rheumatoid arthritis  (Exosome Diagnostics)

Journal: Iranian Journal of Pharmaceutical Research : IJPR

Article Title: Dual TYK2/JAK1 Inhibition by Brepocitinib Reprograms Synoviocyte Pathobiology: Mechanistic Insights Into Targeted Therapy for Rheumatoid Arthritis

doi: 10.5812/ijpr-166019

Figure Lengend Snippet: Effects of brepocitinib on MH7A cell viability and JAK-STAT pathway activation. A, cell viability of MH7A synoviocytes following treatment with brepocitinib at concentrations of 0.5 µM, 1 µM, and 5 µM for 24 hours. MTT assay was performed, and absorbance at 570 nm was normalized to DMSO control (0.1%). Data represent mean ± SD from three independent experiments. * P < 0.05 vs. vehicle. Although small statistical differences were detected at 1 µM and 5 µM, all brepocitinib-treated groups maintained viability above the predefined non-cytotoxic threshold (≥ 85% of vehicle). Therefore, these changes are considered not biologically relevant and confirm that brepocitinib is non-cytotoxic within the tested range. doxorubicin (1 µM) was used as a positive control and showed a marked reduction (~20%) in viability, validating assay responsiveness. B, brepocitinib suppresses phosphorylation of JAK-STAT pathway proteins in MH7A synoviocytes. MH7A cells were treated with brepocitinib at concentrations of 0.5 µM, 1 µM, and 5 µM for 24 hours. Western blot analysis was performed to assess the expression of phosphorylated and unphosphorylated forms of TYK2, JAK1, STAT1, and STAT3. GAPDH was used as a loading control. Protein lysates (30 µg per lane) were resolved by SDS-PAGE and probed with specific antibodies as indicated. Blots are representative of three independent experiments. Regression analysis confirmed a significant concentration-dependent reduction in p-JAK1 and p-STAT3 (R² > 0.90, P < 0.01). C, validation in primary RA-FLS (HFLS-RA). Cells were treated with brepocitinib (0.5, 1, 5 µM, 24 h) or tofacitinib (1 µM) and analyzed by Western blot for p-TYK2, TYK2, p-JAK1, JAK1, p-STAT1, STAT1, p-STAT3, and STAT3. GAPDH served as loading control. Results represent mean ± SD from three independent experiments. Low, medium, and high doses correspond to 0.5, 1, and 5 µM brepocitinib, respectively. Regression analysis also confirmed dose-dependent suppression of p-TYK2, p-JAK1, p-STAT1, and p-STAT3 in RA-FLS (R² > 0.88, P < 0.01). D, primary RA-FLS (HFLS-RA) were treated with brepocitinib (1 µM) for 0, 30, 60, 240 minutes, and 24 hours. Western blot analysis was performed for p-STAT1, STAT1, p-STAT3, and STAT3, with GAPDH serving as the loading control. Values represent mean ± SD from at least three independent experiments. Significance compared with control: * P < 0.05, *** P < 0.001. Two-way ANOVA demonstrated significant effects of treatment, time, and their interaction (P < 0.05). In all experiments, low, medium, and high doses correspond to 0.5 µM, 1 µM, and 5 µM brepocitinib, respectively.

Article Snippet: Primary RA fibroblast-like synoviocytes (RA-FLS; HFLS-RA, Cell Applications, Cat. No. 408RA-05a) were cultured according to the supplier’s instructions in Synoviocyte growth medium (Cell Applications, Cat. No. 415-500).

Techniques: Activation Assay, MTT Assay, Control, Positive Control, Phospho-proteomics, Western Blot, Expressing, SDS Page, Concentration Assay, Biomarker Discovery

Journal: Iranian Journal of Pharmaceutical Research : IJPR

Article Title: Dual TYK2/JAK1 Inhibition by Brepocitinib Reprograms Synoviocyte Pathobiology: Mechanistic Insights Into Targeted Therapy for Rheumatoid Arthritis

doi: 10.5812/ijpr-166019

Figure Lengend Snippet: Analysis of pro-inflammatory cytokine expression in MH7A synoviocytes treated with brepocitinib. A, MH7A cells were treated with brepocitinib at concentrations of 0.5 µM, 1 µM, and 5 µM for 24 hours. Total RNA was extracted using TRIzol reagent, and cDNA was synthesized using a reverse transcription kit. B, RA-FLS cells were treated with brepocitinib at concentrations of 0.5 µM, 1 µM, and 5 µM for 24 hours. Total RNA was extracted using TRIzol reagent, and cDNA was synthesized using a reverse transcription kit. Quantitative real-time PCR (qPCR) was performed using SYBR Green master mix on a QuantStudio 5 thermocycler. Specific primers were used to detect IL-6, TNF-α, and IFN-γ transcripts, with GAPDH serving as the endogenous control. Relative mRNA expression was calculated using the 2 -ΔΔCt method and normalized to DMSO-treated control cells. Results represent the mean fold change from three independent experiments. C, cell culture supernatants of MH7A were collected from the same treatment groups after 24 hours and analyzed by enzyme-linked immunosorbent assay (ELISA). D, cell culture supernatants were collected from RA-FLS after 24 hours of brepocitinib treatment and analyzed by enzyme-linked immunosorbent assay (ELISA). Commercially available ELISA kits were used to quantify the concentrations of IL-6, TNF-α, and IFN-γ according to the manufacturers' protocols. Absorbance was measured at 450 nm using a microplate reader, and cytokine levels were interpolated from standard curves. Data were normalized to total protein content in each well. Results are shown as mean cytokine concentrations (pg/mL) from three independent experiments. Low, medium, and high doses correspond to 0.5, 1, and 5 µM brepocitinib, respectively. Values represent mean ± SD from at least three independent experiments. Significance compared with control: * P < 0.05, ** P < 0.01, *** P < 0.001. Bonferroni-corrected comparisons confirmed significance for all cytokines, and regression analysis supported dose-dependent suppression (P < 0.01). In all experiments, low, medium, and high doses correspond to 0.5 µM, 1 µM, and 5 µM brepocitinib, respectively.

Article Snippet: Primary RA fibroblast-like synoviocytes (RA-FLS; HFLS-RA, Cell Applications, Cat. No. 408RA-05a) were cultured according to the supplier’s instructions in Synoviocyte growth medium (Cell Applications, Cat. No. 415-500).

Techniques: Expressing, Synthesized, Reverse Transcription, Real-time Polymerase Chain Reaction, SYBR Green Assay, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Iranian Journal of Pharmaceutical Research : IJPR

Article Title: Dual TYK2/JAK1 Inhibition by Brepocitinib Reprograms Synoviocyte Pathobiology: Mechanistic Insights Into Targeted Therapy for Rheumatoid Arthritis

doi: 10.5812/ijpr-166019

Figure Lengend Snippet: Evaluation of apoptotic markers in MH7A synoviocytes following brepocitinib treatment. A, MH7A cells were treated with brepocitinib at concentrations of 0.5 µM, 1 µM, and 5 µM for 24 hours. Total cellular protein was extracted using RIPA buffer containing protease inhibitors. Protein lysates (30 µg) were subjected to SDS-PAGE and transferred to PVDF membranes. Membranes were probed with antibodies specific to BAX, BCL-2, total caspase-3, and cleaved caspase-3. GAPDH was used as a loading control. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL), and representative blots from three independent experiments are shown. B, densitometric analysis of Western blot bands was performed using ImageJ software. The ratio of BAX to BCL-2 protein expression was calculated as an indicator of pro-apoptotic signaling. Cleaved caspase-3 levels were quantified relative to total caspase-3 and expressed as a percentage. Data represent the mean ± SD from three independent experiments. Low, medium, and high doses correspond to 0.5, 1, and 5 µM brepocitinib, respectively. Values represent mean ± SD from at least three independent experiments. Significance compared with control: * P < 0.05, ** P < 0.01, *** P < 0.001. In all experiments, low, medium, and high doses correspond to 0.5 µM, 1 µM, and 5 µM brepocitinib, respectively.

Article Snippet: Primary RA fibroblast-like synoviocytes (RA-FLS; HFLS-RA, Cell Applications, Cat. No. 408RA-05a) were cultured according to the supplier’s instructions in Synoviocyte growth medium (Cell Applications, Cat. No. 415-500).

Techniques: SDS Page, Control, Western Blot, Software, Expressing

Journal: Iranian Journal of Pharmaceutical Research : IJPR

Article Title: Dual TYK2/JAK1 Inhibition by Brepocitinib Reprograms Synoviocyte Pathobiology: Mechanistic Insights Into Targeted Therapy for Rheumatoid Arthritis

doi: 10.5812/ijpr-166019

Figure Lengend Snippet: Assessment of MH7A synoviocyte migration using wound healing assay after brepocitinib treatment. A, MH7A synoviocytes were cultured to confluence in 6-well plates and treated with brepocitinib at concentrations of 0.5 µM, 1 µM, and 5 µM, or with 0.1% DMSO as vehicle control. A linear scratch ("wound") was introduced using a sterile 200 µL pipette tip, and wells were washed with PBS to remove detached cells. Cells were incubated in serum-free medium containing the indicated treatments. Phase-contrast images were taken at 0 and 24 hours using an inverted microscope to monitor wound closure. B, quantification of wound closure was performed using ImageJ software. Wound area at 0 and 24 hours was measured for each treatment group, and percentage closure was calculated using the Equation 1. Data represent the mean ± SD from three independent experiments. Low, medium, and high doses correspond to 0.5, 1, and 5 µM brepocitinib, respectively. Values represent mean ± SD from at least three independent experiments. Significance compared with control: * P < 0.05, ** P < 0.01, *** P < 0.001. Regression analysis confirmed a significant linear relationship between concentration and inhibition of wound closure (R 2 > 0.95, P < 0.01). In all experiments, low, medium, and high doses correspond to 0.5 µM, 1 µM, and 5 µM brepocitinib, respectively.

Article Snippet: Primary RA fibroblast-like synoviocytes (RA-FLS; HFLS-RA, Cell Applications, Cat. No. 408RA-05a) were cultured according to the supplier’s instructions in Synoviocyte growth medium (Cell Applications, Cat. No. 415-500).

Techniques: Migration, Wound Healing Assay, Cell Culture, Control, Sterility, Transferring, Incubation, Inverted Microscopy, Software, Concentration Assay, Inhibition

Journal: Iranian Journal of Pharmaceutical Research : IJPR

Article Title: Dual TYK2/JAK1 Inhibition by Brepocitinib Reprograms Synoviocyte Pathobiology: Mechanistic Insights Into Targeted Therapy for Rheumatoid Arthritis

doi: 10.5812/ijpr-166019

Figure Lengend Snippet: Mechanistic summary of brepocitinib’s molecular and functional effects in MH7A synoviocytes. This diagram synthesizes findings from - into an integrated pathway model of brepocitinib action. The illustration highlights brepocitinib’s dual inhibition of TYK2 and JAK1, leading to downstream suppression of STAT1 and STAT3 phosphorylation. These upstream effects result in reduced transcription and secretion of key inflammatory cytokines (IL-6, TNF-α, and IFN-γ), which in turn drive reduced inflammation and impaired synoviocyte migration. Simultaneously, brepocitinib promotes mitochondrial-dependent apoptosis via increased BAX/BCL-2 ratio and activation of caspase-3. Notably, the inhibition of migration is independent of apoptosis, reflecting a cytokine-driven mechanism rather than loss of cell viability.

Article Snippet: Primary RA fibroblast-like synoviocytes (RA-FLS; HFLS-RA, Cell Applications, Cat. No. 408RA-05a) were cultured according to the supplier’s instructions in Synoviocyte growth medium (Cell Applications, Cat. No. 415-500).

Techniques: Functional Assay, Inhibition, Phospho-proteomics, Migration, Activation Assay

Journal: Frontiers in Immunology

Article Title: Galectin-1 levels do not predict outcomes in undifferentiated arthritis: a two-year prospective observational study

doi: 10.3389/fimmu.2026.1757168

Figure Lengend Snippet: Distribution of baseline serum Gal-1 levels stratified by disease category. (A) Proportion of patients by disease category in the study population. (B) Baseline plasma Gal-1 levels (adjusted for plate and frozen storage) across disease categories. Boxplots represent the median and interquartile range (25th–75th percentile), with individual patient values overlaid as dots. Group comparisons were performed using the Kruskal–Wallis test. Significance threshold was set at p < 0.05. CTD, connective tissue diseases; Gal-1, galectin-1; RA, rheumatoid arthritis; SpA, spondyloarthritis; UA, undifferentiated arthritis.

Article Snippet: Galectin-1 (Gal-1), an immunomodulatory lectin with anti-inflammatory properties, has been reported to be elevated in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).

Techniques: Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Galectin-1 levels do not predict outcomes in undifferentiated arthritis: a two-year prospective observational study

doi: 10.3389/fimmu.2026.1757168

Figure Lengend Snippet: Evolution of adjusted serum Gal-1 levels and DAS-28-CRP scores in patients with chronic inflammatory rheumatic diseases (SpA, RA and CTD) along two years of follow-up. Data are presented as boxplots, showing medians, interquartile ranges (IQR), and whiskers extending to 1.5 × IQR, depicting the evolution of adjusted Gal-1 (A) and DAS-28-CRP (B) across four visits over a two-year follow-up. Trends in Gal-1 and DAS-28-CRP were assessed using Cuzick’s non-parametric test, with significance defined as p-trend < 0.05. CTD, connective tissue diseases; DAS-28-CRP, Disease Activity Score in 28 joints using by C reactive protein; Gal-1, galectin-1; RA, rheumatoid arthritis; SpA, spondyloarthritis; UA, undifferentiated arthritis.

Article Snippet: Galectin-1 (Gal-1), an immunomodulatory lectin with anti-inflammatory properties, has been reported to be elevated in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).

Techniques: Activity Assay