Capsaicin acts as a novel NRF2 agonist to suppress ethanol induced gastric mucosa oxidative damage by directly disrupting the KEAP1-NRF2 interaction
- School of Life Sciences, Tianjin University, China
- Tianjin JiAnKang Bio and TCM-technology Development Co, Ltd, China
- CAS and THHDG (Tianjin) Rural Revitalization Industry Development Co, Ltd, China
- Peiyang Park Campus, Tianjin University, China
- Department of Molecular Pharmacology, Tianjin Medical University Cancer Institute & Hospital; National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin; Tianjin's Clinical Research Center for Cancer, China
- Institute for TCM-X, MOE Key Laboratory of Bioinformatics, Bioinformatics Division, BNRist, Department of Automation, Tsinghua University, China
Figures
Figure 1 with 2 supplements
Capsaicin (CAP) reduces reactive oxygen species (ROS) and promotes nuclear factor erythroid 2–related factor 2 (NRF2) expression.
(a) Microscopic examination of gastric mucosal epithelial cells (GES-1) cellular morphology following treatments. Cells were pre-treated with CAP at concentrations of 2 or 8 μM for 2.5 hr, followed by incubation with 5% ethanol (EtOH) for 1.5 hr. 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 superoxide dismutase (SOD) activity in GES-1 cells. (f) Quantification of malondialdehyde (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 heme oxygenase 1 (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 thioredoxin (TXN), HMOX1, and quinone dehydrogenase 1 (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 (UC-MSC). (m) Quantitative analysis of relative mRNA levels of ARE-related genes in HUC-MSCs.
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Figure 1—source data 1
TIFF files that contain original western blots indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig1-data1-v1.zip
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Figure 1—source data 2
Original files for western blot analysis.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig1-data2-v1.zip
Figure 1—figure supplement 1
Capsaicin (CAP) protects gastric mucosal epithelial cells (GES-1) from oxidative stress.
(a) Molecular formula of CAP from PubChem (Compound CID: 1548943). (b) Cell viability of GES-1 after 1.5 hr treatment with ethanol at different concentrations (v/v). (c) Cell viability of GES-1 after pretreatment with different concentrations of CAP for 2.5 hr and adding 5% ethanol for 1.5 hr. (d) Effects of different concentrations of CAP on the viability of GES-1 cells for 24 hr. (e–g) Cell viability was measured by adding CAP (2 μM, 8 μM, or 32 μM) to GES-1 cells for 2.5 hr and then adding three concentrations of ethanol (0.5%, 3.5%, and 5%) for another 1.5 hr. (h) Flow cytometric (FCM) showed CAP inhibited the apoptosis of GES-1 induced by ethanol (EtOH).
Figure 1—figure supplement 2
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.
Figure 2 with 1 supplement
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 .
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Figure 2—source data 1
TIFF files that contain original western blots indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig2-data1-v1.zip
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Figure 2—source data 2
Original files for western blot analysis.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig2-data2-v1.zip
Figure 2—figure supplement 1
Capsaicin (CAP) increases ATP content and inhibits lactic acid (LA) production.
(a) The content of ATP in gastric mucosal epithelial cells (GES-1) cells in different treatment groups. (b) CAP (8 μM) pretreatment for 2.5 hr and 5% ethanol (EtOH) incubation for 1.5 hr. (c) The intracellular LA level was measured. (d) Schematic diagram of intracellular glycolysis. (e and f) Detection of the mRNA level of PKM2 and LDHA in different treatment groups.
Figure 3 with 1 supplement
Capsaicin (CAP) 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 nuclear factor erythroid 2–related factor 2 (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 ethanol (EtOH) using CCK-8 assay. (j) Western blot analysis of NRF2 and heme oxygenase 1 (HO-1) expression in 293T(KO) cells.
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Figure 3—source data 1
TIFF files that contain original western blots indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig3-data1-v1.zip
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Figure 3—source data 2
Original files for western blot analysis.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig3-data2-v1.zip
Figure 3—figure supplement 1
Capsaicin (CAP) protects 293T from oxidative stress.
(a and b) Expression of KEAP1 in human KEAP1 knockout 293T cells (293T KO). (c) Detection of DPP3-CAP interaction in gastric mucosal epithelial cells (GES-1) using cellular thermal shift assay coupled with western blotting (CETSA-WB). (d) The gray analysis of nuclear factor erythroid 2–related factor 2 (NRF2) and HO-1 of each group in 293T. (e) The gray analysis of NRF2 and heme oxygenase 1 (HO-1) of each group in 293T(KO). (f) Microscopic examination of GES-1 cellular morphology following treatment regimens. Cells were pre-treated with CAP or Capsazepine (CAPZ) for 2.5 hr, followed by incubation with 5% ethanol (EtOH) for 1.5 hr. Scale bar, 10 μm. (g) Western blot analysis of NRF2 protein levels under varying concentrations of CAP and CAPZ, with or without EIOH treatment. (h and i) In vitro detection of DPP3 with CAP using bio-layer interferometry assay (BLI).
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Figure 3—figure supplement 1—source data 1
TIFF files that contain original western blots indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original files for western blot analysis.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig3-figsupp1-data2-v1.zip
Figure 4 with 4 supplements
Capsaicin (CAP) specifically interacts with the Kelch domain of KEAP1.
(a) Detection of KEAP1-CAP interaction in gastric mucosal epithelial cells (GES-1) 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 nuclear factor erythroid 2–related factor 2 (NRF2) activation induced by CAP in GES-1 cells. Cells were pre-incubated with 400 μM DTT for 1 hr, followed by a 3 hr incubation with 8 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.
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Figure 4—source data 1
TIFF files that contain original western blots indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for western blot analysis.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig4-data2-v1.zip
Figure 4—figure supplement 1
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).
Figure 4—figure supplement 2
Tandem mass spectrometry (MS/MS) and co-immunoprecipitation (Co-IP) analysis of capsaicin (CAP) and Kelch.
(a) MS/MS of KEAP1 peptide containing Cys257 after CAP treatment. (b) MS/MS of KEAP1 peptide containing Cys273 and Cys288 after CAP treatment. (c) MS/MS of KEAP1 peptide containing Cys434 after CAP treatment. (d) MS/MS of KEAP1 peptide containing Cys613 after CAP treatment. (e) The gray analysis of NRF2 in IP group and in the supernatant of each group.
Figure 4—video 1
Molecular dynamics simulations between nuclear factor erythroid 2–related factor 2 (NRF2) and Kelch.
Figure 4—video 2
Molecular dynamics simulations between nuclear factor erythroid 2–related factor 2 (NRF2) and Kelch with capsaicin (CAP).
Figure 5
Mutation of KEAP1 affects the function of capsaicin (CAP).
(a) In vitro detection of KEAP1-Kelch domain with CAP using bio-layer interferometry assay (BLI). (b) In vitro detection of KEAP1- Mutant Kelch domain (Y334A, R380A, N382A, N414A, R415A, Y572A, and S602A) with CAP using BLI. (c) Co-IP assay to assess the interaction between mutant Kelch and nuclear factor erythroid 2–related factor 2 (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 hydrogen-deuterium exchange mass spectrometry (HDX-MS). Peptides with increased deuterium uptake ratio after CAP treatment are highlighted in red.
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Figure 5—source data 1
TIFF files that contain original western blots indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig5-data1-v1.zip
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Figure 5—source data 2
Original files for western blot analysis.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig5-data2-v1.zip
Figure 6 with 1 supplement
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 human serum albumin (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) Flow cytometric (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.
Figure 6—figure supplement 1
Biosafety and concentration determination of IR-HSA@CAP.
(a and b) IR-HSA@CAP NPs including human serum albumin (HSA) (0.04 mg/ml) and CAP (0.02 mg/ml) were added into GES-1 and Raw264.7 cells for 24 hr to assess biosafety. (c) Standard curve for determination of capsaicin (CAP) by high performance liquid chromatography (HPLC). (d) The concentration of CAP released from IR-HSA@CAP NPs (including 0.4 mg/ml CAP) after a certain period of treatment in simulated gastric fluid (SGF) by HPLC. (e) The concentration of CAP released from IR-HSP@CAP NPs (including 0.4 mg/ml CAP) was measured in SGF and stroke-physiological saline solution (SPSS) for 30 min, respectively.
Figure 7
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.
Figure 8 with 1 supplement
Capsaicin (CAP) also suppressed inflammation in vivo.
(a–c) Immunohistochemical detection of antioxidant proteins and presentation of representative images and statistical analysis results. (a) Nuclear factor erythroid 2–related factor 2 (Nrf2), (b) heme oxygenase 1 (HO-1), and (c) thioredoxin (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 ethanol (EtOH)-only group (marked in blue). (e) Mechanism diagram of CAP alleviating gastric mucosal injury caused by ethanol in rats.
Figure 8—figure supplement 1
Safety evaluation of animal experiments.
(a) A model diagram of safety trial about capsaicin (CAP) and IR-HSA@CAP NPs in rats. (b and c) Organ index of liver and spleen. ns, not significant. Data were presented as mean ± SD, p-values were calculated using t-test. ns, not significant. (d-f) Determination of ALT, AST, and T-Bil in serum to evaluate liver function. (g–i) Determination of BUN, CRE, and UA in serum to evaluate renal function. Data were presented as mean ± SD, p-values were calculated using t-test. ns, not significant. (j–m) Western blot analysis of antioxidant protein expression of nuclear factor erythroid 2–related factor 2 (NRF2), heme oxygenase 1 (HO-1), and thioredoxin (Trx).
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Figure 8—figure supplement 1—source data 1
TIFF files that contain original western blots indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig8-figsupp1-data1-v1.zip
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Figure 8—figure supplement 1—source data 2
Original files for western blot analysis.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig8-figsupp1-data2-v1.zip
Figure 9
Protective effect of capsaicin (CAP) was partially eliminated in nuclear factor erythroid 2–related factor 2 -knockout (Nrf2-KO) mice.
(a) Tissue sections were 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 mice gastric mucosa using hematoxylin and eosin (H&E) staining. Scale bar represents 200 μm. A: Glandular disorder; B: Epithelial exfoliation; C: Inflammatory cell and erythrocyte infiltration. (c) Gastrointestinal bleeding and H&E staining (Zoom) in a representative Nrf2-KO mouse treated with ethanol (EtOH). (d) Western blotting to detect the key proteins of P-p65 and heme oxygenase 1 (HO-1).
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Figure 9—source data 1
TIFF files that contain original western blots indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig9-data1-v1.zip
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Figure 9—source data 2
Original files for western blot analysis.
- https://cdn.elifesciences.org/articles/97632/elife-97632-fig9-data2-v1.zip
Tables
Table 1
The primer sequences of RT-qPCR.
| Genes | Forward Sequences (5’–3’) | Reverse Sequences (5’–3’) |
|---|---|---|
| GAPDH GSS HMOX1 NQO1 TXN KEAP1 PKM2 LDHA r-ACTB r-NQO1 r-HMOX1 | GAA GGT GAA GGT CGG AGT C GGG AGC CTC TTG CAG GAT AAA AAG ACT GCG TTC CTG CTC AAC GAA GAG CAC TGA TCG TAC TGG C GTG AAG CAG ATC GAG AGC AAG CTG GAG GAT CAT ACC AAG CAG G GTG CCG CCT GGA CAT TGA CTC GCT CAT CGT CTC AAA CCC AGT GG GAA GTG TGA CGT TGA CAT CCG AAG CGT CTG GAG ACT TCT GGG AAG AGG CTA AGA CCG CCT TC | GAA GAT GGT GAT GGG ATT TC GAA TGG GGC ATA GCT CAC CAC AAA GCC CTA CAG CAA CTG TCG GGA TAC TGA AAG TTC GCA GGG CGT GGC TGA GAA GTC AAC TAC TA GGA TAC CCT CAA TGG ACA CCA C ATT CAG CCG AGC CAC ATT CAT CC ACT CCC AGC CTT TCT CCC ATC AG TGC TGA TCC ACA TCT GCT GGA CCT CTG GCG AAG AAA CTC TG GCA TAA ATT CCC ACT GCC AC |
Table 2
Experimental method design and parameters.
| Project details | ||
|---|---|---|
| Ligand | Human KEAP-1 Protein | |
| Analyte | Capsaicin, NRF2 | |
| Running buffer | PBS (pH7.4), 1% DMSO PBS (pH7.4) | |
| Regeneration solution | 10 mM Glycine-HCl | |
| Association and dissociation flow rate | 20 μl/min, Association 240 s, Dissociation 360 s | |
| Temperatures | 25 °C | |
| Sensor Chip | Sensor Chip COOH | Nicoya |
| OpenSPR | OpenSPRTM | Nicoya |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Rattus norvegicus) | Sprague Dawley Rats (CD IGS) Rats (SD rats) | Beijing Vital River Laboratory Animal Technology Co., Ltd. | Cat. # 101 | |
| Strain, strain background (Mus musculus) | C57BL/6Smoc (C57BL/6) | Shanghai Model Organisms Center, Inc | Cat. # SM-001 | |
| Strain, strain background (Mus musculus) | Nrf2-knockout (Nrf2-KO) mice | Shanghai Model Organisms Center, Inc | Cat. # NM-KO-190433 | |
| Cell line (Homo sapiens) | 293T cells | The American Type Culture Collection (ATCC) | Cat# CRL-3216 | |
| Cell line (Homo sapiens) | UC-MSC cells | ATCC | Cat#PCS-500–010 | |
| Cell line (Mus musculus) | RAW 264.7 | ATCC | Cat#TIB-7 | |
| Cell line (Homo sapiens) | GES-1 cells | Procell Life Science & Technology Co., Ltd. | Cat#CL-0563 | |
| Cell line (Homo sapiens) | KEAP1 Knockout 293T cells | ABclonal Biotechnology Co., Ltd | Cat#RM02350 | |
| Antibody | Anti-HA-Tag (Mouse monoclonal) | ABclonal Biotechnology Co., Ltd | Cat# AE008 | WB (1:10000) |
| Antibody | Anti-Strep II-Tag (Mouse monoclonal) | ABclonal Biotechnology Co., Ltd | Cat# AE066 | WB (1:10000) |
| Antibody | Anti-MYC-Tag (Mouse monoclonal) | ABclonal Biotechnology Co., Ltd | Cat# AE010 | WB (1:5000) |
| Antibody | Anti-DYKDDDDK -Tag (Rabbit monoclonal) | Proteintech Group, Inc | Cat# 80010–1-RR | WB (1:10000) |
| Antibody | Anti-Actin (Mouse monoclonal) | Proteintech Group, Inc | Cat# 66009–1-Ig | WB (1:1000) |
| Antibody | Anti-GAPDH (Mouse monoclonal) | Proteintech Group, Inc | Cat# 60004–1-Ig | WB (1:20000) |
| Antibody | Anti- NRF2/NFE2L2 (Rabbit polyclonal) | Proteintech Group, Inc | Cat# 16396–1-AP | WB (1:1000) |
| Antibody | Anti-NRF2 (Rabbit monoclonal) | Cell Signaling Technology, Inc | Cat# 12721 | WB (1:1000) |
| Antibody | Anti-GSS (Cat No. Rabbit polyclonal) | Proteintech Group, Inc | Cat# 15712–1-AP | WB (1:1000) |
| Antibody | Anti-HO-1/HMOX1 (Rabbit polyclonal) | Proteintech Group, Inc | Cat# 10701–1-AP | WB (1:1000) |
| Antibody | Anti-Thioredoxin 1/TXN (Rabbit polyclonal) | Proteintech Group, Inc | Cat# 14999–1-AP | WB (1:5000) |
| Antibody | Anti-KEAP1(Rabbit polyclonal) | Proteintech Group, Inc | Cat# 30041–1-AP | WB (1:1000) |
| Antibody | FITC Goat anti-Rabbit IgG (H+L) | SIMUBIOTECH | Cat# S2003 | IF (1:2000) |
| Recombinant DNA reagent | pcDNA3.1-Flag | Addgene | Cat#210342 | |
| Recombinant DNA reagent | pcDNA3.1-KEAP1-FLAG | MiaoLing Plasmid Platform | Cat# P8893 | |
| Recombinant DNA reagent | pCMV-NFE2L2 (human)-HA-Neo | MiaoLing Plasmid Platform | Cat# P45110 | |
| Recombinant DNA reagent | VR1012 | This paper | Construction information described in the Materials and methods section | |
| Recombinant DNA reagent | VR1012- NFE2L2 | This paper | Construction information described in the Materials and methods section | |
| Recombinant DNA reagent | pUb-K48-Myc | This paper | Construction information described in the Materials and methods section | |
| Recombinant DNA reagent | pET-28a-KEAP1(Kelch)-Flag | This paper | Construction information described in the Materials and methods section | |
| Recombinant DNA reagent | pET-28a-KEAP1(Kelch)-Strep | This paper | Construction information described in the Materials and methods section | |
| Recombinant DNA reagent | pET-28a-KEAP1(Mut)-Strep | This paper | Construction information described in the Materials and methods section | |
| peptide, recombinant protein | Recombinant Human KEAP1 Protein | Sino Biological Inc | Cat#11981-HNCB | |
| peptide, recombinant protein | Recombinant Human Nrf2 protein | Abcam | Cat#ab132356 | |
| Commercial assay or kit | pEASY-Basic Seamless Cloning and Assembly Kit | TransGen Biotech, China | Cat#CU201-02 | |
| Commercial assay or kit | BCA Protein Assay Kit | Beyotime Biotechnology | Cat#P0012 | |
| Commercial assay or kit | ATP test kit | Beyotime Biotechnology | Cat#S0026 | |
| Commercial assay or kit | Reactive Oxygen Species Assay Kit | Solarbio Life Science | Cat#CA1410 | |
| Commercial assay or kit | CCK-8 Cell Proliferation And Cytotoxicity Assay Kit | Solarbio Life Science | Cat#CA1210 | |
| Commercial assay or kit | Annexin V-FITC Apoptosis Detection Kit | Solarbio Life Science | Cat#CA1020 | |
| Commercial assay or kit | Anti-HA Affinity Matrix | Roche | Cat#11815016001 | |
| Commercial assay or kit | Anti-Flag affinity matrix | Sigma | Cat#A2220 | |
| Commercial assay or kit | enhanced chemiluminescence (ECL) reagent | Epizyme Biomedical Technology Co., Ltd | Cat#SQ201 | |
| Commercial assay or kit | SOD assay Kit | Solarbio Life Science | Cat#BC0175 | |
| Commercial assay or kit | CAT assay Kit | Solarbio Life Science | Cat#BC0205 | |
| Commercial assay or kit | MDA assay Kit | Solarbio Life Science | Cat#BC0025 | |
| Commercial assay or kit | Mitochondrial Membrane Potential Kit(JC-10 Assay) | Solarbio Life Science | Cat#CA1310 | |
| Commercial assay or kit | PK Mito Orange | Genvivo Biotech, China | Cat#PKMO-1 | |
| Commercial assay or kit | Lactic Acid assay kit | Nanjing Jiancheng Bioengineering Institute | Cat#A019-2-1 | |
| Commercial assay or kit | EasyScript First-Strand cDNA Synthesis SuperMix | TransGen Biotech, China | Cat#AE301-02 | |
| Commercial assay or kit | Hieff UNICON Universal Blue qPCR SYBR Green Master Mix | Yeasen Biotechnology (Shanghai) Co., Ltd. | Cat#11184ES03 | |
| Commercial assay or kit | ELISA kit of Interleukin (IL)- 1β | Solarbio Life Science | Cat#SEKR-0002 | |
| Commercial assay or kit | ELISA kit of IL-6 | Solarbio Life Science | Cat#SEKR-0005 | |
| Commercial assay or kit | ELISA kit of CXCL1/KC(IL-8) | Solarbio Life Science | Cat#SEKR-0014 | |
| Commercial assay or kit | ELISA kit of IL-10 | Solarbio Life Science | Cat#SEKR-0006 | |
| Commercial assay or kit | ELISA kit of tumor necrosis factor-alpha (TNF-α) | Solarbio Life Science | Cat#SEKR-0009 | |
| Chemical compound, drug | Capsaicin (CAP) | Solarbio Life Science | Cat# IC0060 | Used in vitro |
| Chemical compound, drug | Capsaicin (CAP) | MCE | Cat# HY-10448 | Used in vivo |
| Chemical compound, drug | anhydrous ethanol (EtOH) | Tianjin Jiangtian Chemical Technology Co. LTD | Cat#268 | |
| Chemical compound, drug | albumin from human serum (HSA) | Sigma | Cat#A3782 | |
| Chemical compound, drug | DL-dithiothreitol (DTT) | MCE | Cat#HY-15917 | |
| Chemical compound, drug | Cycloheximide (CHX) | MCE | Cat#HY-12320 | |
| Chemical compound, drug | Bortezomib (PS-341) | MCE | Cat#HY-10227 | |
| Chemical compound, drug | IRDye 800CW NHS Ester | LI-COR Biosciences | Cat#929–70020 | |
| Software, algorithm | ImageJ | NIH | RRID:SCR_003070 | |
| Software, algorithm | GraphPad Prism | GraphPad Software, Inc | RRID:SCR_002798 | |
| Other | Dimethyl sulfoxide (DMSO) | Sigma | Cat#D8418 | See Materials and Methods, Section 1 |
| Other | Fetal Bovine Serum (FBS) | Gibco | Cat#10099–141 | See Materials and Methods, Section 1 |
| Other | Lipofectamine 2000 | Thermo Fisher | Cat#11668027 | See Materials and Methods, Section 1 |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/97632/elife-97632-mdarchecklist1-v1.docx
-
Source data 1
Raw quantification data.
- https://cdn.elifesciences.org/articles/97632/elife-97632-data1-v1.xlsx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Capsaicin acts as a novel NRF2 agonist to suppress ethanol induced gastric mucosa oxidative damage by directly disrupting the KEAP1-NRF2 interaction
eLife 13:RP97632.
https://doi.org/10.7554/eLife.97632.4
