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: FEBS letters

Article Title: Structural activity relationship of flavonoids with estrogen-related receptor gamma.

doi: 10.1016/j.febslet.2009.11.026

Figure Lengend Snippet: Fig. 2. Effects of flavonoids on the activities of ERb and estrogen-related receptor c (ERRc). (A) HeLa cells were transfected with an expression plasmid of Gal4-DBD-ERb-LBD together with a luciferase reporter and a control Renilla luciferase plasmid. About 10 nM 17b-estradiol as a positive control or different flavonoids at 5 lM were added for 24 h before luciferase assays. Fold induction by compounds were calculated and shown compared to dimethyl sulfoxide (DMSO) as a vehicle. (B) HeLa cells were transfected with expression plasmids of Gal4-DBD control or Gal4-DBD-ERRc-LBD with or without pcDNA-peroxisome proliferators-activated receptor c coactivator-1a (PGC-1a) together with a luciferase reporter and a control Renilla luciferase plasmid. DMSO or 10 lM 4-hydroxytamoxifen (4-OHT) was added and assays performed as in (A). (C) Transfection were performed as in (B) with different flavonoids (1, 5, and 25 lM); % activity indicates the normalized activities of ERRc under the influences of flavonoids compared to DMSO control set at 100%. (A–C) Results represent mean ± S.E.M. **P < 0.01.

Article Snippet: Membranes were incubated with rabbit anti-human-PGC-1a antibody generated in house or mouse anti-b-actin antibody (Boster) followed by horseradish peroxidase-conjugated secondary antibody (Amersham) and developed with BeyoECL Plus reagent (Beyotime).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Control, Positive Control, Activity Assay

Journal: FEBS letters

Article Title: Structural activity relationship of flavonoids with estrogen-related receptor gamma.

doi: 10.1016/j.febslet.2009.11.026

Figure Lengend Snippet: Fig. 3. Apigenin directly blocks the interaction between ERRc and PGC-1a. (A) 125 nM of purified ERRc-LBD was tested for its interaction with NR1, NR2, and NR3 motifs as described [6]. (B) DMSO, 1 lM 4-OHT, 5 lM DY-131, 25 lM daidzein, or 25 lM luteolin was incubated with 50 nM ERRc-LBD for 1 hr before interaction analysis with NR2 as in (A). (C) Different doses of apigenin were tested as in (B).

Article Snippet: Membranes were incubated with rabbit anti-human-PGC-1a antibody generated in house or mouse anti-b-actin antibody (Boster) followed by horseradish peroxidase-conjugated secondary antibody (Amersham) and developed with BeyoECL Plus reagent (Beyotime).

Techniques: Incubation

Journal: FEBS letters

Article Title: Structural activity relationship of flavonoids with estrogen-related receptor gamma.

doi: 10.1016/j.febslet.2009.11.026

Figure Lengend Snippet: Fig. 4. Effects of flavonoids on the activity of PGC-1a. (A) HeLa cells were transfected with an expression plasmid of Gal4-DBD or Gal4-DBD-PGC-1a together with a luciferase reporter and a control Renilla luciferase plasmid. DMSO or 1, 5, and 25 lM luteolin were added for 24 h; % activity indicates the normalized activities of PGC-1a compared to DMSO control set at 100%. (B) HeLa cell extracts pre-treated with DMSO or 25 lM luteolin was probed with anti-PGC-1a or b-actin antibodies for Western analysis. PGC-1a protein levels normalized to b-actin from three independent experiments were quantified. Results represent mean ± S.E.M. **P < 0.01.

Article Snippet: Membranes were incubated with rabbit anti-human-PGC-1a antibody generated in house or mouse anti-b-actin antibody (Boster) followed by horseradish peroxidase-conjugated secondary antibody (Amersham) and developed with BeyoECL Plus reagent (Beyotime).

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Control, Western Blot