Journal: Frontiers in Endocrinology

Article Title: Integrated causal inference, kidney transcriptomics, and experimental validation identify ChREBP ( MLXIPL ) as a driver of maladaptive metabolic remodeling in diabetic kidney disease

doi: 10.3389/fendo.2026.1809567

Figure Lengend Snippet: Experimental validation of MLXIPL upregulation and metabolic network consistency in diabetic models. (A–C) Assessment of ChREBP expression in kidney tissues from db/m and db/db mice. Representative Western blots (A) and densitometric quantification (B) of ChREBP protein levels. For animal Western blot analyses, densitometric quantification was performed using all 12 mice per group. Full-length immunoblot images are provided in Supplementary Figure 4 . (C) Relative mRNA expression of Mlxipl . (D–F) Assessment of ChREBP expression in primary proximal tubular epithelial cells (PTECs) under normal glucose (NC) and high glucose (HG) conditions. Representative Western blots (D) , protein quantification (E) , and mRNA levels (F) . (G) Representative immunohistochemistry (IHC) staining of ChREBP in mouse kidney sections. Scale bar: 100 μm. (H) Representative immunofluorescence (IF) staining of ChREBP (green) in PTECs; nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. (I) Relative mRNA expression of MLXIPL -interacting metabolic genes in mouse kidneys. (J) Pearson correlation analysis between Mlxipl expression and downstream metabolic targets in kidney tissues.Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Sections were blocked with 1% BSA and incubated with anti-ChREBP antibody (Novus Biologicals, NB400-135; IHC dilution 1:200), followed by appropriate secondary antibodies and chromogenic development.

Techniques: Biomarker Discovery, Expressing, Western Blot, Immunohistochemistry, Immunofluorescence, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: PGC ‐1α Transcriptionally Regulated by ChREBP Mitigates Neuropathic Pain Through Promoting Microglial Fatty Acid Oxidation and Anti‐Inflammatory Response

doi: 10.1002/cns.70744

Figure Lengend Snippet: ChREBP expression is increased in microglia of the NP rat model. (A) Timeline of the SNI surgery and behavioral testing. (B, C) Von Frey tests were conducted to measure the mechanical pain threshold in the ipsilateral (B) and contralateral (C) sides of the Sham group and SNI group before SNI surgery (baseline: BL) and on days 1, 3, 7, 14, and 21 after SNI surgery. (D, E) Representative immunofluorescence staining images (D) and colocalization analysis (E) of ChREBP with Iba‐1, ChREBP with NeuN, and ChREBP with GFAP. (F, G) Representative immunofluorescence staining images and quantitative analysis of the number of Iba‐1 + ChREBP + double‐positive cells in the Sham group and SNI group. (H, I) Representative immunofluorescence staining images and quantitative analysis of the number of NeuN + ChREBP + double‐positive cells in the Sham group and SNI group. (J) Protein band image of ChREBP in HAPI cells. Data are presented as mean ± SD, n = 6, ns p > 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking at room temperature for 1 h, the antibodies were prepared with QuickBlock primary antibody dilution buffer (P0262; Beyotime): goat polyclonal antibody to Iba‐1 (1:250, ab5076; Abcam), mouse monoclonal antibody to GFAP (1:400, 3670S; CST), mouse monoclonal antibody to NeuN (1:400; MAB377, Millipore), rabbit polyclonal antibody to ChREBP (1:200; NB400‐135, Novus Biologicals), mouse monoclonal antibody to PGC‐1α (1:200, 66369‐1‐Ig; Proteintech), rabbit polyclonal antibody to iNOS (1:200, BS1186; Bioworld) and rabbit polyclonal antibody to Arg‐1 (1:200, BS67738; Bioworld), and incubated at 4°C overnight.

Techniques: Expressing, Immunofluorescence, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: PGC ‐1α Transcriptionally Regulated by ChREBP Mitigates Neuropathic Pain Through Promoting Microglial Fatty Acid Oxidation and Anti‐Inflammatory Response

doi: 10.1002/cns.70744

Figure Lengend Snippet: In vivo knockdown of ChREBP reduces the number of anti‐inflammatory microglia and aggravates pain. (A) Experimental timeline of SNI surgery, spinal cord stereotaxic injection, and behavioral tests. (B, C) Representative immunofluorescence staining images (B) and quantitative analysis of cell numbers (C) of ChREBP and Iba‐1‐positive cells in the spinal cord. (D) ChREBP mRNA level in the spinal cord. (E) Representative immunofluorescence staining images for double‐labeled Iba‐1 and pro‐inflammatory microglial marker (iNOS) and double‐labeled Iba‐1 and anti‐inflammatory microglial marker (Arg‐1) in the spinal cord. (F–H) Quantitative analysis of immunofluorescence results, including (F) number of Iba‐1 + iNOS + colocalized cells; (G) number of Iba‐1 + Arg‐1 + colocalized cells; (H) ratio of Iba‐1 + iNOS + to Iba‐1 + Arg‐1 + cells. (I–J) Mechanical pain thresholds on the ipsilateral (I) and contralateral (J) sides were measured before microinjection (BFMI), at baseline (BL), and on days 1, 3, 7, 14, and 21 after SNI surgery in rats. Data are presented as mean ± SD, n = 6, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking at room temperature for 1 h, the antibodies were prepared with QuickBlock primary antibody dilution buffer (P0262; Beyotime): goat polyclonal antibody to Iba‐1 (1:250, ab5076; Abcam), mouse monoclonal antibody to GFAP (1:400, 3670S; CST), mouse monoclonal antibody to NeuN (1:400; MAB377, Millipore), rabbit polyclonal antibody to ChREBP (1:200; NB400‐135, Novus Biologicals), mouse monoclonal antibody to PGC‐1α (1:200, 66369‐1‐Ig; Proteintech), rabbit polyclonal antibody to iNOS (1:200, BS1186; Bioworld) and rabbit polyclonal antibody to Arg‐1 (1:200, BS67738; Bioworld), and incubated at 4°C overnight.

Techniques: In Vivo, Knockdown, Injection, Immunofluorescence, Staining, Labeling, Marker, Microinjection

Journal: CNS Neuroscience & Therapeutics

Article Title: PGC ‐1α Transcriptionally Regulated by ChREBP Mitigates Neuropathic Pain Through Promoting Microglial Fatty Acid Oxidation and Anti‐Inflammatory Response

doi: 10.1002/cns.70744

Figure Lengend Snippet: Overexpression of ChREBP activates the fatty acid oxidation in microglia. (A) KEGG enrichment analysis of the 50 hub genes significantly associated with ChREBP. (B) This panel compares the oxygen consumption rate of HAPI cells in four groups: Empty vector plasmid (±Etomoxir) and ChREBP overexpression plasmid (±Etomoxir). (C–F) Effects of ChREBP overexpression on fatty acid oxidation rate (C), basal respiration (D), maximal respiration (E), and ATP production (F) in the HAPI cells. (G‐N) RT‐qPCR was used to detect the mRNA expression levels of PPARG (G), SCD (H), FASN (I), PGC‐1α (J), ACACA (K), PPARA (L), SREBF1 (M), and HMGCR (N) in HAPI cells. These eight hub genes were selected from the STRING‐derived candidate gene set based on their highest connectivity degrees in the Cytoscape PPI network. Data are presented as mean ± SD, n = 3, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking at room temperature for 1 h, the antibodies were prepared with QuickBlock primary antibody dilution buffer (P0262; Beyotime): goat polyclonal antibody to Iba‐1 (1:250, ab5076; Abcam), mouse monoclonal antibody to GFAP (1:400, 3670S; CST), mouse monoclonal antibody to NeuN (1:400; MAB377, Millipore), rabbit polyclonal antibody to ChREBP (1:200; NB400‐135, Novus Biologicals), mouse monoclonal antibody to PGC‐1α (1:200, 66369‐1‐Ig; Proteintech), rabbit polyclonal antibody to iNOS (1:200, BS1186; Bioworld) and rabbit polyclonal antibody to Arg‐1 (1:200, BS67738; Bioworld), and incubated at 4°C overnight.

Techniques: Over Expression, Plasmid Preparation, Quantitative RT-PCR, Expressing, Derivative Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: PGC ‐1α Transcriptionally Regulated by ChREBP Mitigates Neuropathic Pain Through Promoting Microglial Fatty Acid Oxidation and Anti‐Inflammatory Response

doi: 10.1002/cns.70744

Figure Lengend Snippet: ChREBP directly regulates PGC‐1α expression by binding to its promoter region. (A) Both ChREBP and PGC‐1α colocalize with Iba‐1 in the spinal cord of NP rats. (B‐C) Representative immunofluorescence images of PGC‐1α after in vivo overexpression of ChREBP (B) and quantitative analysis of fluorescence intensity (C). (D) mRNA level of PGC‐1α after in vivo overexpression of ChREBP. (E, F) Effect of ChREBP overexpression on PGC‐1α protein level in HAPI cells (E) and quantitative analysis of protein bands (F). (G, H) mRNA levels of PGC‐1α (G) and ChREBP (H) after overexpression of PGC‐1α in HAPI cells. (I, J) Protein expression of PGC‐1α and ChREBP after overexpression of PGC‐1α in HAPI cells (I), and quantitative analysis of protein bands (J). (K) Two binding sites between ChREBP and the PGC‐1α promoter region were predicted using the JASPAR database. (L) Schematic diagram of the constructed luciferase reporter plasmids for the wild‐type (WT) and mutant (MUT1, MUT2, MUT3) PGC‐1α promoters. (M) Dual‐luciferase reporter assay showing the effect of ChREBP overexpression on luciferase activity of the WT PGC‐1α promoter plasmid. (N) Dual‐luciferase reporter assay comparing the effect of ChREBP overexpression on luciferase activity between the WT and three mutant (MUT1, MUT2, MUT3) PGC‐1α promoter plasmids. (O) ChIP‐qPCR showing the amplification of PGC‐1α promoter fragments in the ChREBP group compared with the IgG group. (P) The enrichment of PGC‐1α promoter fragments by ChREBP immunoprecipitation. Marker: DNA marker; Input: 2% input sample; IgG: Negative control; Histone H3: Positive control. ChREBP: Specific antibody for target detection. Data are represented as mean ± SD, n = 6 for in vivo experiments (A–D), n = 3 for in vitro experiments (E‐P), ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking at room temperature for 1 h, the antibodies were prepared with QuickBlock primary antibody dilution buffer (P0262; Beyotime): goat polyclonal antibody to Iba‐1 (1:250, ab5076; Abcam), mouse monoclonal antibody to GFAP (1:400, 3670S; CST), mouse monoclonal antibody to NeuN (1:400; MAB377, Millipore), rabbit polyclonal antibody to ChREBP (1:200; NB400‐135, Novus Biologicals), mouse monoclonal antibody to PGC‐1α (1:200, 66369‐1‐Ig; Proteintech), rabbit polyclonal antibody to iNOS (1:200, BS1186; Bioworld) and rabbit polyclonal antibody to Arg‐1 (1:200, BS67738; Bioworld), and incubated at 4°C overnight.

Techniques: Expressing, Binding Assay, Immunofluorescence, In Vivo, Over Expression, Fluorescence, Construct, Luciferase, Mutagenesis, Reporter Assay, Activity Assay, Plasmid Preparation, ChIP-qPCR, Amplification, Immunoprecipitation, Marker, Negative Control, Positive Control, In Vitro

Journal: CNS Neuroscience & Therapeutics

Article Title: PGC ‐1α Transcriptionally Regulated by ChREBP Mitigates Neuropathic Pain Through Promoting Microglial Fatty Acid Oxidation and Anti‐Inflammatory Response

doi: 10.1002/cns.70744

Figure Lengend Snippet: PGC‐1α overexpression reverses the microglial metabolism–polarization–inflammation–excitability–pain axis induced by ChREBP knockdown in microglia. (A) Schematic diagram showing the role of key molecules in the fatty acid oxidation pathway. (B‐E) mRNA levels of key fatty acid oxidation molecules CPT1A (B), CPT2 (C), ACADM (D), and HADHA (E) in the spinal cord of rats in each group. (F, G) Representative immunofluorescence staining images for double‐labeled Iba‐1 and pro‐inflammatory microglial marker (iNOS), and double‐labeled Iba‐1 and anti‐inflammatory microglial marker (Arg‐1) in the spinal cord (F), and the ratio of Iba‐1 + iNOS + to Iba‐1 + Arg‐1 + cells (G). (H–J) mRNA levels of inflammatory factors TNF‐α (H), IL‐1β (I), and IL‐6 (J) in the spinal cord of rats in each group. (K) Patch‐clamp electrophysiological recordings performed at the L5 spinal cord segment. (L–N) Representative images of spontaneous excitatory postsynaptic currents (sEPSC) in the spinal cord of rats in each group (L), and statistical analysis of their frequency (M) and amplitude (N). (O, P) Mechanical pain thresholds on the ipsilateral (O) and contralateral (P) sides of rats in each group. Data are represented as mean ± SD; n = 6 for in vivo experiments (B–J, O, P), n = 3 for electrophysiological recordings (K–N), ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking at room temperature for 1 h, the antibodies were prepared with QuickBlock primary antibody dilution buffer (P0262; Beyotime): goat polyclonal antibody to Iba‐1 (1:250, ab5076; Abcam), mouse monoclonal antibody to GFAP (1:400, 3670S; CST), mouse monoclonal antibody to NeuN (1:400; MAB377, Millipore), rabbit polyclonal antibody to ChREBP (1:200; NB400‐135, Novus Biologicals), mouse monoclonal antibody to PGC‐1α (1:200, 66369‐1‐Ig; Proteintech), rabbit polyclonal antibody to iNOS (1:200, BS1186; Bioworld) and rabbit polyclonal antibody to Arg‐1 (1:200, BS67738; Bioworld), and incubated at 4°C overnight.

Techniques: Over Expression, Knockdown, Immunofluorescence, Staining, Labeling, Marker, Patch Clamp, In Vivo

Journal: CNS Neuroscience & Therapeutics

Article Title: PGC ‐1α Transcriptionally Regulated by ChREBP Mitigates Neuropathic Pain Through Promoting Microglial Fatty Acid Oxidation and Anti‐Inflammatory Response

doi: 10.1002/cns.70744

Figure Lengend Snippet: Etomoxir eliminates the protective effect of ChREBP overexpression in microglia on the microglial metabolism–polarization–inflammation–excitability–pain axis by inhibiting microglial fatty acid oxidation. (A) Experimental timeline of SNI surgery, spinal cord stereotaxic injection, intrathecal catheterization, and behavioral tests. (B–E) mRNA levels of key fatty acid oxidation molecules CPT1A (B), CPT2 (C), ACADM (D), and HADHA (E) in the spinal cord of rats in each group. (F, G) Representative immunofluorescence staining images for double‐labeled Iba‐1 and pro‐inflammatory microglial marker (iNOS), and double‐labeled Iba‐1 and anti‐inflammatory microglial marker (Arg‐1) in the spinal cord (F), and the ratio of Iba‐1 + iNOS + to Iba‐1 + Arg‐1 + cells (G). (H–J) mRNA levels of inflammatory factors TNF‐α (H), IL‐1β (I), and IL‐6 (J) in the spinal cord of rats in each group. (K–M) Representative images of sEPSC in the spinal cord of rats in each group (K), and statistical analysis of their frequency (L) and amplitude (M). (N, O) Mechanical pain thresholds on the ipsilateral (N) and contralateral (O) sides of rats in each group. Data are represented as mean ± SD; n = 6 for in vivo experiments (B–J, N, O), n = 3 for electrophysiological recordings (K–M), ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking at room temperature for 1 h, the antibodies were prepared with QuickBlock primary antibody dilution buffer (P0262; Beyotime): goat polyclonal antibody to Iba‐1 (1:250, ab5076; Abcam), mouse monoclonal antibody to GFAP (1:400, 3670S; CST), mouse monoclonal antibody to NeuN (1:400; MAB377, Millipore), rabbit polyclonal antibody to ChREBP (1:200; NB400‐135, Novus Biologicals), mouse monoclonal antibody to PGC‐1α (1:200, 66369‐1‐Ig; Proteintech), rabbit polyclonal antibody to iNOS (1:200, BS1186; Bioworld) and rabbit polyclonal antibody to Arg‐1 (1:200, BS67738; Bioworld), and incubated at 4°C overnight.

Techniques: Over Expression, Injection, Immunofluorescence, Staining, Labeling, Marker, In Vivo