Fig 2: Molecular dynamics simulations of capsaicin (CAP) and Kelch.(a) The binding energy landscapes of the molecular docking of CAP and KEAP1. (b) Nuclear factor erythroid 2–related factor 2 (NRF2) associated with Keap1 in the absence of CAP. Majority of frames throughout the 100 ns trajectory featured NRF2 binding with Keap1. (c) The distribution of NRF2 fragments around Keap1 (gray color) in the presence of CAP (represented as yellow ball/stick). The representative conformations of NRF2 throughout the simulations were superimposed on KEAP1. (d) The averaged distance between NRF2 Asp29 and Keap1 Arg415 (represented as green sticks) were measured and recorded in the MD simulations. (e) The distance between NRF2 Asp29 and KEAP1 Arg415 in the NRF2-KEAP1 complex (black line) and the NRF2-CAP-KEAP1 complex (red line).

Fig 3: Capsaicin (CAP) activated nuclear factor erythroid 2–related factor 2–antioxidant response element (NRF2-ARE) pathway in vivo.(a) Impact of CAP (1 mg/kg) on histopathology of ethanol (EtOH)-induced gastric mucosal injury. Rebamipide (100 mg/kg) was used as a positive control. Tissue sections were stained and evaluated for gastric mucosal ulcer injury using the Guth scoring system. Representative images were shown with a scale bar of 5 mm. The ulcer injury (UI) index was calculated. (b) Histological examination of rat gastric mucosa using hematoxylin and eosin (H&E) staining. Scale bar represents 400 μm. A: Inflammatory cell infiltration; B: Epithelial exfoliation; C: Glandular disorder; D: Gastric edema. Quantitative analysis was conducted using the Masuda scoring system. (c) Visualization of reactive oxygen species (ROS) in rat gastric tissue under various treatments using dihydroethidium (DHE) staining and inverted fluorescence microscopy. Scale bar represents 50 μm. ROS levels were quantified using Image Pro Plus 6.0 software. (d) MDA levels in gastric tissues across different treatment groups. (e) Assessment of catalase (CAT) activity in gastric tissues.

Fig 4: Potential pathways and proteins that capsaicin (CAP) may affect.(a) Heat map showed the up-regulation and down-regulation of all differentially expressed proteins in CAP (8 μM, 2.5 hr) and 5% ethanol (EtOH) (1.5 hr) versus 5% EtOH (1.5 hr) alone. Red and blue are indicative of increased and decreased expression, respectively. (b) Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs). (c) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes (DEGs). (d) Volcano plot illustrating DEGs between two treatment groups in gastric mucosal epithelial cells (GES-1) cells. Genes with significantly increased expression were marked in red, while those with significantly decreased expression were marked in blue. (e–h) Grayscale analysis of western blotting (WB) in GES-1. Statistical results of the expression of total nuclear factor erythroid 2–related factor 2 (Nrf2), heme oxygenase 1 (HO-1), glutathione synthase (GSS), and thioredoxin (Trx) compared with internal reference protein GAPDH. Experiments were repeated three times. (i and j) Grayscale analysis of western blot in HUC-MSC. Statistical results of the expression of total Nrf2, HO-1, GSS, and Trx compared with internal reference protein GAPDH.

Fig 5: Capsaicin (CAP) inhibits the ubiquitination and degradation of nuclear factor erythroid 2–related factor 2 (NRF2).(a) Immunofluorescence detection of NRF2 nuclear localization DAPI was employed to label the cell nuclei for reference. Scale bar, 100 μm. (b) Statistical analysis of NRF2 nuclear translocation following 8 μM CAP pre-treatment. The proportion of NRF2 localized in the nucleus post-CAP treatment was quantitatively assessed. (c and d) Subcellular localization of NRF2 in gastric mucosal epithelial cells (GES-1) cells across different treatment groups. NRF2 levels in both the nucleus and cytoplasm were assessed. GAPDH was used as a cytoplasmic marker, and Histone H3 served as a nuclear marker. Statistical analysis was performed specifically on the nuclear localization of NRF2. (e) Total NRF2 levels induced by PS-341 or CAP. (f) Analysis of total NRF2 levels under the influence of cycloheximide (CHX), with or without 8 μM CAP treatment. NRF2 degradation was semi-quantitatively assessed using ImageJ software to analyze the western blot results. (g–h) Inhibition of K48 ubiquitination on NRF2 protein by 32 μM CAP as assessed by Co-IP assay in 293T cells. (i) Mitochondrial visualization in GES-1 cells following CAP and ethanol (EtOH) treatment regimens. Cells were pre-treated with CAP at concentrations of 2 or 8 μM for 2.5 hr, followed by a 10 min incubation with 5% EtOH. Mitochondria were labeled with Mito Tracker Red CMXRos and detected via the Leica STELLARIS 5 Confocal Microscope Platform. Red fluorescence indicates the mitochondria, while blue fluorescence (DAPI staining) marks the cell nucleus. Scale bar represents 20 μm. (j) Quantitative analysis of mitochondrial branch length in different treatment groups using ImageJ and GraphPad Prism. The branch length of individual mitochondria was analyzed using ImageJ software and the data were plotted using GraphPad Prism. (k) Assessment of mitochondrial membrane potential (ΔΨm) in GES-1 cells using flow cytometric (FCM). Cells were pre-treated with CAP at concentrations of 2 or 8 μM for 2.5 hr, followed by a 10 min incubation with 5% EtOH or a 20 min incubation with CCCP as a positive control. ΔΨm was evaluated using JC-10 staining. (l) Quantitative analysis of ΔΨm across different treatment groups . Figure 2—source data 1.TIFF files that contain original western blots indicating the relevant bands and treatments. Figure 2—source data 2.Original files for western blot analysis.