Fig 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). Figure 8—figure supplement 1—source data 1.TIFF files that contain original western blots indicating the relevant bands and treatments. Figure 8—figure supplement 1—source data 2.Original files for western blot analysis.
Fig 2: 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). Figure 3—figure supplement 1—source data 1.TIFF files that contain original western blots indicating the relevant bands and treatments. Figure 3—figure supplement 1—source data 2.Original files for western blot analysis.
Fig 3: 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 4: 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.
Fig 5: 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.
Supplier Page from Abcam for Recombinant Human Nrf2 protein (GST tag N-Terminus)