Journal: Journal of Neuroinflammation

Article Title: A BRD4/p300/SP1 epigenetic cascade drives microglial P2X4R transcription and promotes neuropathic pain

doi: 10.1186/s12974-026-03965-2

Figure Lengend Snippet: SNI mice exhibit marked microglial P2X4R activation and increased histone acetylation in the spinal cord. a A mouse spared nerve injury (SNI) model of neuropathic pain were established by surgically transecting the tibial and common peroneal nerves of the sciatic nerve, preserving the sural nerve, while control (Ctrl) mice underwent sham surgery. L4-6 segments of spinal cord and dorsal root ganglia (DRGs) were obtained for further analysis (ten mice in each group). b Behavioral analyses assess mechanical and cold allodynia in mice at days 0, 7, 14, and 21 following surgery ( n = 12). * P < 0.05, Two-way ANOVA. PWT, Paw Withdrawal Threshold; LLF, Lifting/Licking Frequency. c Representative traces of action potentials (APs) elicited by current injection in dorsal root ganglion neurons from Ctrl and SNI mice. Quantitative analysis of AP frequency. n = 7 neurons per group. Bar chart with data points. * P < 0.05, Student’s t-test. d Representative photomicrographs of spinal sections from Ctrl and SNI mice stained for P2X4R by immunohistochemistry (IHC) staining. Positively-stained microglia cells were indicated by red arrows. e Representative immunofluorescence images of IBA1 (green) and P2X4R (red) in the spinal cord from Ctrl and SNI mice. Nuclei were stained with DAPI (blue). The boxed region in the merged image is shown at higher magnification in the adjacent panel. f ImageJ quantification of P2X4R/IBA1 colocalization in SNI mouse spinal dorsal horn from panel ( e ). g Western blot analysis of P2X4R, IL-1β, IL-6 and CXCR4 protein levels in spinal tissue homogenates from Ctrl and SNI mice. GAPDH served as a loading control. Blots are representative of two samples per group. Quantitative data (right panel) were presented as means ± SEM, n = 6. * P < 0.05, Student’s t-test. h Western blot analysis of pan-acetylation (Pan-Ace) levels in in spinal tissue homogenates from Ctrl and SNI mice. Histone acetylation is indicated by purple box. H3 is used as a loading control. Quantification is shown on the right. Data are presented as mean ± SEM, n = 6. * P < 0.05, Student’s t-test

Article Snippet: Proteins were transferred to PVDF membranes, blocked with 5% BSA, and probed overnight at 4 °C with primary antibodies: BRD4 (83375 S, CST, 1:1000), SP1 (A19649, ABclonal, 1:1000), p300 (ab235378, Abcam, 1:1000), P2X4R (13534-1-AP, Proteintech, 1:1000), Tip60 (10827-1-AP, Proteintech, 1:1000), CBP (AG1691, Beyotime, 1:1000), GCN5 (CSB- PA002657 , CUSABIO, 1:1000), IL-1β (260481-AP, Proteintech, 1:1000), TNF-α (AF8208, Beyotime, 1:1000), IL-6 (AF7236, Beyotime, 1:1000), CXCR4 ( AF300411 , AiFang Bio, 1:1000), Pan-Acetylation (PTM-105RM, PTM Bio., 1:1000), H3K9ac (ab4441, Abcam, 1:4000), H3K14ac (HY- P80167 , MCE, 1:1000), H3K18ac (HY- P80168 , MCE, 1:1000), H3K27ac (HY- P80169 , MCE, 1:1000), H4K5ac (HY- P80182 , MCE, 1:1000), H4K8ac (HY- P80180 , MCE, 1:1000), H4K12ac (ab177793, Abcam, 1:4000), H4K16ac (HY- P80179 , MCE, 1:1000), Histone H3 (AF7101, Beyotime, 1:2000), Histone H4 (AF7107, Beyotime, 1:2000), GAPDH (FD0063, FDbio, 1:5000).

Techniques: Activation Assay, Preserving, Control, Analysis, Injection, Staining, Immunohistochemistry, Immunofluorescence, Western Blot, Tissue

Journal: Journal of Neuroinflammation

Article Title: A BRD4/p300/SP1 epigenetic cascade drives microglial P2X4R transcription and promotes neuropathic pain

doi: 10.1186/s12974-026-03965-2

Figure Lengend Snippet: Inhibition of the histone acetyltransferase p300 alleviates histone acetylation and microglial P2X4R upregulation.. a Western blotting. Spinal tissue homogenates from Ctrl and SNI mice were assayed for Histone3 acetylation at lysine 9, 14, 18 and 27 (H3K9ac, H3K14ac, H3K18ac, H3K27ac) and Histone3 (the left panel) and Histone4 acetylation at lysine 5, 8, 12 and 16 (H4K16ac, H4K12ac, H4K8ac, H4K5ac) and Histone4 (the right panel). Quantifications are shown on the right. Data are presented as means ± SEM, n = 6. * P < 0.05, Student’s t-test. b Western blotting. Spinal tissue homogenates from Ctrl and SNI mice were assayed for P300, TIP60, CBP, and GCN5 protein levels. Quantifications are shown on the right. Data are presented as means ± SEM, n = 6. * P < 0.05, Student’s t-test. c Western blotting. BV2 cells were treated with LPS (100 ng/ml) and ATP (20 µM) in present with or without JQ1 (1 µM) treatment. Twenty-four hours later, the cell lysates were assayed for H3K9ac, H3K14ac, H3K18ac, H3K27ac, and Histone3 (the left panel) and H4K16ac, H4K12ac, H4K8ac, H4K5ac and Histone4 (the right panel). Quantifications are shown on the right. Data are presented as means ± SEM, n = 3. * P < 0.05, one-way ANOVA followed by Tukey’s post-hoc test. d Chromatin immunoprecipitation (ChIP) assay. BV2 cells were treated with LPS (100 ng/ml) and ATP (20 µM) with or without C646 (10 µM, 24 h). Chromatin was immunoprecipitated with antibodies against H3K9ac, H3K27ac, H4K8ac or H4K5ac. Precipitated DNA was amplified by PCR using primers flanking the SP1 motif region. Agarose gels (1.5%) show results from one representative sample per group. Quantifications are shown on the right. Data are presented as means ± SEM, n = 4. * P < 0.05, one-way ANOVA followed by Tukey’s post-hoc test. e Luciferase assay. BV2 cells were transfected with a P2rx4 promoter luciferase reporter P2rx4 -luc plus a renilla luciferase reporter, and then treated with LPS (100 ng/ml) and ATP (20 µM) with or without C646 (10 µM, 24 h). Cell lysates were assayed for luciferase activities. The relative luciferase activities of fold changes were presented. Data were presented as means ± SEM, n = 3. * P < 0.05, one-way ANOVA followed by Tukey’s post-hoc test. f Western blotting. BV2 cells and primary microglia cells were treated with LPS (100 ng/ml) and ATP (20 µM) with or without C646 (10 µM, 24 h). The cell lysates were assayed for P2X4R protein levels. Quantifications are shown on the right. Data are presented as means ± SEM, n = 3. * P < 0.05, one-way ANOVA followed by Tukey’s post-hoc test

Article Snippet: Proteins were transferred to PVDF membranes, blocked with 5% BSA, and probed overnight at 4 °C with primary antibodies: BRD4 (83375 S, CST, 1:1000), SP1 (A19649, ABclonal, 1:1000), p300 (ab235378, Abcam, 1:1000), P2X4R (13534-1-AP, Proteintech, 1:1000), Tip60 (10827-1-AP, Proteintech, 1:1000), CBP (AG1691, Beyotime, 1:1000), GCN5 (CSB- PA002657 , CUSABIO, 1:1000), IL-1β (260481-AP, Proteintech, 1:1000), TNF-α (AF8208, Beyotime, 1:1000), IL-6 (AF7236, Beyotime, 1:1000), CXCR4 ( AF300411 , AiFang Bio, 1:1000), Pan-Acetylation (PTM-105RM, PTM Bio., 1:1000), H3K9ac (ab4441, Abcam, 1:4000), H3K14ac (HY- P80167 , MCE, 1:1000), H3K18ac (HY- P80168 , MCE, 1:1000), H3K27ac (HY- P80169 , MCE, 1:1000), H4K5ac (HY- P80182 , MCE, 1:1000), H4K8ac (HY- P80180 , MCE, 1:1000), H4K12ac (ab177793, Abcam, 1:4000), H4K16ac (HY- P80179 , MCE, 1:1000), Histone H3 (AF7101, Beyotime, 1:2000), Histone H4 (AF7107, Beyotime, 1:2000), GAPDH (FD0063, FDbio, 1:5000).

Techniques: Inhibition, Western Blot, Tissue, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Luciferase, Transfection

Journal: Journal of Neuroinflammation

Article Title: A BRD4/p300/SP1 epigenetic cascade drives microglial P2X4R transcription and promotes neuropathic pain

doi: 10.1186/s12974-026-03965-2

Figure Lengend Snippet: BRD4 mediates histone acetylation-incurred P2X4R transcriptional activation and neuropathic pain behaviors in SNI mice. SNI mice were treated with or without JQ1 (10 mg/kg, 7 days) (ten mice in each group) ( a ) Real-time PCR analysis of mRNA expression of Brd2 , Brd3 , and Brd4 from the spinal cord of Ctrl and SNI mice. Data are presented as mean ± SEM, n = 6. * P < 0.05, Student’s t-test. b Western blotting. Spinal tissue homogenates were assayed for BRD4 protein levels. Quantifications are shown on the right. Data are presented as means ± SEM, n = 6. * P < 0.05, Student’s t-test. c Beginning on day 7 after SNI surgery, mice were intraperitoneally (i.p.) injected with JQ1 every other day, totaling 7 administrations. d Horizontal movement traces in the open field test (OFT) of the Ctrl, JQ1, SNI, SNI/JQ1 groups. Quantification of the total distance (T.D.) and the time in central (TIC). Data are presented as means ± SEM, n = 6. * P < 0.05, one-way ANOVA followed by Tukey’s post-hoc test. e Behavioral analyses assess mechanical (PWT) and cold allodynia (LLF) in mice at days 0, 7, 14, and 21 following surgeries. Data are presented as means ± SEM, n = 6. * P < 0.05, two-way ANOVA. PWT, Paw Withdrawal Threshold; LLF, Lifting/Licking Frequency. f Representative traces of action potentials (APs) elicited by current injection in dorsal root ganglion neurons from Ctrl, JQ1, SNI, SNI/JQ1 mice. g The number of APs produced in panel ( f ). n = 7 neurons per group. Bar chart with data points. Ca2 + in DRG from Ctrl, JQ1, SNI, SNI/JQ1 mice was measured using test kits. Bar chart with data points. ( n = 5). Data were presented as means ± SEM, * P < 0.05, one-way ANOVA followed by Tukey’s post-hoc test. h Western blotting. Spinal tissue homogenates were assayed for P2X4R, IL-6, CXCR4, and IL-1β protein levels. GAPDH served as a loading control. Blots are representative of two sample per group. Quantifications are shown on the right. Data are presented as means ± SEM, n = 6. * P < 0.05, one-way ANOVA followed by Tukey’s post-hoc test

Article Snippet: Proteins were transferred to PVDF membranes, blocked with 5% BSA, and probed overnight at 4 °C with primary antibodies: BRD4 (83375 S, CST, 1:1000), SP1 (A19649, ABclonal, 1:1000), p300 (ab235378, Abcam, 1:1000), P2X4R (13534-1-AP, Proteintech, 1:1000), Tip60 (10827-1-AP, Proteintech, 1:1000), CBP (AG1691, Beyotime, 1:1000), GCN5 (CSB- PA002657 , CUSABIO, 1:1000), IL-1β (260481-AP, Proteintech, 1:1000), TNF-α (AF8208, Beyotime, 1:1000), IL-6 (AF7236, Beyotime, 1:1000), CXCR4 ( AF300411 , AiFang Bio, 1:1000), Pan-Acetylation (PTM-105RM, PTM Bio., 1:1000), H3K9ac (ab4441, Abcam, 1:4000), H3K14ac (HY- P80167 , MCE, 1:1000), H3K18ac (HY- P80168 , MCE, 1:1000), H3K27ac (HY- P80169 , MCE, 1:1000), H4K5ac (HY- P80182 , MCE, 1:1000), H4K8ac (HY- P80180 , MCE, 1:1000), H4K12ac (ab177793, Abcam, 1:4000), H4K16ac (HY- P80179 , MCE, 1:1000), Histone H3 (AF7101, Beyotime, 1:2000), Histone H4 (AF7107, Beyotime, 1:2000), GAPDH (FD0063, FDbio, 1:5000).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Analysis, Expressing, Western Blot, Tissue, Injection, Produced, Control

Journal: Journal of Neuroinflammation

Article Title: A BRD4/p300/SP1 epigenetic cascade drives microglial P2X4R transcription and promotes neuropathic pain

doi: 10.1186/s12974-026-03965-2

Figure Lengend Snippet: P2X4R inactivation is critical for the neuropathic pain-relieving effects of BRD4 inhibition in SNI mice. All mice were grouped into six groups, vehicle control (Ctrl), SNI, SNI mice treated with JQ1 (10 mg/kg, 7 days), BzATP (5 mg/kg, 7 days), SNI mice treated with BzATP and SNI treated with both JQ1 and BzATP (ten mice in each group). a Representative immunofluorescence images of P2X4R (red) and IBA1 (green) in the spinal cord. b Behavioral analyses for mechanical allodynia (PWT) and cold allodynia (LLF) were assessed in mice at week 3 following surgery. Data were presented as means ± SEM, n = 6. * P < 0.05, two-way ANOVA. PWT, Paw Withdrawal Threshold; LLF, Lifting/Licking Frequency. c Horizontal movement traces in the open field test (OFT). Quantification of the total distance and the time in central. Data were presented as means ± SEM, n = 6. * P < 0.05, two-way ANOVA. d Western blot analysis of P2X4R, IL-6, TNF-α, and CXCR4 protein expression in spinal cord. Blots are representative of two sample per group. The quantitative analysis plot is shown on the right. Data were presented as mean ± SEM, n = 6. * P < 0.05, two-way ANOVA. e Schematic illustrating sequential p300-mediated acetylation of histones H3 and H4, followed by BRD4-dependent assembly of a BRD4–p300–SP1 transcriptional cascade that drives P2rx4 transactivation, leading to dorsal spinal microglial activation and neuropathic pain in the mouse SNI model. Pharmacological inhibition of p300 (C646) or BRD4 (JQ1) disrupts this pathway and attenuates microglial activation and pain

Article Snippet: Proteins were transferred to PVDF membranes, blocked with 5% BSA, and probed overnight at 4 °C with primary antibodies: BRD4 (83375 S, CST, 1:1000), SP1 (A19649, ABclonal, 1:1000), p300 (ab235378, Abcam, 1:1000), P2X4R (13534-1-AP, Proteintech, 1:1000), Tip60 (10827-1-AP, Proteintech, 1:1000), CBP (AG1691, Beyotime, 1:1000), GCN5 (CSB- PA002657 , CUSABIO, 1:1000), IL-1β (260481-AP, Proteintech, 1:1000), TNF-α (AF8208, Beyotime, 1:1000), IL-6 (AF7236, Beyotime, 1:1000), CXCR4 ( AF300411 , AiFang Bio, 1:1000), Pan-Acetylation (PTM-105RM, PTM Bio., 1:1000), H3K9ac (ab4441, Abcam, 1:4000), H3K14ac (HY- P80167 , MCE, 1:1000), H3K18ac (HY- P80168 , MCE, 1:1000), H3K27ac (HY- P80169 , MCE, 1:1000), H4K5ac (HY- P80182 , MCE, 1:1000), H4K8ac (HY- P80180 , MCE, 1:1000), H4K12ac (ab177793, Abcam, 1:4000), H4K16ac (HY- P80179 , MCE, 1:1000), Histone H3 (AF7101, Beyotime, 1:2000), Histone H4 (AF7107, Beyotime, 1:2000), GAPDH (FD0063, FDbio, 1:5000).

Techniques: Inactivation, Inhibition, Control, Immunofluorescence, Western Blot, Analysis, Expressing, Activation Assay

Journal: Journal of Translational Medicine

Article Title: Microbiota-associated metabolite pantothenic acid enhances skeletal muscle contusion repair via epigenetic regulation of macrophage M2 polarization

doi: 10.1186/s12967-026-08087-0

Figure Lengend Snippet: Pantothenic acid synergizes with IL-4 to drive M2 polarization by upregulating Arg1 expression through mediating H3K27ac. ( A ) Intracellular acetyl-CoA levels in each group of cells ( n = 6). ( B - C ) Relative protein expression levels of H3K27ac in each group of cells ( n = 6). ( D ) Enrichment of H3K27ac at the Arg1 gene promoter region in each group of cells ( n = 3). ( E ) Relative mRNA expression of Arg1 in each group of cells ( n = 7). ( F ) Enrichment of H3K27ac at the Arg1 gene promoter region in each group of mice ( n = 3). ( G ) Relative mRNA expression of Arg1 in each group of mice ( n = 6). ( H ) Intracellular acetyl-CoA levels in each group of cells ( n = 6). ( I - J ) Relative protein expression levels of H3K27ac in each group of cells ( n = 6). ( K - M ) Relative protein expression levels of CD206 ( L ) and Arg1 ( M ) in each group of cells ( n = 6). All data are presented as mean ± SEM. Statistical significance among multiple groups was determined by one-way ANOVA. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Chromatin was immunoprecipitated overnight at 4°C with an anti-H3K27ac antibody (Huabio, HA600047 ) or normal Rabbit IgG.

Techniques: Expressing