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

Abstract Mitochondrial dysfunction contributes to the development of secondary brain injury (SBI) following intracerebral hemorrhage (ICH) and represents a promising therapeutic target. Celastrol, the primary active component of Tripterygium wilfordii, is a natural product that exhibits mitochondrial and neuronal protection in various cell types. This study aims to investigate the neuroprotective effects of celastrol against ICH‐induced SBI and explore its underlying mechanisms. Celastrol improves neurobehavioral and cognitive abilities in mice with autologous blood‐induced ICH, reduces neuronal death in vivo and in vitro, and promotes mitochondrial function recovery in neurons. Single‐cell nuclear sequencing reveals that the cyclic adenosine monophosphate (cAMP)/cAMP‐activated exchange protein‐1 (EPAC‐1) signaling pathways are impacted by celastrol. Celastrol binds to cNMP (a domain of EPAC‐1) to inhibit its interaction with voltage‐dependent anion‐selective channel protein 1 (VDAC1) and blocks the opening of mitochondrial permeability transition pores. After neuron‐specific knockout of EPAC1, the neuroprotective effects of celastrol are diminished. In summary, this study demonstrates that celastrol, through its interaction with EPAC‐1, ameliorates mitochondrial dysfunction in neurons, thus potentially improving SBI induced by ICH. These findings suggest that targeting EPAC‐1 with celastrol can be a promising therapeutic approach for treating ICH‐induced SBI.


Figure
Figure S1Refinement of the ICH model in mice and evaluation of the side effects and toxicity profile of celastrol.(A) Representative coronal brain sections of mice from both the sham and ICH (24 h) groups are depicted.(B) Neurons, microglia, and astrocytes were subjected to treatment with or without 10 μM celastrol, followed by the MTT assay for assessing cell viability.(C-E) Mice were subjected to intraperitoneal injections of 5 mg/kg celastrol once daily for 3 consecutive days.After a period of 15 days, the heart, liver, spleen, lungs, and kidneys were examined using HE staining to assess the potential toxicity of celastrol (C).(D) Peripheral venous blood was collected from mice for complete blood count analysis, comparing white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB) levels, and platelet (PLT) count between the two groups.(E)Additionally, serum was isolated to assess levels of alanine transaminase (ALT) and aspartate transaminase (AST).

Figure
Figure S2Celastrol improved neuronal mitochondrial dysfunction under hypoxic conditions.Primary neurons were stimulated with 10 μM Cocl2 for 48 h and subsequently exposed to 50 nM celastrol treatment for 24 h.Afterwards, the cells were collected for assessment of mitochondrial function.(A)The ATP content was measured using a chemiluminescence assay, n = 6.(B) Neurons from various experimental groups were subjected to JC-1 staining and subsequently visualized under a Nikon fluorescence microscope.Scale bar: 100 μm.(C) Neurons from different experimental groups were subjected to JC-1 staining, and the fluorescence intensity was quantified using a fluorescent microplate reader with excitation/emission wavelengths of 514/529 nm for monomers and 585/590 nm for aggregates, n = 6.All data are presented as mean ± SD.Statistical significance was determined using oneor two-way ANOVA with Tukey's multiple comparisons test (***p < 0.0001 vs. control group; ###p < 0.0001 vs. vehicle group).

Figure
Figure S3 Sampling sites and quality control for single-nucleus RNA sequencing analysis.(A) Sampling locations and strata for murine specimens.(B) The quantification of genes per cell, unique molecular identifier (UMI) counts for individual cells, and the percentage of mitochondrial genes are presented.(C) A PCA plot was generated to visualize the single-cell RNA sequencing data.(D) Each data point represents a single cell, with the color scheme indicating distinct cellular clusters.The column labeled "Orig.ident"denotes the original identity or source of each individual cell sample.(E) A quality control plot comparing the distribution of doublet cells before and after removal is presented.

Figure
Figure S4 Top genes identified for each specific cell type.The heatmap displays the top 3 normalized expression levels of cell type-specific genes.

Figure
Figure S5 Enrichment analysis between normal and ICH group.(A) The volcano plot illustrates the differential expression of genes between the ICH+vehicle group and the Normal group.(B) An enrichment analysis of GO was conducted to identify potential signaling pathways with an average log2 fold change greater than 0.25 after ICH.(C) KEGG pathway analysis was performed to identify potential signaling pathways following ICH, with an average log2 fold change greater than 0.25.

Figure
Figure S6 Construction of a truncated EPAC-1 plasmid and purification of cNMP-EPAC1 protein.(A) An illustrative diagram depicting the fragmentation and truncation of EPAC-1 plasmid.(B) Map of the empty vector.(C) 293T cells were transfected with various plasmids for 48 h, and GFP was observed using a Nikon fluorescence microscope.Scale bar = 50 μm.And the transfection efficiency was calculated, n=3 (D) The cNMP-EPAC1 plasmid was constructed in the pGEX-4T vector, where cNMP and pGEX-4T vector GST were fused together, with the GST tag placed at the N-terminal.Then, the prokaryotic expression system was used to express this protein.The lysate, precipitate, flow-through fluid, and lysate supernatant are represented by 1, 2, 3, and 4, respectively.The cleaning process of three different detergents are represented by 0-1, 0-4, 1-1, 1-3, and 2-1, with each tube connected to a volume of 5 mL, while E1, E2, E3, and E4 represent the elution part with each tube connected to a volume of 2 mL.

Figure
Figure S7 Protein-protein interaction (PPI) for EPAC-1.The PPI network obtained from the STRING database (https://string-db.org)was analyzed to identify potential interacting proteins of EPAC-1.

Figure
Figure S8 EPAC-1 knockdown did not affect neuronal cell viability or mitochondrial function.Primary neurons were transfected with siRNA-NC or siRNA-EPAC-1.After 48 h, cell viability was assessed using the MTT assay.Neurons from different experimental groups underwent JC-1 staining, and fluorescence intensity was quantified using a fluorescent microplate reader with excitation/emission wavelengths of 514/529 nm for monomers and 585/590 nm for aggregates.All data are presented as mean ± SD.Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (*p < 0.05, **p < 0.001, ***p < 0.0001 vs. control group; #p < 0.05, ##p < 0.001, ###p < 0.0001 vs. vehicle group; n = 6).