Journal: Advanced Science

Article Title: Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP‐Activated Exchange Protein‐1

doi: 10.1002/advs.202307556

Figure Lengend Snippet: EPAC‐1 contributes to mitochondrial dysfunction induced by ICH through its interaction with VDAC1. A) Samples of cortex surrounding the hematoma in mice were collected at 3, 6, 12, 24, and 48 h post‐ICH induction. The protein levels of EPAC‐1 were assessed via western blot analysis, and the alterations in expression were quantified. β‐Tubulin served as loading controls, with a sham group being normalized to a value of 1.0 for accurate comparisons ( n = 6). B) EPAC‐1 activation was assessed by measuring the levels of activated Rap1‐GTP using a Rap1 Activation Assay Kit. β‐Tubulin served as loading controls, with each sham group being normalized to a value of 1 for accurate comparisons n = 6. C) Neurons underwent a 12 h stimulation with 10 µ m OxyHb. Subsequently, mitochondria and cytoplasm were isolated, and the protein levels of EPAC‐1 in both compartments were evaluated using western blot analysis. β‐Tubulin and Tom20 served as loading controls, with each control group being normalized to a value of 1 for accurate comparisons, n = 6. D) Protein‐protein interaction (PPI) network was queried from the STRING database ( https://string‐db.org ), followed by functional protein enrichment analysis conducted through Cytoscape and ClueGO. E) Rigid protein‐protein docking (ZDOCK) was conducted to investigate the relationship between EPAC‐1 and VDAC1. The PDB format of the protein structural domain was obtained from the Protein Data Bank (PDB) at http://www.rcsb.org/ . The ZDOCK module was used to identify docking sites and calculate ZDOCK scores. F) Cortex samples surrounding the hematoma in mice were collected from sham and ICH 24 h groups, followed by co‐immunoprecipitation to validate the interaction between EPAC‐1 and VDAC‐1. G,H) Following stimulation with 10 µ m OxyHb for 12 h, neurons were treated with the EPAC‐1 inhibitor ESI09 (10 µ m ) or activator 8CPT (10 µ m ) for 24 h. The levels of activated Rap1‐GTP were detected using a Rap1 Activation Assay Kit (G), while EPAC‐1 protein levels in both compartments were assessed via western blot analysis (H). Co‐immunoprecipitation was used to detect the interaction between EPAC‐1 and VDAC‐1 I), and mitochondrial structures were examined by means of transmission electron microscopy with a scale bar of 5 µm J). The ATP content was quantified L), and mitochondrial membrane potential (MMP) was measured by JC‐1 staining K). All data are presented as mean ± SD. Statistical significance was determined using one‐ or two‐way ANOVA with Tukey's multiple comparisons tests ( n = 6, *** p < 0.0001 vs control/ sham group; ### p < 0.0001 vs Vehicle group).

Article Snippet: Rap1‐GTP levels were quantified using the Rap1 Activation Assay Kit (NewEast Biosciences, 81 401) as described previously.

Techniques: Western Blot, Expressing, Activation Assay, Isolation, Control, Functional Assay, Protein Enrichment, Immunoprecipitation, Transmission Assay, Electron Microscopy, Membrane, Staining

Journal: Advanced Science

Article Title: Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP‐Activated Exchange Protein‐1

doi: 10.1002/advs.202307556

Figure Lengend Snippet: Celastrol inhibits the elevation of EPAC‐1 activity and MPTP opening induced by ICH. A) Samples of cortex surrounding the hematoma in mice were collected 48 h after ICH induction and treatment with 1, 2, and 4 mg k −1 g celastrol. EPAC‐1 protein levels were detected by western blot. β‐Tubulin served as loading controls, with a sham group being normalized to a value of 1 for accurate comparisons, n = 6. B) After being stimulated with OxyHb, the neurons were treated with the EPAC‐1 inhibitor ESI09 (10 µ m ) or celastrol at concentrations of 25 and 50 n m . The activation levels of Rap1‐GTP were measured using a Rap1 Activation Assay Kit. β‐Tubulin served as loading controls, with each sham group being normalized to a value of 1 for accurate comparisons, n = 6. C) Following stimulation with OxyHb, the neurons were treated with 50 n m celastrol. Immunofluorescence analysis was performed using an anti‐EPAC‐1 antibody (green) to label neurons and mitotracker staining for mitochondria (red). Nuclei were counterstained with DAPI (blue). Representative images from triplicate experiments are presented, showing co‐localization of celastrol and mitochondria in the proximal neurite, as indicated by arrows. Scale bar: 10 µm. D) The protein levels of EPAC‐1 in the mitochondria and cytoplasm were evaluated by western blot analysis across different groups. The mean value of each protein in the control group was normalized to 1.0, with β‐tubulin and Tom 20 serving as the loading control, n = 6. E) Co‐immunoprecipitation was used to detect the interaction between EPAC‐1 and VDAC‐1. F) Mitochondria were pretreated with celastrol or ESI09 for 30 min, followed by exposure to Ca 2+ for another 10 min. Mitochondria swelling traces were recorded based on the absorbance at 540 nm. The mitochondrial swelling was quantified, n = 6. The neurons were stimulated with 10 µ m OxyHb for 12 h and subsequently exposed to varying doses of celastrol treatment for 24 h. Afterward, the cells were collected for assessment of mitochondrial function. G) Mitochondrial structures in neurons, with or without celastrol treatment following OxyHb stimulation, were observed using transmission electron microscopy. The scale bar was set at 5 µm. H) Neurons from various experimental groups were subjected to MitoSOX staining and subsequently visualized under a Nikon fluorescence microscope. I) Neurons from various experimental groups were subjected to MitoSOX staining, and the fluorescence intensity was quantified using a fluorescent microplate reader with excitation/emission wavelengths of 396/610 nm, n = 6. J) The Fluo3‐AM staining was used to detect calcium in the cytoplasm of neurons, which was subsequently visualized using a Nikon fluorescence microscope. K) The fluorescence intensity of Fluo3‐AM was measured using a fluorescent microplate reader with excitation and emission wavelengths set at 506 and 526 nm, respectively, n = 6. All data are presented as mean ± SD. Statistical significance was determined using one‐or two‐way ANOVA with Tukey's multiple comparisons tests ( * p < 0.05, ** p < 0.001, *** p < 0.0001 vs control group; # p < 0.05, ## p < 0.001, ### p < 0.0001 vs vehicle group).

Article Snippet: Rap1‐GTP levels were quantified using the Rap1 Activation Assay Kit (NewEast Biosciences, 81 401) as described previously.

Techniques: Activity Assay, Western Blot, Activation Assay, Immunofluorescence, Staining, Control, Immunoprecipitation, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy