Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) alleviates collagen-induced arthritis (CIA)-associated chronic pain by reversing central sensitization. (A) The schematic of CIA model establishment and intervention with different doses of LUT. (B) Mechanical pain threshold in each group ( n = 8 per group). (C) Thermal withdrawal latency in each group ( n = 8 per group). (D) Immunofluorescence was used to detect the expression of cFos proto-oncogene (cFos) and calcitonin gene-related peptide (CGRP) in the spinal dorsal horn (SDH) of mice across groups. Specifically, c-Fos expression was quantified by counting positive cells per mm 2 , while CGRP levels were assessed based on fluorescence intensity ( n = 4 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with control group; # P < 0.05 and ## P < 0.01, compared with CIA model group, by repeated-measures one-way analysis of variance (ANOVA) or two-way ANOVA followed by post hoc Dunnett's multiple comparisons test. i.g. q.d.: intragastric administration once daily; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: : PTM-1419RM, PTM-1406RM, and PTM-1413RM; PTM Bio Inc.), rabbit anti-histone H3 (Cat. No.: ab1791; Abcam), mouse anti-CD68 (Cat. No.: sc-20060; Santa Cruz Biotechnology), rabbit anti-cFos proto-oncogene (cFos) antibody (Cat. No.: ab222699; Abcam), rabbit anti-calcitonin gene-related peptide (CGRP) antibody (Cat. No.: 14959; Cell Signaling Technology, Inc.), rabbit anti-IL-1β antibody (Cat. No.: 12242; Cell Signaling Technology, Inc.), rabbit anti-IL-6 antibody (Cat. No.: 12912; Cell Signaling Technology, Inc.), rabbit anti-TNF-α antibody (Cat. No.: 11948; Cell Signaling Technology, Inc.), rabbit anti-NFATC2 antibody (Cat. No.: 5861; Cell Signaling Technology, Inc.), rabbit anti-CD68 (Cat. No.: 97778; Cell Signaling Technology, Inc.), rabbit anti-CD40 (Cat. No.: 86165; Cell Signaling Technology, Inc.), mouse anti-NFATC2 antibody (Cat. No.: sc-7296; Santa Cruz Biotechnology), mouse anti-PRKCE antibody (Cat. No.: sc-1681; Santa Cruz Biotechnology), mouse anti-CCR6 antibody (Cat. No.: MAB590; Bio-Techne, Minneapolis, MN, USA), rabbit anti-CCL20 antibody (Cat. No.: ab9829; Abcam), rabbit anti-β-actin monoclonal antibody (Cat. No.: 4970; Cell Signaling Technology, Inc.), goat anti-rabbit IgG heavy and light chains (H&L) (Alexa Fluor® 488) (Cat. No.: ab150077; Abcam), and goat anti-mouse IgG H&L (Alexa Fluor® 647) (Cat. No.: ab150115; Abcam).

Techniques: Immunofluorescence, Expressing, Fluorescence, Control

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) can inhibit microglia activation in a collagen-induced arthritis (CIA)-induced rheumatoid arthritis (RA) model. (A) The protein level of ionized calcium-binding adapter molecule 1 (IBA1) was conducted by Western blot (top) and quantification and statistics (below) ( n = 5 per group). (B) The double-immunofluorescence staining (left) and quantitative analysis (right) of IBA1 and cluster of differentiation 68 (CD68) ( n = 4 per group). The arrows indicate CD68 + IBA1 + cells. The inset provides a detailed view of cells where CD68 and IBA1 are co-localized. (C) Representative pictures of immunofluorescence staining and the relative fluorescence for interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) ( n = 4 per group). (D) Enzyme-linked immunosorbent assay (ELISA) assay detected the level of IL-1β, IL-6, and TNF-α in the spinal dorsal horn (SDH) of CIA mice with or without LUT gavage ( n = 6 per group). (E) Determination of LUT concentration of mouse SDH by liquid chromatography-mass spectrometry (LC-MS). The chromatogram of LUT negative control (up) and sample (down). (F) The double-immunofluorescence staining of IBA1 and CD68 in the SDH of CIA mice with or without injection of 1.5 ng/mL LUT via the implanted catheter ( n = 4 per group). The arrows indicate CD68 + IBA1 + cells. (G) The immunofluorescence of IL-1β, IL-6, and TNF-α in the SDH of CIA mice with or without injection of 1.5 ng/mL LUT via the implanted catheter ( n = 4 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with control group; # P < 0.05, compared with CIA model group, by two-tailed Student's t -test or repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. ns: not significant. ns: not significant. DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: : PTM-1419RM, PTM-1406RM, and PTM-1413RM; PTM Bio Inc.), rabbit anti-histone H3 (Cat. No.: ab1791; Abcam), mouse anti-CD68 (Cat. No.: sc-20060; Santa Cruz Biotechnology), rabbit anti-cFos proto-oncogene (cFos) antibody (Cat. No.: ab222699; Abcam), rabbit anti-calcitonin gene-related peptide (CGRP) antibody (Cat. No.: 14959; Cell Signaling Technology, Inc.), rabbit anti-IL-1β antibody (Cat. No.: 12242; Cell Signaling Technology, Inc.), rabbit anti-IL-6 antibody (Cat. No.: 12912; Cell Signaling Technology, Inc.), rabbit anti-TNF-α antibody (Cat. No.: 11948; Cell Signaling Technology, Inc.), rabbit anti-NFATC2 antibody (Cat. No.: 5861; Cell Signaling Technology, Inc.), rabbit anti-CD68 (Cat. No.: 97778; Cell Signaling Technology, Inc.), rabbit anti-CD40 (Cat. No.: 86165; Cell Signaling Technology, Inc.), mouse anti-NFATC2 antibody (Cat. No.: sc-7296; Santa Cruz Biotechnology), mouse anti-PRKCE antibody (Cat. No.: sc-1681; Santa Cruz Biotechnology), mouse anti-CCR6 antibody (Cat. No.: MAB590; Bio-Techne, Minneapolis, MN, USA), rabbit anti-CCL20 antibody (Cat. No.: ab9829; Abcam), rabbit anti-β-actin monoclonal antibody (Cat. No.: 4970; Cell Signaling Technology, Inc.), goat anti-rabbit IgG heavy and light chains (H&L) (Alexa Fluor® 488) (Cat. No.: ab150077; Abcam), and goat anti-mouse IgG H&L (Alexa Fluor® 647) (Cat. No.: ab150115; Abcam).

Techniques: Activation Assay, Binding Assay, Western Blot, Double Immunofluorescence Staining, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Negative Control, Injection, Control, Two Tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) can inhibit the stimulation of cluster of differentiation 4 positive T (CD4 + T) cells on microglia activation in collagen-induced arthritis (CIA). (A) The expression level of CD4 was conducted by Western blot (left) and blot quantification and statistics (right) ( n = 3 per group). (B) Transwell assay showed the cell number across the upper chamber in CD4 + T cells from different groups ( n = 15 per group). (C) The double-immunofluorescence staining of ionized calcium-binding adapter molecule 1 (IBA1) and CD4 ( n = 3 per group). (D) A schematic illustration of the microglia intervened with supernatants of CD4 + T cells derived from different groups of mice. (E) Representative pictures of immunofluorescence staining and the relative fluorescence for IBA1, CD68, and CD40 of the microglia intervened with supernatants of CD4 + T cells in different groups of mice ( n = 3 per group). (F) The level of interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) conducted by enzyme-linked immunosorbent assay (ELISA) ( n = 6 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with control or control CD4 + T cell group; # P < 0.05, compared with CIA or CIA/rheumatoid arthritis (RA) CD4 + T cell group, by repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: : PTM-1419RM, PTM-1406RM, and PTM-1413RM; PTM Bio Inc.), rabbit anti-histone H3 (Cat. No.: ab1791; Abcam), mouse anti-CD68 (Cat. No.: sc-20060; Santa Cruz Biotechnology), rabbit anti-cFos proto-oncogene (cFos) antibody (Cat. No.: ab222699; Abcam), rabbit anti-calcitonin gene-related peptide (CGRP) antibody (Cat. No.: 14959; Cell Signaling Technology, Inc.), rabbit anti-IL-1β antibody (Cat. No.: 12242; Cell Signaling Technology, Inc.), rabbit anti-IL-6 antibody (Cat. No.: 12912; Cell Signaling Technology, Inc.), rabbit anti-TNF-α antibody (Cat. No.: 11948; Cell Signaling Technology, Inc.), rabbit anti-NFATC2 antibody (Cat. No.: 5861; Cell Signaling Technology, Inc.), rabbit anti-CD68 (Cat. No.: 97778; Cell Signaling Technology, Inc.), rabbit anti-CD40 (Cat. No.: 86165; Cell Signaling Technology, Inc.), mouse anti-NFATC2 antibody (Cat. No.: sc-7296; Santa Cruz Biotechnology), mouse anti-PRKCE antibody (Cat. No.: sc-1681; Santa Cruz Biotechnology), mouse anti-CCR6 antibody (Cat. No.: MAB590; Bio-Techne, Minneapolis, MN, USA), rabbit anti-CCL20 antibody (Cat. No.: ab9829; Abcam), rabbit anti-β-actin monoclonal antibody (Cat. No.: 4970; Cell Signaling Technology, Inc.), goat anti-rabbit IgG heavy and light chains (H&L) (Alexa Fluor® 488) (Cat. No.: ab150077; Abcam), and goat anti-mouse IgG H&L (Alexa Fluor® 647) (Cat. No.: ab150115; Abcam).

Techniques: Activation Assay, Expressing, Western Blot, Transwell Assay, Double Immunofluorescence Staining, Binding Assay, Derivative Assay, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) inhibits T helper 17 (Th17) differentiation and spinal infiltration. (A) Volcano plot of differentially expressed genes (DEGs) between cluster of differentiation 4 positive T (CD4 + T) cells of control and collagen-induced arthritis (CIA) group. (B) Volcano plot of DEGs between CD4 + T cells of CIA mice with or without LUT treatment. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated genes in CIA CD4 + T cells. (D) KEGG pathway enrichment analysis of downregulated genes in CIA + LUT CD4 + T cell. (E) Representative flow cytometry plots of spleen CD4 + interleukin-17 (IL-17) + T cells measured as CD4 + IL-17A + cell percentages (left), and the percentage of spleen CD4 + IL-17A + T cell was calculated by CytExpert 2.4.0.28 ( n = 3 per group) (right). (F) Enzyme-linked immunosorbent assay (ELISA) was employed to quantify IL-17A and IL-22 concentrations in spinal dorsal horn (SDH) samples collected from mice in each experimental group ( n = 6 per group). (G) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to quantify IL-17 a and IL-22 messenger ribonucleic acid (mRNA) in SDH samples collected from mice in each experimental group ( n = 6 per group). (H) The Venn diagram showed the intersection of the genes upregulated in CIA mice, genes downregulated after LUT treatment, and genes enriched in the Th17 differentiation pathway. (I) The heat map showed the mRNA expression profile of the overlapping genes ( n = 3 per group). (J) qRT-PCR validation of mRNA expression of the overlapping genes (nuclear factor of activated T cells 2 ( Nfatc2 ) , IL23α , IL1 receptor accessory protein ( IL1rap ), signal transducer and activator of transcription 5A ( Stat5a ), and zeta chain of T cell receptor associated protein kinase 70 ( Zap70 )) ( n = 4 per group). (K) The immunofluorescence intensity of NFATC2 in peripheral CD4 + T cells from each group was detected ( n = 15 per group). (L) The double-immunofluorescence staining was used to verify the level of co-localization of NFATC2 and CD4 in SDH ( n = 4 per group). The insets and white arrows indicate representative images of NFATC2-CD4 co-localization and cells exhibiting NFATC2-CD4 co-localization, respectively. ∗ P < 0.05 and ∗∗ P < 0.01, compared with control group; # P < 0.05 vs. CIA or rheumatoid arthritis (RA) group, by repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. CIA + LUT-H: CIA + LUT high dose; HIF-1: hypoxia inducible factor-1; NF-κB: nuclear factor kappa B; FoxO: forkhead box O; VEGF: vascular endothelial growth factor; PE-A: phycoerythrin-area; APC-A: allophycocyanin-area; UL: upper left; UR: upper right; LL: lower left; LR: lower right; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: : PTM-1419RM, PTM-1406RM, and PTM-1413RM; PTM Bio Inc.), rabbit anti-histone H3 (Cat. No.: ab1791; Abcam), mouse anti-CD68 (Cat. No.: sc-20060; Santa Cruz Biotechnology), rabbit anti-cFos proto-oncogene (cFos) antibody (Cat. No.: ab222699; Abcam), rabbit anti-calcitonin gene-related peptide (CGRP) antibody (Cat. No.: 14959; Cell Signaling Technology, Inc.), rabbit anti-IL-1β antibody (Cat. No.: 12242; Cell Signaling Technology, Inc.), rabbit anti-IL-6 antibody (Cat. No.: 12912; Cell Signaling Technology, Inc.), rabbit anti-TNF-α antibody (Cat. No.: 11948; Cell Signaling Technology, Inc.), rabbit anti-NFATC2 antibody (Cat. No.: 5861; Cell Signaling Technology, Inc.), rabbit anti-CD68 (Cat. No.: 97778; Cell Signaling Technology, Inc.), rabbit anti-CD40 (Cat. No.: 86165; Cell Signaling Technology, Inc.), mouse anti-NFATC2 antibody (Cat. No.: sc-7296; Santa Cruz Biotechnology), mouse anti-PRKCE antibody (Cat. No.: sc-1681; Santa Cruz Biotechnology), mouse anti-CCR6 antibody (Cat. No.: MAB590; Bio-Techne, Minneapolis, MN, USA), rabbit anti-CCL20 antibody (Cat. No.: ab9829; Abcam), rabbit anti-β-actin monoclonal antibody (Cat. No.: 4970; Cell Signaling Technology, Inc.), goat anti-rabbit IgG heavy and light chains (H&L) (Alexa Fluor® 488) (Cat. No.: ab150077; Abcam), and goat anti-mouse IgG H&L (Alexa Fluor® 647) (Cat. No.: ab150115; Abcam).

Techniques: Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Biomarker Discovery, Immunofluorescence, Double Immunofluorescence Staining

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) inhibited nuclear factor of activated T cells 2 (NFATC2)-drived T helper 17 (Th17) differentiation and spinal infiltration in collagen-induced arthritis (CIA) model. (A) Representative flow cytometry plots of cluster of differentiation 4 (CD4) + interleukin-17 (IL-17) + T cells measured as IL-17A + CD4 + cell percentages (left), and the percentage of spleen Th17 cells was calculated by CytExpert 2.4.0.28 ( n = 3 per group) (right). (B) Quantitative live cell tracking of motility reveals significant increases in migration speed and distance of Th17 polarization cytokine cocktail-stimulated CD4 + T cells by transfected with NFATC2 short hairpin RNA (shRNA) plasmid in vitro (top). Visual narrative of cell morphology through time-lapse imaging and tracking (middle). Distinct cellular structures such as lamellipodia and uropodia are annotated with red arrow ( n = 20 per group) (bottom). (C) The double-immunofluorescence staining was used to verify the level of co-localization of IL-17A and CD4 in spinal dorsal horn (SDH) ( n = 4 per group). The arrows indicate IL-17A + CD4 + cells. The inset provides a detailed view of cells where IL-17A and CD4 are co-localized. (D) The Spearman correlation analysis of the percentage of IL-17A + CD4 + T cells and the expression level of NFATC2 ( n = 6). (E–G) The immunofluorescence staining and Western blot were used to verify the level of IL-17A (E), phosphorylated signal transducer and activator of transcription 3 (pSTAT3) (F), and ionized calcium-binding adapter molecule 1 (IBA1) (G) in SDH ( n = 3–4 per group). (H) Mechanical pain threshold and thermal withdrawal latency in different groups ( n = 8 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with scramble shRNA, control, or CD4 c yclization recombination enzyme (Cre) + scramble shRNA groups; # P < 0.05 and ## P < 0.01, compared with CIA, or CD4 Cre + scramble shRNA + CIA group, by two-tailed Student's t -test or repeated-measures one-way analysis of variance (ANOVA) or two-way ANOVA followed by post hoc Dunnett's multiple comparisons test. SSC-A: side scatter-area; PE-A: phycoerythrin-area; DAPI: 4′,6-diamidino-2-phenylindole; CIA + LUT-H: CIA + LUT high dose; mRNA: messenger RNA; AAV: adeno-associated virus.

Article Snippet: : PTM-1419RM, PTM-1406RM, and PTM-1413RM; PTM Bio Inc.), rabbit anti-histone H3 (Cat. No.: ab1791; Abcam), mouse anti-CD68 (Cat. No.: sc-20060; Santa Cruz Biotechnology), rabbit anti-cFos proto-oncogene (cFos) antibody (Cat. No.: ab222699; Abcam), rabbit anti-calcitonin gene-related peptide (CGRP) antibody (Cat. No.: 14959; Cell Signaling Technology, Inc.), rabbit anti-IL-1β antibody (Cat. No.: 12242; Cell Signaling Technology, Inc.), rabbit anti-IL-6 antibody (Cat. No.: 12912; Cell Signaling Technology, Inc.), rabbit anti-TNF-α antibody (Cat. No.: 11948; Cell Signaling Technology, Inc.), rabbit anti-NFATC2 antibody (Cat. No.: 5861; Cell Signaling Technology, Inc.), rabbit anti-CD68 (Cat. No.: 97778; Cell Signaling Technology, Inc.), rabbit anti-CD40 (Cat. No.: 86165; Cell Signaling Technology, Inc.), mouse anti-NFATC2 antibody (Cat. No.: sc-7296; Santa Cruz Biotechnology), mouse anti-PRKCE antibody (Cat. No.: sc-1681; Santa Cruz Biotechnology), mouse anti-CCR6 antibody (Cat. No.: MAB590; Bio-Techne, Minneapolis, MN, USA), rabbit anti-CCL20 antibody (Cat. No.: ab9829; Abcam), rabbit anti-β-actin monoclonal antibody (Cat. No.: 4970; Cell Signaling Technology, Inc.), goat anti-rabbit IgG heavy and light chains (H&L) (Alexa Fluor® 488) (Cat. No.: ab150077; Abcam), and goat anti-mouse IgG H&L (Alexa Fluor® 647) (Cat. No.: ab150115; Abcam).

Techniques: Flow Cytometry, Cell Tracking Assay, Migration, Transfection, shRNA, Plasmid Preparation, In Vitro, Imaging, Double Immunofluorescence Staining, Expressing, Immunofluorescence, Staining, Western Blot, Binding Assay, Control, Two Tailed Test, Virus

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Effects of luteolin (LUT) treatment on nuclear factor of activated T cells 2 (NFATC2) transcription and lactic acid levels. (A, B) The total ion chromatogram of metabolites between cluster of differentiation 4 positive T (CD4 + T) cells derived from collagen-induced arthritis (CIA) mice with or without LUT (50 mg/kg) treatment: positive (A) and negative (B) ion models. (C, D) The principal component analysis (PCA) score plot of CD4 + T cells. It represents samples in the groups were closely cluster to one another: positive (C) and negative (D) ion models ( n = 3 per group). (E) Heatmap of differential metabolites derived from CIA mice with or without LUT treatment. The upregulated metabolites were marked in red, and the downregulated metabolites levels were presented in blue ( n = 3 per group). (F) Bubble map of the impact values of metabolic pathways, where the horizontal coordinate is the impact values enriched into different metabolic pathways, and the vertical coordinate is the enrichment pathway. The size of the dots indicates the corresponding number of metabolites on the pathway. The color of the dot reflects the P value, where the redder the color, the smaller the P value, and the bluer the color, the larger the P value. (G) The lactate level of CD4 + T cells derived from CIA mice treated with LUT or not based on global untargeted metabolomics ( n = 3 per group). (H) The lactate level in peripheral CD4 + T cells from each group was detected ( n = 15 per group). (I) Lactate content assay kit and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to detect the lactate level and Nfatc2 expression in T helper 17 (Th17) polarization cytokine cocktail-induced CD4 + T cells at different LUT concentrations (10, 20, and 40 μM) ( n = 6 per group). (J) The correlation of the lactate and the expression level of Nfatc2 in Th17 polarization cytokine cocktail-induced CD4 + T cells at 40 μM LUT concentrations ( n = 6). ∗ P < 0.05, compared with control CD4 + T cell group; # P < 0.05, compared with rheumatoid arthritis (RA) CD4 + T cell group; && P < 0.01, compared with CIA CD4 + T cell group, by two-tailed Student's t -test or repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. PC: principal component; HIF-1: hypoxia-inducible factor-1; mTOR: mammalian target of rapamycin.

Article Snippet: : PTM-1419RM, PTM-1406RM, and PTM-1413RM; PTM Bio Inc.), rabbit anti-histone H3 (Cat. No.: ab1791; Abcam), mouse anti-CD68 (Cat. No.: sc-20060; Santa Cruz Biotechnology), rabbit anti-cFos proto-oncogene (cFos) antibody (Cat. No.: ab222699; Abcam), rabbit anti-calcitonin gene-related peptide (CGRP) antibody (Cat. No.: 14959; Cell Signaling Technology, Inc.), rabbit anti-IL-1β antibody (Cat. No.: 12242; Cell Signaling Technology, Inc.), rabbit anti-IL-6 antibody (Cat. No.: 12912; Cell Signaling Technology, Inc.), rabbit anti-TNF-α antibody (Cat. No.: 11948; Cell Signaling Technology, Inc.), rabbit anti-NFATC2 antibody (Cat. No.: 5861; Cell Signaling Technology, Inc.), rabbit anti-CD68 (Cat. No.: 97778; Cell Signaling Technology, Inc.), rabbit anti-CD40 (Cat. No.: 86165; Cell Signaling Technology, Inc.), mouse anti-NFATC2 antibody (Cat. No.: sc-7296; Santa Cruz Biotechnology), mouse anti-PRKCE antibody (Cat. No.: sc-1681; Santa Cruz Biotechnology), mouse anti-CCR6 antibody (Cat. No.: MAB590; Bio-Techne, Minneapolis, MN, USA), rabbit anti-CCL20 antibody (Cat. No.: ab9829; Abcam), rabbit anti-β-actin monoclonal antibody (Cat. No.: 4970; Cell Signaling Technology, Inc.), goat anti-rabbit IgG heavy and light chains (H&L) (Alexa Fluor® 488) (Cat. No.: ab150077; Abcam), and goat anti-mouse IgG H&L (Alexa Fluor® 647) (Cat. No.: ab150115; Abcam).

Techniques: Derivative Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Control, Two Tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) could inhibit the expression and activity of lactate dehydrogenase A (LDHA). (A) Venn gram showed the intersection of upregulated genes in spleen cluster of differentiation 4 positive T (CD4 + T) cells of collagen-induced arthritis (CIA) mice and downregulated genes in spleen CD4 + T cells of CIA mice with LUT treatment and the genes enriched in the glycolytic pathway according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. (B) Heat map of differential shows the messenger ribonucleic acid (mRNA) level of hexokinase domain containing 1 ( Hkdc1 ) , phosphoglycerate kinase 1 ( Pgk1 ), Ldha , pyruvate kinase m1/2 ( Pkm ), bisphosphoglycerate mutase ( Bpgm ), phosphofructokinase platelet ( Pfkp ), phosphoglucomutase 1 ( Pgm1 ), and glucose-6-phosphate isomerase 1 ( Gpi1 ) in CD4 + T cells of CIA mice with or without LUT administration ( n = 3 per group). (C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) monitors the mRNA expression of Hkdc1 , Pgk1 , Ldha , Bpgm , Pkm , Pfkp , Pgm1 , and Gpi1 in CD4 + T cells of CIA mice with or without LUT administration ( n = 4 per group). The dashed lines serves to highlight the genes that were significantly upregulated in CD4 + T cells of the CIA group and significantly downregulated upon LUT treatment, thereby drawing clearer attention to the key findings. (D) The immunofluorescence intensity of LDHA in peripheral CD4 + T cells from each group was detected ( n = 15 per group). (E–H) A model of LUT binding with LDHA protein generated by molecular docking: surface diagram of LUT and LDHA binding (E), enlarged view of the surface diagram of LUT and LDHA binding (F), three-dimensional (3D) diagram of the interaction between LUT and LDHA (G), and 2D diagram of the interaction between LUT and LDHA (H). (I–K) The surface plasmon resonance (SPR) analysis of LUT-LDHA interaction (1.9–62.5 μmol/L): steady-state affinity fitting curve, showing the binding signal as a function of analyte concentration (I), kinetic fitting using a 1:1 binding model, where the affinity is calculated from the ratio of association to dissociation rates (J), and sensogram depicting real-time binding responses, with data points recorded every 0.1 s (K). (L) The root mean square deviation (RMSD) analysis of 100 ns-length molecular dynamics (MD) simulation results for LDHA and LUT. (M) Radius of gyration (Rg) graph of LDHA docked with LUT from 0 to 100 ns time scale. (N) The 3D free energy landscape of LDHA docked with LUT. (O) The 2D free energy landscape of LDHA docked with LUT. (P) The average number of hydrogen bonds between each ligand throughout 100 ns simulations. (Q) LDHA activity was measured in CD4 + T cells derived from control, CIA, and CIA + LUT mice ( n = 6 per group). (R) LDHA activity was measured in CD4 + T cells derived from different group ( n = 15 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with control CD4 + T cell group; # P < 0.05 and ## P < 0.05, compared with CIA or rheumatoid arthritis (RA) CD4 + T cell group, by repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. CIA + LUT-H: CIA + LUT high dose; DAPI: 4′,6-diamidino-2-phenylindole; K D : dissociation constant.

Article Snippet: : PTM-1419RM, PTM-1406RM, and PTM-1413RM; PTM Bio Inc.), rabbit anti-histone H3 (Cat. No.: ab1791; Abcam), mouse anti-CD68 (Cat. No.: sc-20060; Santa Cruz Biotechnology), rabbit anti-cFos proto-oncogene (cFos) antibody (Cat. No.: ab222699; Abcam), rabbit anti-calcitonin gene-related peptide (CGRP) antibody (Cat. No.: 14959; Cell Signaling Technology, Inc.), rabbit anti-IL-1β antibody (Cat. No.: 12242; Cell Signaling Technology, Inc.), rabbit anti-IL-6 antibody (Cat. No.: 12912; Cell Signaling Technology, Inc.), rabbit anti-TNF-α antibody (Cat. No.: 11948; Cell Signaling Technology, Inc.), rabbit anti-NFATC2 antibody (Cat. No.: 5861; Cell Signaling Technology, Inc.), rabbit anti-CD68 (Cat. No.: 97778; Cell Signaling Technology, Inc.), rabbit anti-CD40 (Cat. No.: 86165; Cell Signaling Technology, Inc.), mouse anti-NFATC2 antibody (Cat. No.: sc-7296; Santa Cruz Biotechnology), mouse anti-PRKCE antibody (Cat. No.: sc-1681; Santa Cruz Biotechnology), mouse anti-CCR6 antibody (Cat. No.: MAB590; Bio-Techne, Minneapolis, MN, USA), rabbit anti-CCL20 antibody (Cat. No.: ab9829; Abcam), rabbit anti-β-actin monoclonal antibody (Cat. No.: 4970; Cell Signaling Technology, Inc.), goat anti-rabbit IgG heavy and light chains (H&L) (Alexa Fluor® 488) (Cat. No.: ab150077; Abcam), and goat anti-mouse IgG H&L (Alexa Fluor® 647) (Cat. No.: ab150115; Abcam).

Techniques: Expressing, Activity Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Binding Assay, Generated, SPR Assay, Concentration Assay, Derivative Assay, Control