consequently eased inflammation-related hypoxia, and hampered pathological neovascularization.