BACKGROUND

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammatory arthritis and extra-articular involvement. Comorbidities are highly prevalent in patients with RA, in particular cardiovascular disease (CVD), which is responsible for over 50% of premature deaths. This study aimed to describe cardiovascular diseases and their risk factors among patients with rheumatoid arthritis in the Philippine General Hospital (PGH).

To describe cardiovascular (CV) diseases and their risk factors among patients with rheumatoid arthritis.

A retrospective descriptive cross-sectional study was done in the University of the Philippines – Philippine General Hospital (UP-PGH) inpatient and outpatient services. The study included patients 18 years old and above diagnosed with RA and fulfills the 1987 American College of Rheumatology or 2010 American College of Rheumatology-European League Against Rheumatism (ACR/EULAR) classification criteria with no overlap features with other autoimmune connective tissue diseases and with complete records of the information required for the study from January 2019-December 2022. The primary outcomes of interest were the prevalence of CV diseases and CV risk factors. Descriptive statistics were used to summarize the data.

There were 123 patients in the study, 93.4% outpatients, and 95.1% females, with a mean age and disease duration of 51.3 and 9.8 years, respectively. Disease activity was moderate in 35% and high in 9.7%, based on disease activity score (DAS 28) or clinical disease activity index (CDAI) scores. Methotrexate (54%) was the most commonly used conventional synthetic disease-modifying antirheumatic drug (csDMARD). Glucocorticoid use was observed in 51.2%. None of the patients were receiving a biologic DMARD. There were 24 (19.5%) patients with CV diseases, namely myocardial infarction, heart failure, and stroke. There were 87 (70%) patients with at least one CV risk factor and 62 (50.4%) with multiple risk factors. The risk factors identified were: dyslipidemia (43.1%), hypertension (40.7%), elevated body mass index (35.7%), and diabetes mellitus (15.4%). There were f ive deaths in the hospitalized patients (4%), one due to a myocardial infarction.

The majority (70%) in our cohort had at least one CV risk factor, 19.5% had an identified CV disease, and one died from a myocardial infarction. Dyslipidemia was the most common CV risk factor. The high proportion of patients with CV disease and CV risk factors highlights the need to add the screening and management of CV diseases and risk factors as a priority among patients with rheumatoid arthritis.

OBJECTIVES: To investigate the role of the BNIP3-PI3K/Akt signaling pathway in mediating the inhibitory effect of Buyang Huanwu Decoction (BYHWT) on mitochondrial autophagy in human synovial fibroblasts from rheumatoid arthritis patients (FLS-RA) cultured under a hypoxic condition. METHODS: Forty normal Wistar rats were randomized into two groups (n=20) for daily gavage of BYHWT or distilled water for 7 days to prepare BYHWT-medicated or control sera. FLS-RA were cultured in routine condition or exposed to hypoxia (10% O₂) for 24 h wigh subsequent treatment with IL-1β, followed by treatment with diluted BYHWT-medicated serum (5%, 10% and 20%) or control serum. AnnexinV-APC/7-AAD double staining and T-AOC kit were used for detecting apoptosis and total antioxidant capacity of the cells, and the changes in ROS, ATP level, mitochondrial membrane potential and Ca²⁺ homeostasis were analyzed. The changes in mRNA and protein expressions of BNIP3, PI3K and AKT and mRNA expressions of LC3, Beclin-1 and P62 were detected using RT-qPCR and Western blotting. RESULTS: Treatment with BYHWT-medicated serum dose-dependently lowered apoptosis rate of IL-1β-induced FLS-RA with hypoxic exposure. The treatment significantly decreased T-AOC concentration, increased ROS production, autophagosome formation and ATPase levels, and lowered mitochondrial membrane potential and Ca²⁺ level in the cells. In IL-1β-induced FLS-RA with hypoxic exposure, treatment with BYHWT-medicated serum significantly increased BNIP3 protein expression, decreased the protein expressions of PI3K and AKT, increased the mRNA expressions of BNIP3 and P62, and lowered the mRNA expressions of PI3K, AKT, LC3 and Beclin-1 without significantly affecting Beclin-1 protein expression. The cells treated with 5% and 10% BYHWT-medicated serum showed no significant changes in LC3 expression. CONCLUSIONS BYHWT inhibits mitochondrial autophagy in IL-1β-induced FLS-RA with hypoxic exposure possibly by inhibiting BNIP3-mediated PI3K/AKT signaling pathway.
Drugs, Chinese Herbal/pharmacology* ; Arthritis, Rheumatoid/pathology* ; Animals ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Autophagy/drug effects* ; Humans ; Fibroblasts/cytology* ; Rats, Wistar ; Membrane Proteins/metabolism* ; Rats ; Phosphatidylinositol 3-Kinases/metabolism* ; Mitochondria/metabolism* ; Cells, Cultured ; Proto-Oncogene Proteins/metabolism* ; Apoptosis/drug effects* ; Cell Hypoxia ; Synovial Membrane/cytology* ; Male ; Mitochondrial Proteins

OBJECTIVES: To investigate the regulatory effect of LINC00837/miR-671-5p/SERPINE2 functional axis on pathological processes of fibroblast-like synovial cells (FLS) in rheumatoid arthritis (RA). METHODS: RA-FLS were transfected with a LINC00837 overexpression plasmid (pcDNA3.1-LINC00837), a LINC00837 interference plasmid (siRNA-LINC00837), or their respective negative control plasmids (pcDNA3.1-NC and siRNA-NC). Dual luciferase was used to verify the targeting relationship between LINC00837 and miR-671-5p and between miR-671-5p and SERPINE2. RT-qPCR was used to detect the expression levels of LINC00837, miR-671-5p and SERPINE2 in normal FLS or the transfected cells, whose proliferation and migration abilities were assessed using Edu assay and scratch healing assay and by detecting the expression levels of Ki-67, PCNA, E-cadherin and N-cadherin with Western blotting. The changes in cellular secretion of the inflammatory cytokines (TNF‑α, IL-17, IL-4 and IL-10) were examined using ELISA. RESULTS: Dual luciferase reporter gene assay showed that LINC00837 was capable of binding to the 3'-UTR of miR-671-5p, and the latter bound to the 3-UTR of SERPINE2 at specific binding sites between them. Compared with normal FLS, RA-FLS showed significantly increased expressions of LINC00837 and SERPINE2, lowered miR-671-5p expression and enhanced proliferation and migration abilities with increased expressions of pro-inflammatory cytokines and reduced expressions of anti-inflammatory cytokines. Transfection of RA-FLS with pcDNA-LINC00837 further enhanced cell proliferation and migration and the changes in the inflammatory cytokines, while transfection with si-LINC00837 produced the opposite changes. CONCLUSIONS RA-FLS have a LINC00837/miR-671-5p/SERPINE2 functional axis, which regulates cell proliferation, migration and secretion of inflammatory factors, and interventions targeting LINC00837 may provide a potential strategy to regulate the pathological processes in RA-FLS.
Arthritis, Rheumatoid/metabolism* ; MicroRNAs/metabolism* ; Humans ; Cell Proliferation ; Cell Movement ; Synovial Membrane/pathology* ; RNA, Long Noncoding/genetics* ; Fibroblasts/metabolism* ; Synoviocytes/metabolism* ; Cells, Cultured ; Transfection

OBJECTIVES: To explore the role of oxidative stress and immune infiltration in rheumatoid arthritis (RA). METHODS: RA datasets GSE55235 (10 RA vs 10 normal samples) and GSE55457 (13 RA vs 10 normal samples) from the GEO database were merged as the test set to identify the differentially expressed genes (DEGs) in RA using R. The DEGs were intersected with oxidative stress-related genes to obtain oxidative stress-associated DEGs. KEGG and GO enrichment analyses of the DEGs were performed, and the RA-related pathways and biological processes were analyzed using GSEA. A protein-protein interaction (PPI) network was constructed using STRING and Cytoscape, and the top 10 key genes were obtained using the Degree algorithm. The validation dataset GSE1919 from GEO database was used for ROC analysis of the key genes to obtain the core genes, and their correlations with infiltrating immune cells were analyzed using CIBERSORT. The results were verified by RT-qPCR for detecting expression levels of the core genes in RA and normal joint samples. RESULTS: We identified 89 oxidative stress-associated DEGs. Enrichment analysis suggested that these DEGs were involved in the biological processes including oxidative stress, chemical stress response, reactive oxygen species response, and lipopolysaccharide response. ROC analysis showed that the 5 core genes (STAT1, MMP9, MYC, CCL5, and JUN) all had AUC values >0.7, indicating their high diagnostic sensitivity and specificity for RA. These genes were closely correlated with immune cells, particularly T cells. RT-qPCR confirmed significant differential expressions of the core genes between RA and normal samples. CONCLUSIONS Oxidative stress and diverse immune responses are features of RA, and the immune responses contribute to activation of oxidative stress. The identified core genes can potential serve as new diagnostic markers for RA.
Arthritis, Rheumatoid/genetics* ; Oxidative Stress/genetics* ; Humans ; Computational Biology ; Protein Interaction Maps ; Gene Expression Profiling ; Gene Regulatory Networks

OBJECTIVES: To investigate the causal associations between autoimmune diseases and aplastic anemia (AA) using Mendelian randomization analysis. METHODS: Publicly available genome-wide association study (GWAS) data were utilized to obtain single nucleotide polymorphisms (SNPs) associated with autoimmune diseases and AA for analysis. The inverse variance weighted (IVW) method was employed as the primary analytical approach, with MR Egger, Weighted Mode, Weighted Median, and Simple Mode methods serving as complementary analyses. Heterogeneity and pleiotropy analyses were conducted using designated functions, and the robustness of Mendelian randomization results was assessed using leave-one-out analysis. RESULTS: The two-sample Mendelian randomization analysis using the IVW method revealed significant positive causal associations of rheumatoid arthritis (OR=1.094, 95% CI: 1.023-1.170, P=0.009, adjusted P=0.042), systemic lupus erythematosus (OR=1.111, 95% CI: 1.021-1.208, P=0.015, adjusted P=0.036), Hashimoto thyroiditis (OR=1.206, 95% CI: 1.049-1.387, P=0.009, adjusted P=0.029), and Sicca syndrome (OR=1.173, 95% CI: 1.054-1.306, P=0.004, adjusted P=0.035) with AA, which was supported by the results from the Weighted Median method. Sensitivity analyses indicated no evidence of pleiotropy or heterogeneity, and leave-one-out analysis confirmed the robustness of the causal relationships. No direct evidence was found linking Graves' disease, ulcerative colitis, Crohn's disease, autoimmune hepatitis, primary biliary cholangitis, or primary sclerosing cholangitis with AA (P>0.05, adjusted P>0.05), indicating a lack of causal association. Reverse Mendelian randomization results and multiple corrections indicated that AA was not an influencing factor for autoimmune diseases (adjusted P>0.05). CONCLUSIONS Our findings support at the genetic level that rheumatoid arthritis, systemic lupus erythematosus, Hashimoto thyroiditis, and Sicca syndrome are risk factors for AA, and confirm a causal association of the these 4 autoimmune diseases with an increased risk of AA.
Humans ; Mendelian Randomization Analysis ; Anemia, Aplastic/genetics* ; Autoimmune Diseases/complications* ; Polymorphism, Single Nucleotide ; Genome-Wide Association Study ; Arthritis, Rheumatoid/genetics* ; Lupus Erythematosus, Systemic/genetics* ; Genetic Predisposition to Disease

OBJECTIVES: To investigate the effect of Qianggu Kangshu Formula (QGKSF) for alleviating osteoclast differentiation in rheumatoid arthritis and the underlying mechanism. METHODS: RAW264.7 cells cultured under hypoxic conditions were treated with RANKL to induce osteoclast differentiation and incubated with normal rat serum or sera from rats medicated with methotrexate (MTX) or QGKSF at low and high doses. Cell viability, TRAP-positive multinucleated cells and F-actin ring formation in the treated cells were assessed with CCK-8 assay, TRAP staining and Phalloidin staining, respectively. Autophagy and autophagosomes in the cells were observed with MDC staining and transmission electron microscopy. ELISA was used to measure IL-6 and TNF-α levels in the culture supernatant, and the expressions of HIF-1α, BNIP3, Bcl-2, Beclin1, LC3-I, LC3-II, P62 and TRAP mRNAs and proteins were analyzed using RT-qPCR and Western blotting. RESULTS: In hypoxia- and RANKL-induced RAW264.7 cells treated with normal rat serum, significant increments of TRAP-positive cells and F-actin ring formation were observed with an enhanced autophagic fluorescence intensity and increased autophagosomes. Treatment of the induced cells with rat sera medicated with MTX and low- and high-dose QGKSF obviously reduced the TRAP-positive cells, F-actin rings and autophagosomes as well as the autophagic fluorescence intensity. RANKL treatment significantly increased IL-6 and TNF-α levels in RAW264.7 cells, which were obviously decreased by treatment with MTX- and QGKSF-medicated sera. RANKL also significantly increased the mRNA and protein expression levels of HIF-1α, BNIP3, Bcl-2, Beclin1, LC3 and TRAP and lowered P62 expressions, and these changes were effectively reversed by treatment with MTX- and QGKSF-medicated sera. CONCLUSIONS QGKSF attenuates RANKL-induced osteoclast differentiation in hypoxic RAW264.7 cells by inhibiting the HIF-1α/BNIP3 autophagy signaling pathway, suggesting its potential for treatment of bone destruction in rheumatoid arthritis.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Osteoclasts/drug effects* ; Autophagy/drug effects* ; Mice ; Signal Transduction/drug effects* ; Rats ; Cell Differentiation/drug effects* ; Arthritis, Rheumatoid/pathology* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; RAW 264.7 Cells ; Membrane Proteins/metabolism* ; Mitochondrial Proteins

OBJECTIVES: To explore the therapeutic mechanism of puerarin for alleviating synovitis in rats with collagen-induced arthritis (CIA). METHODS: In a SD rat model of CIA, we tested the effects of daily gavage of puerarin at low, moderate and high doses (10, 30, and 100 mg/kg, respectively) for 3 weeks, with tripterygium glycosides (GTW, 10 mg/kg) as the positive control, on swelling in the hind limb joints regions evaluated by arthritis index scoring. Mass fraction of the liver of the rats was calculated, and pathologies in joint synovial membrane were observed with HE staining. The expressions of transforming growth factor β‑activated kinase-1 (TAK1), Toll-like receptor 4 (TLR4), and nuclear factor kappa-Bp65 (NF‑κB p65) at the mRNA and protein levels in the synovial tissues were detected using Real-time PCR and Western blotting. RESULTS: Compared with those in the model group, the rats in GTW group and high-dose puerarin group showed significantly reduced mass fraction of the liver. Treatment with GTW and puerarin at the 3 doses all significantly alleviated plantar swelling, lowered arthritis index scores, and improved synovitis in CIA rats (P<0.05), and the effects of puerarin showed an obvious dose dependence. Both GTW and puerarin treatments significantly lowered TAK1, TLR4, and NF‑κB p65 mRNA and protein expressions in the synovium of CIA rats. CONCLUSIONS Puerarin alleviates synovium damages in CIA rats possibly by suppressing the TLR4/NF‑κB signaling pathway via downregulating TAK1 expression.
Animals ; Toll-Like Receptor 4/metabolism* ; Rats, Sprague-Dawley ; Rats ; MAP Kinase Kinase Kinases/metabolism* ; Signal Transduction/drug effects* ; Arthritis, Rheumatoid/drug therapy* ; NF-kappa B/metabolism* ; Isoflavones/therapeutic use* ; Male ; Arthritis, Experimental/drug therapy* ; Transcription Factor RelA/metabolism* ; Synovial Membrane/metabolism*

OBJECTIVES: To analyze the molecular mechanism of Xixian Pills for treatment of rheumatoid arthritis (RA). METHODS: Forty-eight rats were randomized into 6 groups (n=8), including a normal control group, a collagen-induced arthritis (CIA) model group, 3 Xixian Pills treatment (200, 400 and 800 mg/kg) groups, and a Tripterygium glycosides tablet (TGT) treatment group. In the latter 4 groups, the rats were treated with daily gavage of Xixian Pills or TGT 2 weeks after CIA modeling for 3 consecutive weeks. The differentially expressed proteins in high-dose Xixian Pills group and the model group compared with the normal control group were screened based on the tandem mass spectrometry tag (TMT) technology, and the core targets and signaling pathways were analyzed. The immune cell infiltration and gene expression data were analyzed using ggplot2 and tidyverse packages, and the correlation coefficients between the core targets and the immune cells were calculated. RESULTS: The CIA rats showed significantly increased serum levels of TNF-α and IL-6 and lowered serum IL-10 level. Treatments with high- and medium-dose Xixian Pills and TGT all significantly reduced serum TNF‑α and IL-6 and increased IL-10 levels in CIA rats. Proteomic analysis identified 160 differential proteins between the model group and high-dose Xixian Pills group, and the core targets included CCL5, STAT1, GZMB and IL7R. The areas under the ROC curve of CCL5 and STAT1 were both greater than 0.9. Immunohistochemical and immunofluorescence staining revealed increased levels of CCL5 and STAT1 in the ankle joints of CIA rats, which were significantly decreased after treatment with Xixian Pills. CONCLUSIONS Treatment with Xixian Pills offers protection of the joints in CIA rats possibly by inhibiting joint inflammation via regulating protein expressions of CCL5 and STAT1.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Rats ; Arthritis, Rheumatoid/metabolism* ; Proteomics ; Tripterygium/chemistry* ; Arthritis, Experimental/metabolism* ; Tumor Necrosis Factor-alpha/blood* ; Interleukin-10/blood* ; Interleukin-6/blood* ; Male ; Rats, Sprague-Dawley ; Signal Transduction

INTRODUCTION: Hydroxychloroquine (HCQ), originally an antimalarial drug, is currently used to treat multiple disorders, especially rheumatic diseases. Given its good efficacy and safety, HCQ is widely administered in pregnant patients. However, the safety profile of HCQ during pregnancy remains controversial due to limited research. In addition, HCQ has been reported to reduce preeclampsia in patients with systemic lupus erythematosus (SLE) and could potentially alleviate the symptom of preeclampsia. However, the clinical profile and molecular mechanism of HCQ in preeclampsia is yet to be fully understood. METHOD: We reviewed the literature on HCQ treatment in pregnancy with rheumatic diseases and preeclamp-sia in PubMed and Web of Science. We also discussed the safety of long-term therapy with HCQ during pregnancy. RESULTS: HCQ mainly modulates autoimmune response through inhibition of lysosomal function, toll-like receptor (TLR) signalling, nicotinamide adenine dinucleotide phosphate-mediated oxidative stress and autophagy. Benefits of HCQ in treating rheumatic diseases, including antiphospholipid syndrome, rheumatoid arthritis and Sjogren's syndrome during pregnancy, has been demonstrated in clinics. In particular, multiple clinical guidelines recommend HCQ as an indispensable therapeutic drug for pregnant patients with SLE. Additionally, it may potentially function in preeclampsia to improve clinical symptoms. CONCLUSION HCQ is effectively used for rheumatic diseases during pregnancy. The benefits of HCQ treatment in rheumatic diseases outweigh the risk of adverse reactions it induces in pregnant women.
Humans ; Hydroxychloroquine/pharmacology* ; Pregnancy ; Female ; Antirheumatic Agents/pharmacology* ; Rheumatic Diseases/drug therapy* ; Pregnancy Complications/drug therapy* ; Pre-Eclampsia/prevention & control* ; Lupus Erythematosus, Systemic/drug therapy* ; Arthritis, Rheumatoid/drug therapy* ; Antiphospholipid Syndrome/drug therapy* ; Sjogren's Syndrome/drug therapy*

BACKGROUND: G protein-coupled receptor kinase 2 (GRK2) could participate in the regulation of diverse cells via interacting with non-G-protein-coupled receptors. In the present work, we explored how paroxetine, a GRK2 inhibitor, modulates the differentiation and activation of immune cells in rheumatoid arthritis (RA). METHODS: The blood samples of healthy individuals and RA patients were collected between July 2021 and March 2022 from the First Affiliated Hospital of Anhui Medical University. C57BL/6 mice were used to induce the collagen-induced arthritis (CIA) model. Flow cytometry analysis was used to characterize the differentiation and function of dendritic cells (DCs)/T cells. Co-immunoprecipitation was used to explore the specific molecular mechanism. RESULTS: In patients with RA, high expression of GRK2 in peripheral blood lymphocytes, accompanied by the increases of phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In animal model, a decrease in regulatory T cells (T regs ), an increase in the cluster of differentiation 8 positive (CD8 + ) T cells, and maturation of DCs were observed. Paroxetine, when used in vitro and in CIA mice, restrained the maturation of DCs and the differentiation of CD8 + T cells, and induced the proportion of T regs . Paroxetine inhibited the secretion of pro-inflammatory cytokines, the expression of C-C motif chemokine receptor 7 in DCs and T cells. Simultaneously, paroxetine upregulated the expression of programmed death ligand 1, and anti-inflammatory cytokines. Additionally, paroxetine inhibited the PI3K-AKT-mTOR metabolic pathway in both DCs and T cells. This was associated with a reduction in mitochondrial membrane potential and changes in the utilization of glucose and lipids, particularly in DCs. Paroxetine reversed PI3K-AKT pathway activation induced by 740 Y-P (a PI3K agonist) through inhibiting the interaction between GRK2 and PI3K in DCs and T cells. CONCLUSION Paroxetine exerts an immunosuppressive effect by targeting GRK2, which subsequently inhibits the metabolism-related PI3K-AKT-mTOR pathway of DCs and T cells in RA.
G-Protein-Coupled Receptor Kinase 2/metabolism* ; Arthritis, Rheumatoid/immunology* ; Animals ; Dendritic Cells/metabolism* ; Paroxetine/therapeutic use* ; Proto-Oncogene Proteins c-akt/metabolism* ; Mice ; Humans ; Mice, Inbred C57BL ; Signal Transduction/drug effects* ; Male ; Phosphatidylinositol 3-Kinases/metabolism* ; Lymphocyte Activation/drug effects* ; Female ; T-Lymphocytes/metabolism* ; Middle Aged