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: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

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: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

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: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

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: Nuclear factor of activated T cells 2 (NFATC2) orchestrates T helper 17 (Th17) pathogenicity via a protein kinase C epsilon (PRKCE)-signal transducer and activator of transcription 3 (STAT3) cascade. (A) The binding density of NFATC2 was visualized by deepTools: the heatmap presents the cleavage under targets and tagmentation (CUT&Tag) counts on the different NFATC2 binding peaks in cluster of differentiation 4 positive T (CD4 + T) cells derived from control and collagen-induced arthritis (CIA) mice ( n = 3 per group). (B) Genome-wide distribution of upregulated and downregulated NFATC2-binding peaks in CD4 + T cells derived from control and CIA mice. (C) Gene Ontology biological process (GO-BP) analysis of the NFATC2 binding peaks at promoter genes. (D) Genome browser tracks of CUT&Tag signal at the representative target gene loci ( n = 3 per group). (E) Chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (ChIP-qPCR) assay detected NFATC2 levels at the PRKCE promoter regions in CD4 + T cells derived from control and CIA mice ( n = 3 per group). (F) The co-localization of PRKCE and pSTAT3 was detected by laser confocal microscopy ( n = 3 per group). (G) The levels of interleukin-17A (IL-17A) and retinoic acid-related orphan receptor gamma t (RORγt) were detected by enzyme-linked immunosorbent assay (ELISA) ( n = 6 per group). (H, I) Transwell assay (H) and quantification (I) showed the cell number across the upper chamber in cells treated with scramble short hairpin RNA (shRNA) or NFATC2 shRNA ( n = 5 per group). (J) The co-localization of C–C chemokine receptor type 6 (CCR6) and C–C motif chemokine ligand 20 (CCL20) was detected by laser confocal microscopy ( n = 3 per group). (K) The levels of CCR6 and CCL20 were detected by ELISA ( n = 6 per group). (L) The levels of IL-17A and RORγt were detected by ELISA under different interventions ( n = 6 per group). (M, N) Transwell assay (M) and quantification (N) showed the cell number across the upper chamber in cells under different interventions ( n = 5 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with scramble shRNA or control group; # P < 0.05, compared with NFATC2 shRNA 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. TSS: transcription start site; TTS: transcription termination site; bp: base pairs; Ifna11 : interferon alpha 11; Msh2 : mutator S (MutS) homolog 2; Psen2 : presenilin 2; Sanbr : switch 3 (SWI3), alteration/deficiency in activation 2 (ADA2), nuclear receptor co-repressor (N-CoR), transcription factor III B (TFIIIB) and bric-a-brac, tramtrack, broad complex domain regulator of class switch recombination (CSR); Tmem98 : transmembrane protein 98; DAPI: 4′,6-diamidino-2-phenylindole; OE: overexpression.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Binding Assay, Derivative Assay, Control, Genome Wide, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Transwell Assay, shRNA, Two Tailed Test, Activation Assay, Over Expression

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: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

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 suppress nuclear factor of activated T cells 2 (NFATC2) transcription by decreasing histone H3 lysine 9 lactylation (H3K9la) in cluster of differentiation 4 positive T (CD4 + T) cells. (A) Western blot analysis showed the level of global lactylation in spleen CD4 + T cells of collagen-induced arthritis (CIA) mice with or without LUT treatment ( n = 5 per group). (B) The immunofluorescence staining was used to verify the localization of global lactylation and 4′,6-diamidino-2-phenylindole (DAPI) in CD4 + T cells. The white arrow shows the nucleus. (C–E) The Western blot was used to verify the expression of H3K9la (C), histone h3 lysine 18 lactylation (H3K18la) (D), and histone h3 lysine 23 lactylation (H3K23la) (E) in spleen CD4 + T cells of CIA mice with or without LUT treatment ( n = 4 per group). (F) The immunofluorescence intensity of H3K9la in peripheral CD4 + T cells from each group was detected ( n = 15 per group). (G) Chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (ChIP-qPCR) detected the H3K9la level at the NFATC2 promoter regions in CD4 + T cells with or without sodium lactate (NaLa) treatment ( n = 3 per group). (H) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays monitoring expression of the NFATC2 in CD4 + T cells treated with different concentrations of NaLa (0, 5, 10, and 25 mM) for 24 h ( n = 6 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with control CD4 + T cell group; # P < 0.05, compared with CIA or rheumatoid arthritis (RA) 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 or Tukey multiple comparisons test. Pan Kla: pan-lysine lactylation.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, 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: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

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

Journal:

Article Title: Naturally Occurring Deletional Mutation in the C-Terminal Cytoplasmic Tail of CCR5 Affects Surface Trafficking of CCR5

doi: 10.1128/JVI.75.7.3462-3468.2001

Figure Lengend Snippet: Expression of CCR5 on peripheral blood CD4+ cells. (A) Typical staining of CCR5 on CD4+ T cells. CD4+ cells stained only with the FITC-conjugated goat antibody directed against rat IgG served as a control (shaded). Cells shown by a bar are regarded as CCR5+. (B) Proportion of CCR5+ cells in CD4+ HLA-DR+ cells and in CD4+ HLA-DR− cells. Ex. 1 and Ex. 2 denote the first and second sets of experiments, respectively. Closed circles denote cells obtained from heterozygotes for CCR5-893(−), and open circles denote cells from individuals who do not possess CCR5-893(−). Statistical significance of difference between heterozygotes for CCR5-893(−) and individuals who do not possess CCR5-893(−) are shown only in the second set of experiments. (C) Mean fluorescence intensity of CCR5 in CCR5+ cells. For details, see the legend to panel B. (D) Infectability of donor CD4+ cells. CD4+ cells obtained from two heterozygotes (open circles and squares) and two nonmutant individuals (closed circles and squares) were stimulated with phytohemagglutinin and inoculated with HIV-1 strain SF162. Culture supernatants of infected cells were periodically assayed for levels of p24 core antigen.

Article Snippet: CD4 + cells were positively selected from peripheral blood mononuclear cells by using MACS CD4 MicroBeads (Miltenyi Biotec, Nordrhein-Westfalen, Germany) and stained with T227 rat monoclonal antibody directed against CCR5 followed by FITC-conjugated goat antibody directed against rat IgG (American Qualex Antibodies) and a peridinin chlorophyll protein (PerCP)-conjugated mouse monoclonal antibody directed against HLA-DR (Becton Dickinson).

Techniques: Expressing, Staining, Control, Fluorescence, Infection

Journal: Journal of Immunotoxicology

Article Title: Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

doi: 10.1080/1547691x.2016.1193574

Figure Lengend Snippet: Figure 1. Colonies of proliferating CD4þ T-cells during in vitro human TH17 cell differentiation. For stimulation and expansion of the T-cells, polyclonal stimulators (includ- ing anti-CD3 and anti-CD28 antibodies) were used (see ‘‘Methods’’ section). Representative micro-images are shown with different magnifications.

Article Snippet: From each sample, CD4þ T-cells were isolated by magnetic activated cell sorting (MACS) using human CD4 MicroBeads (Miltenyi Biotec, London, UK), according to the manufacturer instructions.

Techniques: In Vitro, Cell Differentiation

Journal: Journal of Immunotoxicology

Article Title: Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

doi: 10.1080/1547691x.2016.1193574

Figure Lengend Snippet: Figure 2. IL-1b, IL-6, and IL-23 – but not TGFb – are required for optimal in vitro differentiation of human CD4þ TH17 cells. (A) Flow cytometric dot-plot analysis of intra- cellular IL-17 expression by CD4þ T-cells. Representative data of five independent experiments (i.e. PBMC from five different donors) are shown. (B) Columns represent mean percentage (±SEM) of T-cells expressing intracellular IL-17 in each culture condition. ns: Non-significant (*p < 0.05).

Article Snippet: From each sample, CD4þ T-cells were isolated by magnetic activated cell sorting (MACS) using human CD4 MicroBeads (Miltenyi Biotec, London, UK), according to the manufacturer instructions.

Techniques: In Vitro, Expressing

Journal: Journal of Immunotoxicology

Article Title: Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

doi: 10.1080/1547691x.2016.1193574

Figure Lengend Snippet: Figure 3. IFNc production by human T-cells differentiated in presence of IL-1b, IL-6, and IL-23 – with or without TGFb as well. (A) Flow cytometric analysis of intracellu- lar expression of IFNc by TH17 cells derived from CD4þ T-cells. (B) Mean expression levels (±SEM) from five independent experiments (i.e. PBMC from five different donors) using various culture conditions (i.e. different TGFb concentration and inhibitors of TGFb signaling pathways) are shown. ns: Non-significant (*p < 0.05).

Article Snippet: From each sample, CD4þ T-cells were isolated by magnetic activated cell sorting (MACS) using human CD4 MicroBeads (Miltenyi Biotec, London, UK), according to the manufacturer instructions.

Techniques: Expressing, Derivative Assay, Concentration Assay, Protein-Protein interactions

Journal: Journal of Immunotoxicology

Article Title: Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

doi: 10.1080/1547691x.2016.1193574

Figure Lengend Snippet: Figure 4. TGFb effects on Foxp3 expression by CD4þ T-cells differentiated under TH17 cell-promoting conditions. (A) Intracellular staining of Foxp3. (B) Columns are mean (±SEM) Foxp3 expression levels in five independent experiments (i.e. PBMC from five different donors) (*p < 0.05).

Article Snippet: From each sample, CD4þ T-cells were isolated by magnetic activated cell sorting (MACS) using human CD4 MicroBeads (Miltenyi Biotec, London, UK), according to the manufacturer instructions.

Techniques: Expressing, Staining

Journal: Journal of Immunotoxicology

Article Title: Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

doi: 10.1080/1547691x.2016.1193574

Figure Lengend Snippet: Figure 6. TH17 cell-derived cytokines; modulation by TGFb and blocking of TGFb signaling. Cytokine levels in supernatants of CD4þ T-cells differentiated toward TH17 states in the presence or absence of TGFb and/or inhibitors of signaling pathways. Data showed means± SEM from five independent experiments (i.e. PBMC from five different donors). ND: not detected (*p < 0.05).

Article Snippet: From each sample, CD4þ T-cells were isolated by magnetic activated cell sorting (MACS) using human CD4 MicroBeads (Miltenyi Biotec, London, UK), according to the manufacturer instructions.

Techniques: Derivative Assay, Blocking Assay, Protein-Protein interactions