Capsaicin reduces ROS and promotes NRF2 expression.

(a) Microscopic examination of GES-1 cellular morphology following treatment regimens. Cells were pre-treated with Capsaicin (CAP) at concentrations of 2 or 8 μM for 2.5 hours, followed by incubation with 5% EtOH for 1.5 hours. Scale bar, 50 μm. (b) Detection of ROS-positive GES-1 cells using DCFH-DA staining. Scale bar, 200 μm. (c) Flow cytometric (FCM) analysis of ROS-positive GES-1 cells labeled with DCFH-DA. (d) Statistical analysis of ROS levels measured by FCM. The experiment was conducted in triplicate. (e) Assessment of SOD activity in GES-1 cells. (f) Quantification of MDA levels of GES-1 cells. (g) A network targets of CAP. Purple, blue, pink nodes represent proteins or genes in the predicted biological effect profile of CAP related to ROS, inflammation or immune, respectively. (h) Heatmap depicting differentially expressed genes (DEGs) enriched in antioxidant activity based on proteomic analysis. Each row represents the expression level of a single gene, while each column corresponds to an individual experimental sample. (i) Western blot analysis of NRF2 and HO-1 expression in GES-1 cells. (j) Western blot assessment of antioxidant protein expression in GES-1 cells. (k) Quantitative analysis of relative mRNA levels of antioxidant response elements (ARE)-related genes in GES-1 cells. Cells were treated with or without CAP and EtOH, and the mRNA levels of TXN, HMOX1, and NQO1 were measured. Each group consisted of three replicates. (l) Western blot analysis of NRF2 and HO-1 expression in human umbilical cord mesenchymal stem cells (HUC-MSCs). (m) Quantitative analysis of relative mRNA levels of ARE-related genes in HUC-MSCs. The mRNA levels of TXN, HMOX1, and NQO1 were measured in different treatment groups. Data were presented as mean ± SD. P-values were calculated using a T-test. Significance levels were indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Capsaicin inhibits the ubiquitination and degradation of 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 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 EtOH treatment regimens. Cells were pre-treated with CAP at concentrations of 2 or 8 μM for 2.5 hours, followed by a 10-minute incubation with 5% EtOH. Mitochondria were labeled with Mito Tracker Red CMXRos and detected via immunofluorescence. Red fluorescence indicates the mitochondria, while blue fluorescence (DAPI staining) marks the cell nucleus. Scale bar represents 100 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 FCM. Cells were pre-treated with CAP at concentrations of 2 or 8 μM for 2.5 hours, followed by a 10-minute incubation with 5% EtOH or a 20-minute incubation with CCCP as a positive control. ΔΨm was evaluated using JC-10 staining. (l) Quantitative analysis of ΔΨm across different treatment groups using ImageJ Software. Data were presented as mean ± SD. P-values were calculated using a T-test. Significance levels were indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Capsaicin disrupted KEAP1-NRF2 interaction.

(a-b) Assessment of KEAP1 transcription and expression levels using RT-qPCR and western blot analyses. (c) KEAP1-NRF2 interaction was detected with Surface plasmon resonance (SPR) in vitro. (d) Disruption of KEAP1-NRF2 interaction by CAP as assessed by SPR. (e) Disruption of KEAP1-NRF2 interaction by 32 μM CAP as assessed by Co-Immunoprecipitation (Co-IP) in 293T Cells. (f) Quantitative analysis of relative NRF2 expression in IP samples using ImageJ software. (g) Cell viability assessment of 293T cells treated with CAP and EtOH using CCK-8 assay. (h) Western blot analysis of NRF2 and HO-1 expression in 293T cells. (i) Cell viability assessment of 293T(KO) cells treated with CAP and EtOH using CCK-8 assay. (j) Western blot analysis of NRF2 and HO-1 expression in 293T(KO) cells. Data were presented as mean ± SD. P-values were calculated using a T-test. Significance levels were indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

CAP specifically interacts with the Kelch domain of KEAP1.

(a) Detection of KEAP1-CAP interaction in GES-1 cells using cellular thermal shift assay coupled with western blotting (CETSA-WB) and the melting curve generated from CETSA was analyzed using ImageJ software. The red fold line represents cells treated with CAP, while the black fold line represents cells treated with DMSO as a control. (b) Computational docking of CAP molecule to KEAP1 surface pockets. The Keap1 protein is represented in gray, while the CAP molecule is shown in yellow. The seven key amino acids predicted to be crucial for the interaction are highlighted in blue. (c) Partial overlap of CAP-binding pocket with KEAP1-NRF2 interface. The KEAP1-NRF2 interaction interface is represented in purple. (d) Influence of dithiothreitol (DTT) on NRF2 activation induced by CAP in GES-1 cells. Cells were pre-incubated with 400 μM DTT for 1 hour, followed by a 3-hour incubation with 32 μM CAP. (e) Tandem mass spectrometry (MS/MS) of analysis of KEAP1 peptide containing Cys151 following CAP treatment. (f) Dose-dependent examination of Kelch-NRF2 interaction in the presence of CAP in 293T cells. Cells were treated with varying concentrations of CAP (0, 62.5, 125, 250, and 500 μM) to assess the impact on the interaction between exogenously purified Kelch protein and NRF2 in the total cell lysate. (g) Pull-Down assay demonstrating the direct inhibition of Kelch-NRF2 Interaction by CAP. Data were presented as mean ± SD. P-values were calculated using a T-test. Significance levels were indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Mutation of KEAP1 affects the action of CAP.

(a) In vitro detection of KEAP1-Kelch domain with CAP using BLI. (b) In vitro detection of KEAP1-Kelch domain (Mut) with CAP using BLI. (c) Co-IP assay to assess the interaction between mutant Kelch and NRF2, and the Impact of 32 μM CAP on Kelch-NRF2 binding in 293T cells. (d) Docking analysis reveals encirclement of CAP’s vanillyl headgroup by main chain atoms of the Kelch domain. (e) Left side.: existing binding sites of two common ligands with Kelch (PDB:4IQK and 5FNQ); Right side: newly discovered binding sites of CAP with Kelch. (f) CAP allosterically regulated the conformation of Kelch by HDX-MS. Peptides with increased deuterium uptake ratio after CAP treatment are highlighted in red.

Preparation and characterization of IR-HSA@CAP NPs.

(a) Schematic illustration of the synthesis process for IR-HSA@CAP nanoparticles (NPs). (b) Determination of IRDye800 binding with HSA by UV-Vis spectra. (c) The morphology of IR-HSA@CAP NPs was investigated by transmission electron microscope. Scale bar, 0.5 μm. (d-e) Particle size comparison of HSA@CAP and IR-HSA@CAP NPs. (f) Zeta-Potential analysis of nanomaterials. The zeta-potential values of the different materials were measured: H representing HSA, HC representing HSA@CAP NPs, and IHC representing IR-HSA@CAP NPs. (g) FCM analysis demonstrates endocytosis of IR-HSA@CAP NPs by GES-1 cells. (h-i) Detection of CAP release in simulated gastric fluid (SGF) and stroke-physiological saline solution (SPSS) Using high-performance liquid chromatography (HPLC). (j) In vivo imaging reveals the localization of IR-HSA@CAP NPs. (k) Observing the distribution of IR-HSA@CAP NPs in major organs of rats. Data were presented as mean ± SD. P-values were calculated using a T-test. Significance levels were indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

CAP activated NRF2/ARE pathway in vivo.

(a) Impact of CAP (1 mg/kg) on histopathology of 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 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 ROS in rat gastric tissue under various treatments using 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. (f) Measurement of HMOX1 and NQO1 mRNA levels in gastric tissues using RT-qPCR. Data were presented as mean ± SD. Significance levels were indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared to the EtOH-only group (marked in blue).

CAP also suppressed inflammation in vivo.

(a-c) Immunohistochemical detection of antioxidant proteins and presentation of representative images and statistical analysis results. (a) Nrf2, (b) HO-1, and (c) Trx protein expression levels were assessed. (d) ELISA measurement of IL-1β, TNF-α, IL-6, CXCL1/KC (IL-8), and IL-10 Levels in gastric tissues. Data were presented as mean ± SD. Significance levels were indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared to the EtOH-only group (marked in blue). (e) Mechanism diagram of capsaicin alleviating gastric mucosal injury caused by ethanol in rats.

The primer sequences of RT-qPCR

Experimental method design and parameters

ic diagram