Unveiling the Nexus: Cellular Metabolomics Unravels the Impact of Estrogen on Nicotinamide Metabolism in Mitigating Rheumatoid Arthritis Pathogenesis.

Rheumatoid arthritis (RA) is a metabolic joint disorder influenced by hormonal regulation, notably estrogen, which plays a cytoprotective role against inflammation. While estrogen's impact on RA pathogenesis has been studied, the altered metabolite expression under estrogen's influence remains unexplored. This study investigated the changes in the metabolome of synovial fibroblasts isolated from RA patients under 17ß-estradiol (E2) using the liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach followed by multivariate and biological pathway analysis along with in vitro validation. Results identified 3624 m/z, among which eight metabolites were significant (p < 0.05). Nicotinate and nicotinamide metabolism was found to be highly correlated with the treatment of E2, with metabolites NAD+ and 1-methynicotinamide (1-MNA) upregulated by E2 induction in RA-FLS. PharmMapper analysis identified potential gene targets of 1-MNA, which were further matched with RA gene targets, and thus, STAT1, MAPK14, MMP3, and MMP9 were concluded to be the common targets. E2 treatment affected the expression of these gene targets and ameliorated the development of oxidative stress associated with RA inflammation, which can be attributed to increased concentration of 1-MNA. Thus, an LC-MS/MS-based metabolomics study revealed the prominent role of estrogen in preventing inflammatory progression in RA by altering metabolite concentration, which can support its therapeutic capacity in remitting RA.

Targeting TNF-α-induced expression of TTR and RAGE in rheumatoid arthritis: Apigenin's mediated therapeutic approach.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease induced by TNF-α, which increases fibroblast-like synoviocytes inflammation, resulting in cartilage destruction. The current work sought to comprehend the pathophysiological importance of TNF-α stimulation on differential protein expression and their regulation by apigenin using in-vitro and in-vivo models of RA. METHODS: The human RA synovial fibroblast cells were stimulated with or without TNF-α (10 ng/ml) and treated with 40 µM apigenin. In-silico, in-vitro and in-vivo studies were performed to confirm the pathophysiological significance of apigenin on pro-inflammatory cytokines and on differential expression of TTR and RAGE proteins. RESULTS: TNF-α induced inflammatory response in synoviocytes revealed higher levels of IL-6, IL-1ß, and TNF-α cytokines and upregulated differential expression of TTR and RAGE. In-silico results demonstrated that apigenin has a binding affinity towards TNF-α, indicating its potential effect in the inflammatory process. Both in-vitro and in-vivo results obtained by Western Blot analysis suggested that apigenin reduced the level of p65 (p = 0.005), TTR (p = 0.002), and RAGE (p = 0.020). CONCLUSION: The findings of this study suggested that TNF-α promotes the differential expression of pro-inflammatory cytokines, TTR, and RAGE via NF-kB pathways activation. Anti-inflammatory effect of apigenin impedes TNF-α mediated dysregulation or expression associated with RA pathogenesis.

Functional Significance of miR-4693-5p in Targeting HIF1α and Its Link to Rheumatoid Arthritis Pathogenesis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes joint inflammation and destruction with an unknown origin. Our study aims to elucidate the molecular mechanism behind HIF1α overexpression in RA. Dysregulated miRNA expressions are known to influence gene behavior, thereby enhancing cell proliferation, inflammation, and resistance to apoptosis, contributing to RA development. Our earlier finding indicated that exogenous miRNA similar to miR-4693-5p may modulate RA-related targets. However, the specific role of miR-4693-5p and its targets in RA remain unexplored. In this study, we found that miR-4693-5p was significantly reduced in PBMCs of RA patients, with evidence suggesting it targets the 3' UTR of HIF1α, thereby potentially contributing to its overexpression in RA. In vitro overexpression of miR-4693-5p leads to the knockdown of HIF1α, resulting in inhibited expression of Survivin to disrupt apoptosis resistance, inflammation suppression, and a reduction in the total cellular ROS response in SW982 and RAFLS cells. The results were validated using the CIA Rat model. In conclusion, this study provides a crucial foundation for understanding the functional role of miR-4693-5p. These findings improve our understanding and provide novel insights into the molecular mechanisms underlying RA pathogenesis.

Mitochondrial functioning in Rheumatoid arthritis modulated by estrogen: Evidence-based insight into the sex-based influence on mitochondria and disease.

Alteration of immune response and synovium microvasculature in Rheumatoid arthritis (RA) progression has been suggested to be associated with mitochondrial functioning. Mitochondria, with maternally inherited DNA, exhibit differential response to the female hormone estrogen. Various epidemiological evidence has also shown the prominence of RA in the female population, depicting the role of estrogen in modulating the pathogenesis of RA. As estrogen regulates the expression of differential proteins and associated signaling pathways of RA, its influence on mitochondrial functioning seems evident. Thus, in this review, the studies related to mitochondria and their relation with estrogen and Rheumatoid arthritis were retrieved. We analyzed the different mitochondrial activities that are altered in RA and the possibility of their estrogenic control. The study expands to in silico analysis, revealing the differential mitochondrial proteins expressed in RA and examining these proteins as potential estrogenic targets. It was found that ALDH2, CASP3, and SOD2 are the major mitochondrial proteins involved in RA progression and are also potent estradiol targets. The analysis establishes the role of mitochondrial proteins in RA progression, which were found to be direct or indirect targets of estrogen, depicting its potential for regulating mitochondrial functions in RA.

Interrelated grid of non-coding RNA: An important aspect in Rheumatoid Arthritis pathogenesis.

Inflammation and autoimmunity are the root cause of rheumatoid arthritis, a destructive disease of joints. Multiple biomolecules are involved in the pathogenesis of RA and are related to various events of molecular biology. RNA is a versatile biomolecule, playing numerous roles at structural, functional, and regulatory stages to maintain cellular homeostasis. The involvement of RNA (coding/non-coding) in disease development and progression has left a wide whole to fill with newer approaches. Non-coding RNAs belong to the housekeeping and regulatory categories and both have their specific roles, and their alteration causes specific implications in disease pathogenesis. Housekeeping RNAs, rRNA, tRNA and regulatory RNA, micro-RNA, circular RNA, piRNA and long non-coding RNA were found to be important regulators of inflammation. They work at the pre-and post-transcriptional levels and were found to be more intriguing to study their regulatory impact on disease pathogenesis. The review addresses a question on how the non-coding RNA gets involved in early RA pathogenesis and can be utilized to know their targets to understand the disease better and make way towards the unresolved mystery of RA development.

Anti-inflammatory potential of selective small compounds by targeting TNF-α & NF-kB signaling: a comprehensive molecular docking and simulation study.

Tumor necrosis factor alpha (TNF-α) is the major cause of inflammation in autoimmune diseases like rheumatoid arthritis (RA). It's mechanisms of signal transduction through nuclear factor kappa B (NF-kB) pathway via small molecules such as metabolite crosstalk are still elusive. In this study, we have targeted TNF-α and NF-kB through metabolites of RA, to inhibit TNF-α activity and deter NF-kB signaling pathways, thereby mitigating the disease severity of RA. TNF-α and NF-kB structure was obtained from PDB database and metabolites of RA were selected from literature survey. In-silico studies were carried out by molecular docking using AutoDock Vina software and further, known TNF-α and NF-kB inhibitors were compared and revealed metabolite's capacity to targets the respective proteins. Most suitable metabolite was then validated by MD simulation to verify its efficiency against TNF-α. Total 56 known differential metabolites of RA were docked with TNF-α and NF-kB compared to their corresponding inhibitor compounds. Four metabolites such as Chenodeoxycholic acid, 2-Hydroxyestrone, 2-Hydroxyestradiol (2-OHE2), and 16-Hydroxyestradiol were identified as a common TNF-α inhibitor's having binding energies ranging from -8.3 to -8.6 kcal/mol, followed by docking with NF-kB. Further, 2-OHE2 was selected because of having binding energy -8.5 kcal/mol, found to inhibit inflammation and the effectiveness was validated by root mean square fluctuation, radius of gyration and molecular mechanics with generalized born and surface area solvation against TNF-α. Thus 2-OHE2, an estrogen metabolite was identified as the potential inhibitor, attenuated inflammatory activation and can be utilized as a therapeutic target to disseminate severity of RA.

Estrogen-mediated differential protein regulation and signal transduction in rheumatoid arthritis.

Exploration of the dual and opposing facets of estrogen necessitates a clear understanding to diminish the controversy of estrogen regulation in averting the systemic, autoimmune, joint degrading disorder, and rheumatoid arthritis (RA). Experimental evidences consider estrogen as a pivotal enzyme to modulate the disease progression via managing several cellular mechanisms targeting inflammatory markers such as TNF, ILs, nuclear factor kappa B, and other regulatory proteins like matrix metalloproteinases impeding joint erosion and cartilage degradation. Estrogen modulates cellular signaling associated with inflammation, oxidative stress, related cardiovascular risk, and miRNA regulation during RA progression. Studies determining estrogen regulation in RA complicate the resemblance of the outcome as they represent both hyper and hypo level of estrogen is linked to the disease. Although some reports deliver estrogen as malign, there is now increasing evidence of rendering protection dose dependently. Variation in estrogen level causes differential expression of certain proteins and their related signaling which is directly or indirectly linked to RA pathogenesis. This review summarizes the variations in protein expression levels by focusing on the in vitro, in vivo,and clinical studies of estrogen deficiency and treatment. Construction of protein-protein interaction network, GO, and KEGG pathway enrichment analysis of the differentially expressed proteins assist in hypothesizing a potential molecular mechanism of estrogen in RA via in silico studies. Targeting these differential proteins can emerge a new path for developing advanced therapeutic strategies.

Anti-inflammatory activity of nicotine isolated from Brassica oleracea in rheumatoid arthritis.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune disease, associated with chronic inflammation of synoviocytes. Tumor necrosis factor α (TNF-α) plays a crucial role in the pathogenesis of RA through pro-inflammatory cytokines. Nicotine, an alkaloid used as herbal medicine, often worked as an anti-inflammatory agent. In the present study, we tried to uncover the anti-inflammatory impact of nicotine against RA. MATERIALS AND METHODS: Nicotine was isolated from Brassica oleracea, purified by high profile/phase liquid chromatography (HPLC). In-silico docking was carried out using bioinformatics tools SwissADME (absorption, distribution, metabolism and excretion), PASS, and Drug-induced Gene Expression Profile (DIGEP)-Pred to determine drug likeliness of nicotine. The in-vitro study was performed in TNFα-induced SW982 synoviocytes by qPCR. mRNA expression of pro-inflammatory cytokines (TNF, IL6, IL1ß) and proteins (TRAF2, P50, P65) were analyzed followed by validation of P65 (RELA), pP65, IkBα by Western blot analysis. RESULTS: Nicotine compound was extracted from Brassica oleracea and purified by HPLC method (Rt values at 2.67 min). The physicochemical, pharmacokinetic properties and drug-likeliness of nicotine were studied by in-silico analysis. In-vitro studies revealed that nicotine lowers the expression of inflammatory cytokines (TNF, IL6, IL1ß) and proteins (TNF receptor-associated factor 2 (TRAF2), P50, P65) at 1 µg/ml in TNFα-induced SW982 cells. CONCLUSION: Nicotine from natural sources (Brassica oleracea) has been found to be an effective anti- inflammatory compound at a low dosage; thus, identifying the role of nicotine present in the natural sources as a therapeutic option for RA, may be recommended as remedial drug instead of synthetic drug.

AGE/Non-AGE Glycation: An Important Event in Rheumatoid Arthritis Pathophysiology.

Rheumatoid arthritis (RA) is a chronic inflammatory, autoimmune disease that gradually affects the synovial membrane and joints. Many intrinsic and/or extrinsic factors are crucial in making RA pathology challenging throughout the disease. Substantial enzymatic or non-enzymatic modification of proteins driving inflammation has gained a lot of interest in recent years. Endogenously modified glycated protein influences disease development linked with AGEs/non-AGEs and is reported as a disease marker. In this review, we summarized current knowledge of the differential abundance of glycated proteins by compiling and analyzing a variety of AGE and non-AGE ligands that bind with RAGE to activate multi-faceted inflammatory and oxidative stress pathways that are pathobiologically associated with RA-fibroblast-like synoviocytes (RA-FLS). It is critical to comprehend the connection between oxidative stress and inflammation generation, mediated by glycated protein, which may bind to the receptor RAGE, activate downstream pathways, and impart immunogenicity in RA. It is worth noting that AGEs and non-AGEs ligands play a variety of functions, and their functionality is likely to be more reliant on pathogenic states and severity that may serve as biomarkers for RA. Screening and monitoring of these differentially glycated proteins, as well as their stability in circulation, in combination with established pre-clinical characteristics, may aid or predict the onset of RA.

Transthyretin and Receptor for Advanced Glycation End Product's Differential Levels Associated with the Pathogenesis of Rheumatoid Arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic autoimmune, inflammatory joint disease. The identification of multifaceted etiological changes at the protein level in RA remains an important need. We aimed to identify differential proteins (DPs) and gene profiles to uncover inflammatory indicators and their association to RA pathogenesis. METHODS: 2-DE and SWATH-MS were used to identify DPs in RA and healthy control plasma. Fluorescence phenylboronate gel electrophoresis (Flu-PAGE) with mass spectrometry was used for protein glycation in RA plasma. Disease specificity of identified DPs was confirmed by ELISA and Western blot analysis. The gene expressions of selected DPs were evaluated by qRT-PCR in PBMCs of RA, systemic lupus erythematosus (SLE), spondyloarthritis (SpA), and osteoarthritis (OA). The functional implication of glycated protein was determined by in- silico and validated by in vitro analysis in fibroblast-like synoviocytes. RESULTS: A total of 150 DPs (127 increased and 23 decreased) were identified by 2-DE and SWATH-MS analysis in RA plasma compared to healthy control (HC). Nine proteins were identified as glycated by Flu-PAGE LC-MS/MS. Transthyretin (TTR), serotransferrin, and apolipoprotein-A1 (Apo-A1) were found to be differential and glycated. ELISA and Western blot results revealed the disease-specific increased expression of TTR and RAGE in RA. The qRT-PCR results signify the aberrant gene expression of TTR and RAGE, found to be associated with RA when compared with SLE, SpA, and OA PBMCs. TTR-RAGE interactions were predicted by in-silico and validated by in- vitro analysis using RA-FLS. The increased levels of pro-inflammatory cytokines IL-6, IL-1ß, TNF-α, and differently expressed TTR and RAGE were confirmed in fibroblast-like synoviocytes under inflammatory conditions. CONCLUSION: Our findings showed that the level of TTR was increased in RA plasma, along with an altered glycation rate. TTR and RAGE aberrant gene expression in PBMCs are the key events associated with RA, and TNF-α activates the NF-KB pathways and promote TTR and RAGE differential expressions that may have pathogenic/inflammatory significance.

Exogenous miRNA: A Perspective Role as Therapeutic in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease that causes joint deformation. Till now several studies has been carried out promising its cure, but curing has not yet achieved to the satisfactory levels. Herbal approach to treat disease by a cross-kingdom mechanism via exogenous miRNA is an emerging trend to target associated genes with RA pathogenesis as a therapeutic potential. The concept of acquired/exogenous miRNA into pathophysiological prospect provides an opportunity to explore inter-species kingdom like regulation of plant miRNAs on human health. The change in gene expression was attributed by a short 22-24 nucleotide long sequence that binds to its complementary region to suppress/silence the gene expression. This makes exogenous miRNA a novel approach for targeted therapy to treat complex chronic inflammatory diseases. Here, aim of the review was to address significance of plant derived miRNA based targeted therapy to regulate inflammation in RA.

Differential Metabolome in Rheumatoid Arthritis: a Brief Perspective.

PURPOSE OF REVIEW: Rheumatoid arthritis (RA) is a chronic autoimmune, inflammatory disease of the synovium that affects the movable joints. It develops due to the infiltration and invasion of the synovial joints by immune cells. Metabolism is anabolic or catabolic chemical reactions occurring in a cell. The biochemical pathways in synovial and immune cells are altered affecting the downstream metabolite formation. Changes in the metabolite levels alter signaling cascades which further intensify the disease. Despite current knowledge of metabolomics, there remain certain features that need to be elucidated to correlate the differential metabolite levels with RA. RECENT FINDINGS: Metabolite profiling can be used to find altered patterns of metabolites in RA. Glucose, lipid, amino acid, and estrogen metabolism are the key pathways that are altered and contribute to the aggravation of RA. The altered metabolic pathways involved in different cells in RA results in complex interactions between metabolites and biomacromolecules; thus, it generates autoantigens. Moreover, understanding the correlation between differential metabolites and disease severity might help reveal potential new biomarkers and therapeutic targets for RA pathogenesis. So, considering the multi-faceted role of altered metabolites in the pathogenesis of RA, metabolic pathways of different cells are needed to be studied for a better understanding of their functions in the disease and thus, improving the present therapeutic strategies.

Post Translational Modification and Its Pathologic Association in Rheumatoid Arthritis: A Brief Perspective.

Post Translational Modification (PTM) is a process in which covalent addition of functional groups on protein occurs to maintain their structure, function and stability. Every PTM process in our living system occurs to enhance the functional diversity of a protein. But sometimes, it occurs without any regulation and that might lead to autoimmunity. Rheumatoid arthritis (RA) is one such chronic, inflammatory, autoimmune disease that affects the joints. Proper treatment can make the symptoms manageable for RA, but it is not curable. Delayed diagnosis of RA can cause severe bone pain, stiffness, inflammation, redness in joints and affect other parts of the body such as the liver, kidney, etc. Early diagnosis of RA is necessary to manage the aggressive symptoms. Currently, Rheumatoid factor (RF) and anti-citrullinated cyclic peptide (Anti-CCP) are considered as biomarkers to diagnose RA. Besides citrullination, several other PTMs are also involved in the generation of autoantibodies, such as carbamylation, glycosylation, glycation, acetylation, ubiquitination, proteolysis, phosphorylation, and lipidation. The aim of this review is to elucidate several changes in the form, nature, and function of PTMs in RA. This review will give a recent overview on the role of PTMs in the pathogenesis of RA with a focus on the post-translational modifications.

Plasma Proteome Profiling of Coronary Artery Disease Patients: Downregulation of Transthyretin-An Important Event.

Coronary artery disease (CAD) is a prevalent chronic inflammatory cardiac disorder. An early diagnosis is likely to help in the prevention and proper management of this disease. As the study of proteomics provides the potential markers for detection of a disease, in the present investigation, attempt has been made to identify disease-associated differential proteins involved in CAD pathogenesis. For this study, a total of 200 selected CAD patients were considered, who were recruited for percutaneous coronary intervention (PCI) treatment. The proteomic analysis was performed using two-dimensional gel electrophoresis (2-DE) and MALDI-TOF MS/MS. Samples were also subjected to Western blot analysis, enzyme-linked immunosorbent assay (ELISA), peripheral blood mononuclear cells isolation immunofluorescence (IF) analysis, analytical screening by fluorescence-activated cell sorting (FACS), and in silico analysis. The representative data were shown as mean ± SD of at least three experiments. A total of 19 proteins were identified. Among them, the most abundant five proteins (serotransferrin, talin-1, alpha-2HS glycoprotein, transthyretin (TTR), fibrinogen-α chain) were found to have altered level in CAD. Serotransferrin, talin-1, alpha-2HS glycoprotein, and transthyretin (TTR) were found to have lower level, whereas fibrinogen-α chain was found to have higher level in CAD plasma compared to healthy, confirmed by Western blot analysis. TTR, an important acute phase transport protein, was validated low level in 200 CAD patients who confirmed to undergo PCI treatment. Further, in silico and in vitro studies of TTR indicated a downexpression of CAD in plasma as compared to the plasma of healthy individuals. Lower level of plasma TTR was determined to be an important risk marker in the atherosclerotic-approved CAD patients. We suggest that the TTR lower level predicts disease severity and hence may serve as an important marker tool for CAD screening. However, further large-scale studies are required to determine the clinical significance of TTR.

Synovial Fluid Cell Proteomic Analysis Identifies Upregulation of Alpha-Taxilin Proteins in Rheumatoid Arthritis: A Potential Prognostic Marker.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease affecting the joints and surrounding tissue. Identification of novel proteins associated with the progression of a disease is a prerequisite for understanding the pathogenesis of RA. The present study was undertaken to identify the potential biomarkers from a less explored biological sample such as synovial fluid (SF) cells which is specific for RA and to analyze their functional aspects using proteomic approach. Two-dimensional gel electrophoresis (2-DE) was performed using synovial fluid cells of RA and osteoarthritis (OA) patients, and 7 differentially expressed proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS). Αlpha-Taxilin (α-Taxilin) has been found as one of the novel, significantly up regulated protein in RA. It has been validated in the synovium, synovial fluid (SF), SF cells, and plasma samples by Western blot, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), and real-time PCR. The identification of autoantibody against α-Taxilin and in silico studies has further helped us to understand its involvement in disease mechanism. The present study will therefore provide knowledge towards the etiology of RA that pave the way for suitable prognostic marker identification along with other clinical parameters.