Capsaicin acts as a novel NRF2 agonist to suppress ethanol induced gastric mucosa oxidative damage by directly disrupting the KEAP1-NRF2 interaction

  1. Xiaoning Gao
  2. WuYan Guo
  3. Peiyuan Liu
  4. Mingyue Yuwen
  5. Hongyu Ren
  6. Shengtao Hu
  7. Zixiang Liu
  8. Ruyang Tan
  9. Kairui Liu
  10. Zhiru Yang
  11. Junli Ba
  12. Xue Bai
  13. Shiti Shama
  14. Cong Tang
  15. Kai Miao
  16. Haozhi Pei
  17. Liren Liu
  18. Cheng Zhu
  19. Tao Wang
  20. Bo Zhang
  21. Jun Kang

  • Curated by eLife

    eLife logo

    eLife Assessment

    This valuable study suggests that capsaicin nanoparticle administration in rats activates the transcription factor Nrf2 by directly binding to its repressor, KEAP1, leading to the induction of cytoprotective genes and preventing alcohol-induced gastric damage, offering a potential avenue for treating alcoholism-related gastric disorders. The authors provide solid evidence through a wealth of biochemical experiments in vitro, in cultured cells as well as in a rat model. The work will be of great interest to researchers studying oxidative damage in a variety of different diseases and the exploitation of molecules for therapeutic approaches.

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Excessive alcohol consumption poses significant health risks and is closely associated with oxidative damage. The KEAP1-NRF2-ARE signaling pathway serves as the primary antioxidant system. However, current small molecule inhibitors are all covalently bound to KEAP1, meaning that once bound, they are not easily dissociated, while continuous inhibition of KEAP1 exhibits severe side effects. In this study, BLI, CETSA, Pull-down, Co-IP and HDX-MS assay analysis were conducted to detect the KEAP1 binding behavior of natural product, capsaicin (CAP), both in vitro and in cells. The ethanol-induced acute gastric mucosal damage rat model was also established to evaluate the therapeutic effect of CAP. Our findings demonstrated that CAP mitigated mitochondrial damage, facilitated the nuclear translocation of NRF2, leading to the up-regulation of downstream antioxidant response elements, HMOX1, TXN, GSS and NQO1 in GES-1 cells. Furthermore, CAP directly bind to KEAP1 and inhibit the interaction between KEAP1 and NRF2. In the KEAP1-knockout 293T cells, CAP failed to activate NRF2 expression. We identified that CAP non-covalently bound to Kelch domain and allosterically regulated three specific regions of KEAP1 : L342-L355, D394-G423 and N482-N495. To improve drug solubility and delivery efficiency, we developed IR-Dye800 modified albumin coated CAP nanoparticles. The nanoparticles significantly reduced the gastric mucosal inflammation and activated NRF2 downstream genes in vivo. Our hypothesis was further verified our hypothesis in Nfe2l2-knockout mice. This study provides new insights that CAP is a safe and novel NRF2 agonist by allosterically regulating KEAP1, which may contribute to the development of lead drugs for oxidative stress-related illness, e.g. aging, cancer, neurodegenerative and cardiovascular diseases.

Article activity feed

  1. Version published to 10.7554/elife.97632.3 on eLife
  2. Version published to 10.7554/elife.97632 on eLife
  3. eLife

    eLife Assessment

    This valuable study suggests that capsaicin nanoparticle administration in rats activates the transcription factor Nrf2 by directly binding to its repressor, KEAP1, leading to the induction of cytoprotective genes and preventing alcohol-induced gastric damage, offering a potential avenue for treating alcoholism-related gastric disorders. The authors provide solid evidence through a wealth of biochemical experiments in vitro, in cultured cells as well as in a rat model. The work will be of great interest to researchers studying oxidative damage in a variety of different diseases and the exploitation of molecules for therapeutic approaches.

  4. eLife

    Reviewer #1 (Public review):

    The paper by Gao et al. describes the effect of capsaicin on the NRF2/KEAP1 pathway. The authors carried out a set of in vitro and in vivo experiments that addressed the mechanisms of the protective effect of capsaicin on ethanol-induced cytotoxicity.

    The authors conclude that capsaicin activates NRF2, which leads to the induction of cytoprotective genes, preventing oxidative damage. The paper shows that capsaicin may directly bind to KEAP1 and that it is a noncovalent modification of the Kelch domain.

    The authors also designed new albumin-coated capsaicin nanoparticles, which were tested for the therapeutic effect in vivo.

    Comments on latest version:

    The manuscript has been substantially improved. I have no further comments.

  5. eLife

    Reviewer #2 (Public review):

    Summary:

    The paper by Gao et al. describes that capsaicin (CAP) might act as a novel NRF2 agonist capable of suppressing ethanol (EtOH)-induced oxidative damage in the gastric mucosa by disrupting the KEAP1-NRF2 interaction. Initially the authors established and validated a cell model for EtOH-induced oxidative stress which they used to experiment with different CAP concentrations and to determine changes in a variety of parameters such as cell morphology, ROS production, status of redox balance to mitochondrial function, amongst others.

    The proposed mechanism by which CAP activates NRF2 and mitigates oxidative stress is thought to be via non-covalent binding to the Kelch-domain of KEAP1. A variety of assays such as BLI, CETSA, Pull-down, Co-IP, and HDX-MS were employed to investigate the KEAP1 binding behavior of CAP both in vitro and in GES1 cells. Consequently, the authors developed in vivo nanoparticles harboring CAP and tested those in a rat model. They found that pretreatment with the CAP-nanoparticles led to significant upregulation of NRF2 and subsequent effects on pro- (suppression of IL-1β, TNF-α, IL-6 and CXCL1) and anti-inflammatory (activation of IL-10) cyotkines pointing to a resolved state of inflammation and oxidative stress.

    Strengths:

    The work comprises a comprehensive approach with a variety of in vitro assays as well as cell culture experiments to investigate CAP binding behaviour to KEAP1. In addition, the authors employ in vivo validation by establishing an ethanol-induced acute gastric mucosal damage rat model and providing evidence of the potential therapeutic effect of CAP.

    The study further provides novel insights into the mode of CAP action by elucidating the mechanism by which CAP promotes NRF2 expression and downstream antioxidant target gene activation.

    The design of IR-Dye800 modified albumin-coated CAP nanoparticles for enhanced drug solubility and delivery efficiency demonstrates a valuable practical application of the research findings.

    In summary the study's findings suggest that CAP could be a safe and novel NRF2 agonist with implications for the development of lead drugs for oxidative stress-related diseases. Collectively, the data support the significance and potential impact of CAP as a therapeutic agent for oxidative stress-related conditions.

    Weaknesses:

    While the study provides valuable insights into the molecular mechanisms and in vivo effects of CAP, further clinical studies are needed to validate its efficacy and safety in human subjects. The study primarily focuses on the acute effects of CAP on ethanol-induced gastric mucosa damage. Long-term studies are necessary to assess the sustained therapeutic effects and potential side effects of CAP treatment.

    While the design of CAP nanoparticles is innovative, further research is needed to optimize the nanoparticle formulation for enhanced efficacy and targeted delivery to specific tissues.

    Addressing these weaknesses through additional research and clinical trials can strengthen the validity and applicability of CAP as a therapeutic agent for oxidative stress-related conditions.

  6. eLife

    Author response:

    The following is the authors’ response to the previous reviews

    Public Reviews:

    Reviewer #1 (Public review):

    The paper by Gao et al. describes the effect of capsaicin on the NRF2/KEAP1 pathway. The authors carried out a set of in vitro and in vivo experiments that addressed the mechanisms of the protective effect of capsaicin on ethanol-induced cytotoxicity.

    The authors conclude that capsaicin activates NRF2, which leads to the induction of cytoprotective genes, preventing oxidative damage. The paper shows that capsaicin may directly bind to KEAP1 and that it is a noncovalent modification of the Kelch domain.

    The authors also designed new albumin-coated capsaicin nanoparticles, which were tested for.

    I appreciate the authors' experimental efforts to strengthen the study's conclusions. However, in my opinion, the paper is still not fully technically sound, which weakens the strength of the evidence.

    Thank you very much for your constructive review. We are truly gratified by your recognition of our key findings—that capsaicin activates NRF2 by disrupting the KEAP1–NRF2 interaction, as conclusively demonstrated through multiple methods including Pull-down, Co-IP, CETSA, SPR, BLI, deuterium exchange MS, CETSA, MS simulations and other target gene expression assays, and that albumin-coated capsaicin nanoparticles exhibit therapeutic effects in vivo. Your technical suggestions were particularly valuable. In this revised version, We have carefully and thoroughly addressed the points raised by you and the other reviewer by providing additional data, including nuclear-cytoplasmic fractionation assays performed with an alternative NRF2 antibody to strengthen and clarify the supporting evidence. We believe this revision have significantly enhanced the overall quality and rigor of the manuscript. Regarding the limitation of the insufficient number of animals used in this article, we have also explained it in the main text. This is the revision we have made with our utmost efforts, and we hope it can meet your expectations to a certain extent.

    Reviewer #2 (Public review):

    Summary:

    In this paper the authors wanted to show that capsaicin can disrupt the interaction between Keap1 and Nrf2 by directly binding to Keap1 at an allosteric site. The resulting stabilization of Nrf2 would protect CAP-treated gastric cells from alcohol- induced redox stress and damage as well as inflammation (both in vitro and in vivo)

    Strengths:

    One major strength of the study is the use of multiple methods (CoIP, SPR, BLI, deuterium exchange MS, CETSA, MS simulations, target gene expression) that consistently show for the first time that capsaicin can disrupt the Nrf2/Keap1 interaction at an allosteric site and lead to stabilization and nuclear translocation of Nrf2.

    Moreover, efforts to show causal involvement of the Keap/Nrf2 axis for the made cellular observations as well as addressing potential off target effects of the polypharmacological CAP appreciated.

    One point that still hampers a bit of full appreciation of the capsaicin effect in cells is that capsaicin is not investigated alone, but mostly in combination with alcohol only.

    Moreover, the true add-on value of the developed nanoparticles remains obscure.

    The partly relatively high levels of NRF2 in putatively unstressed cells question the validity of used models.

    The rationale for switching between different CAP concentrations is unclear /not entirely convincing.

    The language and introduction could be improved.

    Overall, the authors are convinced that capsaicin (although weakly) can bind to Keap1 and releases Nrf2 from degradation, with relevance for biological settings. With this, the authors provide a significant finding with marked relevance for the redox/Nrf2 as well as natural products /hit discovery communities.

    Thank you very much for your positive assessment of our work and for the constructive suggestions to make it better. We also believe that capsaicin (CAP) offers new insights into the activation of NRF2. In this revision, we have addressed the shortcomings with the following efforts:

    (1) The inclusion of a capsaicin (CAP)-only treatment group—covering the same doses and time points as the ethanol co-treatment—revealed that CAP alone can directly inhibit the KEAP1–NRF2 interaction (Figure 3d,3e and Figure 4g), and promote the entry of NRF2 into the nucleus (Figure 2c), resulting in moderate NRF2 activation (Figure 3h and Figure 4d) after carefully revision. However, this effect was significantly enhanced in the presence of ethanol. We attribute the results to the ROS-enriched environment generated by ethanol. Given that KEAP1 is a sensor highly susceptible to oxidative modification, the combination of CAP's allosteric regulation and ethanol-induced oxidative stress promotes a more robust and persistent dissociation of the KEAP1–NRF2 complex. These findings align fully with the established model in which KEAP1–NRF2 dissociation is markedly facilitated under oxidative stress conditions.

    (2) From a translational and industrial perspective, nanoparticle formulations offer improved palatability compared with CAP itself; based on firsthand experience, the nano formulation is more tolerable than CAP. When preparing pure CAP, the pungency often causes irritation, whereas HSA@CAP nanoparticles are milder and demonstrate superior safety in mice following oral gavage. Moreover, ELISA results indicate that HSA@CAP nanoparticles exhibit enhanced anti-inflammatory activity compared with CAP alone (Figure 8d). In light of these findings, we prefer to retain this part of the data.

    (3) Your question is highly professional and well taken. In GES-1 (Fig. 1i) and UC-MSC (Fig. 1l), the expression of NRF2 was low in unstressed conditions, and the transcription and translation of its downstream targets indicate no functional activation, supporting the validity of our model. Accordingly, the control groups in some experiments were suboptimal. We repeated these experiments with additional biological replicates and used cells with early-passage; the discrepancies likely relate to high passage numbers and serum batch effects, but they do not affect our main conclusions. We have replaced the relevant data in the revised manuscript (Fig. 2c and Fig. 3h) and hope this addresses your concern.

    (4) In GES-1 cells, 8 μM consistently yielded the optimal effect, and we therefore maintained this concentration in other experiments in the same cells. And for other experiments, we needed to co-transfect multiple plasmids. Transfection efficiency was poor in GES-1 cells, so we switched to the commonly used HEK-293T cell line. In 293T cells, 2 and 8 μM were suboptimal, so we ultimately used 32 μM (Figure 3h), consistent with other 293T experiments (Co-IP and Pull-down) that also used 32 μM. Therefore, 8 μM were insufficient in Fig. 2g as we repeated many times. This likely reflects cell line–specific differences and the experimental context in 293T cells, including transfection and overexpression of NRF2 and Ub-K48-Myc, which necessitated a relatively higher CAP concentration.

    (5) Thank you very much for noting that the language and Introduction could be further improved. We have rechecked the manuscript for grammar and style and revised the Introduction with a more comprehensive, up-to-date description of the NRF2 pathway. The main changes include rewriting the third and forth paragraph of the Introduction, consolidating/removing irrelevant sections for greater clarity and concision. We hope these updates meet your expectations.

    Figure 2C: It is still not clear why naïve (unstressed /untreated cells) already show rather high nuclear abundance of Nrf2 (shouldn´t Nrf2 be continuously tagged for degradation by Keap1)

    Thank you for your constructive comments. In response to the concern raised, we repeated the nuclear–cytoplasmic fractionation experiments in early-passage GES‑1 cells and performed three independent replications using an alternative, widely recognized NRF2 antibody (Cell Signaling Technology, Cat. No. 12721). The results showed low nuclear NRF2 levels under basal conditions, consistent with the KEAP1-mediated continuous degradation mechanism. Accordingly, we have updated the relevant figure in Figure 2C. Nevertheless, NRF2 could still be detected in the control group, which is basically consistent with the reported baseline levels of NRF2 observed in GES - 1 cells and other cell lines [1,2,3]. Therefore, this does not indicate the failure of model construction.

    References：

    (1) Wang, R. et al. Costunolide ameliorates MNNG-induced chronic atrophic gastritis through inhibiting oxidative stress and DNA damage via activation of Nrf2. Phytomedicine 130, 155581, doi:10.1016/j.phymed.2024.155581 (2024).

    (2) Li, Y. F. et al. Construction of Magnolol Nanoparticles for Alleviation of Ethanol-Induced Acute Gastric Injury. J Agric Food Chem 72, 7933-7942, doi:10.1021/acs.jafc.3c09902 (2024).

    (3) Li, M., Wang, J., Xu, Z., Lin, Y. & Dong, J. Atraric acid attenuates chronic intermittent hypoxia-induced brain injury via AMPK-mediated Nrf2 and FoxO3a antioxidant pathway activation. Phytomedicine 148, 157261, doi:10.1016/j.phymed.2025.157261 (2025).

    Figure 2G-H: Why switch to rather high concentrations?

    To validate ubiquitin-mediated degradation in Figure 2G-H, we needed to co-transfect multiple plasmids. Transfection efficiency was poor in GES-1 cells, so we switched to the commonly used HEK-293T cell line. In 293T cells, 2 and 8 μM were suboptimal, so we ultimately used 32 μM, consistent with other 293T experiments (Co-IP and Pull-down) that also used 32 μM. These choices reflect intrinsic cell line properties and protein overexpression in 293T, but do not affect our investigation of capsaicin’s mechanism.

    Figure 2I: in the pics of mitochondria the control mitochondria look way more punctuated (likely fissed) than the ones treated with EtOH or EtOH + CAP. Wouldn´t one expect that EtOH leads to mitochondrial fission and CAP can prevent it?

    Thank you very much for your comments. We re-acquired and analyzed mitochondrial morphology by the Leica STELLARIS 5 Confocal Microscope Platform, which our school didn't have at that time. The earlier wide-field fluorescence images lacked sufficient magnification and resolution, which obscured details and may have caused confusion. In the revised manuscript, we have replaced them with confocal images showing EtOH-induced mitochondrial abnormalities, whereas CAP treatment restored the reticular network, as expected. We also added a CAP-only group, which shows no discernible effect on mitochondrial morphology.

    Figure 3H: High basal Nrf2 levels in unstressed/untreated HEK WT cells, why?

    Thank you for raising this concern. We repeated the experiments in HEK-293T (WT) cells in better condition, and validated the results using an alternative, widely recognized NRF2 antibody (Cell Signaling Technology, Cat. No. 12721). The data consistently show relatively low NRF2 expression under basal conditions, in line with the KEAP1-mediated continuous degradation mechanism. We have corrected the corresponding figures accordingly.

    Figure 4a: Inclusion of an additional Keap1 binding protein (one with a ETGE motif) would have been desirable (to get information on specificity/risks of off-target (unwanted) effects of CAP).

    Thank you for this valuable suggestion. We have added CETSA experiments for DPP3, which contains an ETGE motif. The results show that endogenous DPP3 expression was low in GES-1 cells and does not bind CAP in vitro that BLI experiments indicated the KD was above 1 mM in Supplementary Figure 4h and 4i, and thus CAP does not thermally stabilize DPP3 at the cellular level. Therefore, the risk of off-target effects via binding to ETGE-containing proteins like DPP3 appears minimal.

    Figure 4D: Why is there no stabilization of Nrf2 by CAP in lane 2?

    Thank you for raising this concern. We repeated the experiment in GES‑1 cells and performed three independent replicates using an alternative, widely recognized Nrf2 antibody (Cell Signaling Technology, Cat. No. 12721). The data show that CAP alone increases NRF2 expression to some extent. We have updated the corresponding figures accordingly in Figure 4D.

    Figure 4f: 5% DMSO is a rather high solvent concentration, why so high (the solvent alone seems to have quite marked effects!)

    Thank you for raising this concern. Our original figure legend was misleading and has been corrected. Only the highest CAP concentration (500 μM) contained 5% DMSO as the vehicle; the other CAP concentrations, prepared by serial dilution in complete medium, did not contain such high solvent levels (e.g., 65.5 μM CAP contained 0.625% DMSO). This experiment included transient overexpression of NRF2-HA as purified recombinant NRF2 protein is prohibitively expensive, 10 ug needs about 900 GBP from Abcam. We therefore conducted a preliminary assay by incubating purified Kelch-Flag protein with cell lysates overexpressing NRF2-HA and measured NRF2 levels in the supernatant and pellet in Figure 4f. Nevertheless, the conclusion that CAP disrupts the NRF2–KEAP1 interaction is better supported by SPR (Figure 3d), Co-IP (Figure 3e) and Pull-down (Figure 4g).

    Figure 6/7: not expert enough to judge formulations and histology scores. However, the benefit of the encapsulated capsaicin does not become entirely clear to me, as CAP and IRHSA@CAP mostly do not significantly differ in their elicited response.

    Thank you very much for the valuable suggestion. Although histopathology suggests only modest differences between the two treatments, the nanoparticle group showed markedly lower inflammatory cytokine levels than pure CAP: IL-1β, IL-6, TNF-α, and CXCL-1 were significantly reduced, while the anti-inflammatory cytokine IL-10 was significantly increased (Figure 8d). These changes are important for maintaining a healthy gastric environment and may better support digestive function in vivo. Accordingly, from a translational and industrial perspective, nanoparticle formulations also offer improved palatability compared with capsaicin itself. Based on firsthand experience, the nano formulation is more tolerable than CAP. When preparing pure CAP, the pungency often causes irritation, whereas HSA@CAP nanoparticles are milder and demonstrate superior safety in mice following oral gavage.

    Figure 7: Rebamipide was introduced as positive control in the text with an activating effect on Nrf2, but there is no induction of hmox and nqo in Figure 7f, why? It does not look as the positive control was wisely chosen.

    Thank you for your insightful comment. We agree that this result was suboptimal and sincerely apologize for the oversight. We are currently facing significant constraints: the original cDNA is depleted, and funding cuts have severely limited our resources for reagents and animal studies. A full repetition of the rat experiment at the original scale and quality is not feasible in the short term. To ensure the scientific rigor of the paper, we have made the difficult decision to remove Figure 7f. We believe this is necessary to base our conclusions on the most robust evidence. We apologize for any inconvenience and hope this solution is acceptable. We have revised the manuscript accordingly and appreciate your understanding.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    （1) The authors did not provide data validating the NRF2 antibody for in vitro studies, particularly for IF data where there is no molecular mass indication for NRF2. The IF data suggest that NRF2 is primarily located in the cytoplasm under control conditions (Fig. 2A), whereas the WB data show a strong band in the nucleus (Fig. 2C). What is the reason for this inconsistency?

    We sincerely appreciate your valuable comments. Previously, we used an NRF2 antibody (Cat. No. 16396-1-AP, Proteintech); the vendor’s data show that shRNA knockdown in HeLa cells markedly reduces NRF2 at the expected molecular weight and IF data in HepG2 cells show a trace amount of cytoplasmic localization in controls and clear nuclear translocation after MG-132 treatment, which indicates that this antibody can be used for immunofluorescence (IF) to indicate the subcellular localization of NRF2, and our experimental results are also in line with expectations in Figure 2A. In addition, to address the reviewer's concern, we purchased another NRF2 antibody (Cat. No. 12721, Cell Signaling Technology), which was also highly validated. In this version, we repeated nuclear-cytoplasmic fractionation experiments and other important experiments using this antibody. Together, these data confirm the low basal level of NRF2 in the absence of stress and also show that CAP could improve the expression of NRF2. We have corrected the Figure 2C so that the WB and IF results are now consistent. We wish to reiterate our deep appreciation for the professionalism and rigor of your review.

    (2) Additionally, I could not find Supplementary Figure 4F-I, which concerns TRPV1. These figures are mentioned in the response to reviewers but are missing from the manuscript-please double-check.

    The supplementary figures were initially submitted as a compressed archive. We recognize that there might have been an issue with the transfer of this file to the reviewers. As shown in Supplement Figure 4f to Supplement Figure 4i, we further explored the TRPV1 and DPP3 to detect the potential off-target effects of CAP respectively. Capsazepine (CAPZ), which is TRPV1 receptor antagonist did not affect the protection of CAP against GES-1 (Fig S4f and S4g), which may indicate that CAP activation of NRF2 does not have to depend on TRPV1. The binding of CAP with DPP3, containing an ETGE motif and can bind to KEPA1, was detected by BLI, and we found that the KD between CAP and DPP3 was 1.653 mM(＞100 μM), which may indicate the potential off-target effect of CAP is low because CAP had a relatively strong binding force with KEAP1 about 31.45 μM (Fig S4h and S4i).

    (3) I am also somewhat unconvinced by the data obtained from NRF2 KO mice. First, it appears that some NRF2 KO mice respond to CAP treatment well while others do not, resulting in a high standard deviation. To strengthen the conclusions, it would be advisable to use a larger number of animals to confirm or exclude the effect. This is precisely why I still believe that three rats per group are insufficient for the in vivo studies. Please emphasize in the manuscript that a limitation of this study is the use of only three rats per group for the in vivo experiments.

    Thank you very much for your question and suggestions. As for the rat experiments in Figure 7 and Figure 8, there are many other references available, as noted in the introduction: “Recent experiments conducted in rats have demonstrated that red pepper/capsaicin (CAP) possesses significant protective effects on ethanol-induced gastric mucosal damage , and the mechanisms involved may relate to the promotion of vasodilation[6,7], increased mucus secretion[8] and the release of calcitonin gene-related peptide (CGRP)[9,10]. However, it is important to note that the specific role of the antioxidant activity of CAP has not been thoroughly investigated.” Therefore, we conducted extensive literature research and preliminary experiments to ensure that our formal experiment with 8 groups could yield relatively stable results. Of course, we admit that using more rats in vivo would make the conclusion more reliable. Unfortunately, the project was delayed due to funding issues. We are currently facing significant resource constraints: reductions in research funding from the National Natural Science Foundation have severely limited our funding for reagents and animal experiments in this study. As a result, it has become impossible to fully repeat all animal experiments at the original scale and quality in the short term. Regrettably, to supplement the NRF2 knockout animal-related experiments (n=6), we have already spent approximately 70,000 RMB (about 10,000 USD). We have made tremendous efforts to ensure the scientific rigor of the paper. We sincerely apologize for any inconvenience caused. At the same time, we fully recognize the importance of increasing the sample size in animal experiments for this study. We have explicitly acknowledged this as a limitation of our work in the Discussion Section and have revised the manuscript accordingly. We greatly appreciate your understanding.

    (4) Furthermore, please double-check the blot in Fig. 9D. Tubulin and P-p65 bands appear very similar, and tubulin disappears in response to EtOH and EtOH/CAP treatment in KO mice. Is it the case? I am not sure the quantitative data reflect the WB bands. Please verify that.

    We sincerely appreciate your valuable feedback on our manuscript. Indeed, we may have included bands that do not meet the requirements due to our eagerness, and we are very grateful for your pointing this out; it was indeed a significant oversight on our part. I will definitely pay more attention to careful checking in the future. In response to this, we have re-conducted the experiments using the preserved tissue samples and have accordingly updated Figure 9d. Thank you for your insightful suggestions.

    Reviewer #2 (Recommendations for the authors):

    Presentation:

    The data with the encapsulated CAP appear a little as side arm that does not bolster your main message (maybe take out and elaborate on this topic more extensively in another manuscript)

    We sincerely thank the reviewer for this suggestion. However, based on the ELISA results demonstrating that nano-capsaicin exerts a significantly stronger anti-inflammatory effect than pure capsaicin (CAP), and considering its superior sensory profile for industrial applications (confirmed by our sensory evaluations), we believe these data provide valuable insights. Therefore, we would prefer to retain this section in the manuscript and hope for your understanding.

    Revise the introduction on the Nrf2 signaling pathway ...as it is written at the moment, someone outside the Nrf2 field might have trouble to understand

    Thank you for the valuable suggestion again. We have rewritten the introduction to the NRF2 signaling pathway to improve accessibility for readers outside the field.

    “The Kelch-like ECH-associated protein 1 (KEAP1)–Nuclear factor erythroid 2–related factor 2 (NRF2)–antioxidant response element (ARE) pathway is a core defense mechanism against oxidative and electrophilic stress[11]. Under homeostatic conditions, KEAP1 acts as a linker protein for the Cul3-E3 ubiquitin ligase complex, continuously promoting the ubiquitination and proteasomal degradation of NRF2, thereby maintaining NRF2 at basal levels[12]. When oxidative or electrophilic stress occurs, critical cysteine residues in KEAP1 are modified, or the interaction between the ETGE/DLG motifs on NRF2 and the Kelch domain of KEAP1 is disrupted, allowing NRF2 to escape degradation, accumulate, and translocate to the nucleus. There, NRF2 forms heterodimers with small Maf proteins and binds to ARE, inducing the expression of antioxidant and cytoprotective genes such as those involved in glutathione metabolism, NADPH regeneration, phase II detoxifying enzymes, and drug efflux transporters, thereby restoring redox balance within the cell and reducing oxidative damage[13].

    Classical NRF2 agonists, such as sulforaphane, are small molecules that bind to KEAP1 and covalently modify its cysteine residues, thereby altering the binding affinity between KEAP1 and NRF2 [14]. However, traditional covalent agonists may induce sustained overactivation of NRF2, leading to adverse side effects and limiting clinical application [15]. Consequently, recent efforts have shifted toward the development of non-covalent NRF2 agonists, which are generally associated with lower toxicity and greater translational potential, enabling more controlled enhancement of NRF2 activity and offering new insights and therapeutic opportunities in antioxidant-related interventions.”

    The authors should check and review extensively for improvements to the use of English to get rid of awkward phrases /wording.

    Thank you very much for this helpful comment. We sincerely appreciate the suggestion and have carefully re‑read and further polished the entire manuscript to remove awkward phrasing and improve the readability of expressions and phrases. We hope these revisions address your concern, and we remain grateful for your guidance.

  7. eLife

    eLife Assessment

    This valuable study suggests that capsaicin nanoparticle administration in rats activates the transcription factor Nrf2 by directly binding to its repressor, KEAP1, leading to the induction of cytoprotective genes and preventing alcohol-induced gastric damage, offering a potential avenue for treating alcoholism-related gastric disorders. Although improvements were made following the first revision, the evidence supporting capsaicin as an Nrf2 activator remains incomplete, as some methodological aspects still require revision and the interpretation of key data needs further clarification.

  8. eLife

    Reviewer #1 (Public review):

    The paper by Gao et al. describes the effect of capsaicin on the NRF2/KEAP1 pathway. The authors carried out a set of in vitro and in vivo experiments that addressed the mechanisms of the protective effect of capsaicin on ethanol-induced cytotoxicity.

    The authors conclude that capsaicin activates NRF2, which leads to the induction of cytoprotective genes, preventing oxidative damage. The paper shows that capsaicin may directly bind to KEAP1 and that it is a noncovalent modification of the Kelch domain.

    The authors also designed new albumin-coated capsaicin nanoparticles, which were tested for the therapeutic effect in vivo.

    I appreciate the authors' experimental efforts to strengthen the study's conclusions. However, in my opinion, the paper is still not fully technically sound, which weakens the strength of the evidence.

  9. eLife

    Reviewer #2 (Public review):

    Summary:

    In this paper the authors wanted to show that capsaicin can disrupt the interaction between Keap1 and Nrf2 by directly binding to Keap1 at an allosteric site. The resulting stabilization of Nrf2 would protect CAP-treated gastric cells from alcohol- induced redox stress and damage as well as inflammation (both in vitro and in vivo)

    Strengths:

    One major strength of the study is the use of multiple methods (CoIP, SPR, BLI, deuterium exchange MS, CETSA, MS simulations, target gene expression) that consistently show for the first time that capsaicin can disrupt the Nrf2/Keap1 interaction at an allosteric site and lead to stabilization and nuclear translocation of Nrf2.
    Moreover, efforts to show causal involvement of the Keap/Nrf2 axis for the made cellular observations as well as addressing potential off target effects of the polypharmacological CAP appreciated.

    One point that still hampers a bit of full appreciation of the capsaicin effect in cells is that capsaicin is not investigated alone, but mostly in combination with alcohol only.
    Moreover, the true add-on value of the developed nanoparticles remains obscure.
    The partly relatively high levels of NRF2 in putatively unstressed cells question the validity of used models.

    The rationale for switching between different CAP concentrations is unclear /not entirely convincing.

    The language and introduction could be improved.

    Overall, the authors are convinced that capsaicin (although weakly) can bind to Keap1 and releases Nrf2 from degradation, with relevance for biological settings. With this, the authors provide a significant finding with marked relevance for the redox/Nrf2 as well as natural products /hit discovery communities.

    - Figure 2C: It is still not clear why naïve (unstressed /untreated cells) already show rather high nuclear abundance of Nrf2 (shouldn´t Nrf2 be continuously tagged for degradation by Keap1)
    - Figure 2G-H: Why switch to rather high concentrations?
    - Figure 2I: in the pics of mitochondria the control mitochondria look way more punctuated (likely fissed) than the ones treated with EtOH or EtOH + CAP. Wouldn´t one expect that EtOH leads to mitochondrial fission and CAP can prevent it?
    - Figure 3H: High basal Nrf2 levels in unstressed/untreated HEK WT cells, why?
    - Figure 4a: Inclusion of an additional Keap1 binding protein (one with a ETGE motif) would have been desirable (to get information on specificity/risks of off-target (unwanted) effects of CAP)
    - Figure 4D: Why is there no stabilization of Nrf2 by CAP in lane 2 ?
    - Figure 4f: 5% DMSO is a rather high solvent concentration , why so high (the solvent alone seems to have quite marked effects !)
    - Figure 6/7: not expert enough to judge formulations and histology scores. However, the benefit of the encapsulated capsaicin does not become entirely clear to me, as CAP and IRHSA@CAP mostly do not significantly differ in their elicited response.
    - Figure 7: Rebamipide was introduced as positive control in the text with an activating effect on Nrf2, but there is no induction of hmox and nqo in Figure 7f, why? It does not look as the positive control was wisely chosen.

  10. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Reviewer #1 (Public Review):

    Major concerns:

    For studies investigating capsaicin binding to KEAP1, the authors used capsaicin concentrations that are toxic to cells (Figures S1D and 4F, G). In vivo studies were performed only in 3 rats per group. The T-test was used for the comparison of more than two groups. Given the well-known issues with the specificity of the NRF2 antibody, the authors should provide appropriate controls, especially for IF and IHC staining.

    We sincerely appreciate your valuable comments. We repeated the experiments about CCK8 (Figure S1d) and Pull-down (Figure 4g), and then updated the results. In September 2022, GES-1 cells were more sensitive to capsaicin (CAP) because Gibco serum from North America was used. Later, in 2024, we changed the serum from Australia(Gibco: 10099-141), and we found that such GES-1 cells raised better, so we re-ran the test, and the IC50 was seen to be 304.8 μM, so concentrations used in this paper has no obvious toxicity to cells. What’s more, we repeated the Pull-down experiment with more reasonable concentrations of 32 μM and 100 μM, and the results were still in line with expectations. In summary, we concluded that the effect of CAP on GES-1 cells is closely related to the cell state, and that treatments of CAP from 32 to 100 μM can hinder the interaction between NRF2 and the Kelch domain of KEPA1. What’s more, at the cellular level, the experimental concentration of CAP was not more than 32 μM, which is a relatively safe concentration for cells.

    Thank you very much for your comments. We also pay attention to using more repetitions to increase the reliability of the experimental results in animal experiments. Therefore, recently we supplemented the experiment of Nfe2l2Knockout mice in Figure 9 (6 mice per group). Additionally, thank you very much for your comments on the use of T-test analysis, we reviewed the statistics and changed them by one-way ANOVA.

    Finally, thanks to your concern about the specificity of NRF2 antibody, we used commercialized NRF2 antibody which have been KO/KD validated (Cat No. 16396-1-AP, Proteintech) and can be used for IF and IHC staining. Each of our fluorescence result was equipped with Western Blotting in its active form at the size of 105-110 KDa for statistical analysis, the trend was consistent with the experimental results of IF and IHC, which fully proves the correctness of the results presented (Figure 2c and Figure S8j).

    Reviewer #2 (Public Review):

    Weaknesses:

    One major weakness of the study is that plausibility is taken as proof for causality. The finding that capsaicin directly binds to Keap1 and releases Nrf2 from its fate of degradation (in vitro) is taken for granted as the sole explanation for the observed improved gastric health upon alcohol exposure (in vivo). There is no consideration or exclusion of any potential unrelated off-target effect of capsaicin, or proteins other than Nrf2 that are also controlled by Keap1.

    Another point that hampers full appreciation of the capsaicin effect in cells is that capsaicin is not investigated alone, but mostly in combination with alcohol only.

    Thank you very much for this comment. In the introduction, we clarified as follows: “Currently, experiments conducted in rats have demonstrated that red pepper/capsaicin (CAP) had significant protective effects on ethanol-induced gastric mucosal damage, and the mechanism may be related to the promotion of vasodilation(6,7), increased mucus secretion(8) and the release of calcitonin gene-related peptide (CGRP)(9,10). However, it is noteworthy that whether the antioxidant activity of CAP works has not been fully investigated.” Therefore, we also recognize that CAP does not exert its effects through the KEAP1-NRF2 pathway alone. Your advice is very useful. We further explored the TRPV1 and DPP3 to detect the potential off-target effects of CAP respectively. Capsazepine (CAPZ), which is TRPV1 receptor antagonist did not affect the protection of CAP against GES-1 (Fig S4f and S4g), which may indicate that CAP activation of NRF2 does not have to depend on TRPV1. The binding of CAP with DPP3, containing an ETGE motif and can bind to KEPA1, was detected by BLI, and we found that the KD between CAP and DPP3 was 1.653 mM(＞100 μM), which may indicate the potential off-target effect of CAP is low because CAP had a strong binding force with KEAP1 about 31.45 μM (Fig S4h and S4i).

    Thank you very much for the comment of another point. Multiple experiments have shown that CAP significantly up-regulates NRF2 in the presence of additional stimuli such as EtOH (Figure 1i), H2O2 (Figure 1l), PS-341(Figure 2e) and DTT (Figure 4d), which pattern is consistent with our understanding of allosteric regulation and as expected. Especially for the experiments of PS-341 and DTT, we had a group that only adds CAP, and it can be seen that the addition of CAP alone did not significantly up-regulate NRF2, which is completely different from traditional NRF2 activators (especially artificially designed covalent binding peptides which have serious side effects).

    Reviewer #3 (Public Review):

    Weaknesses:

    While the study provides valuable insights into the molecular mechanisms and in vivo effects of CAP, further clinical studies are needed to validate its efficacy and safety in human subjects. The study primarily focuses on the acute effects of CAP on ethanol-induced gastric mucosa damage. Long-term studies are necessary to assess the sustained therapeutic effects and potential side effects of CAP treatment.

    Furthermore, the study primarily focuses on the interaction between CAP and the KEAP1-NRF2 axis in the context of ethanol-induced gastric mucosa damage. It may be beneficial to explore the broader effects of CAP on other pathways or conditions related to oxidative stress. CAP has been known for its interaction with the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel and subsequent NRF2 signaling pathway activation. Those receptors are also expressed within the gastric mucosa and could potentially cross-react with CAP leading to the observed outcome. Including experiments to investigate this route of activation could strengthen the present study.

    While the design of CAP nanoparticles is innovative, further research is needed to optimize the nanoparticle formulation for enhanced efficacy and targeted delivery to specific tissues.

    Addressing these weaknesses through additional research and clinical trials can strengthen the validity and applicability of CAP as a therapeutic agent for oxidative stress-related conditions.

    Thank you very much for these suggestions. We also believe that CAP is very valuable and promising for protecting EtOH induced gastric mucosal injury, and actively promote patent applications and if conditions permit, longer drug research for biosecurity is essential. Because of the inherently new discovery of the binding of CAP and KEAP1, and the important role of NRF2 in various oxidative stress-related diseases, we used Human umbilical cord mesenchymal stem cells (HUC-MSCs) and H2O2 to explore the potential broader effects of CAP related to oxidative stress in cells (Figure 1l and 1m). At the same time, we also explored TRPV1 related experiments, and we were surprised to find that inhibiting TRPV1 did not affect the effect of CAP (Supplementary Figure 4f and 4g). We hope that more people can read this article and do more interesting research together.

    Recommendations for the authors:

    Reviewing Editor (Recommendations For The Authors):

    Although this study has been conducted in rats, a direct proof that albumin-coated capsaicin nanoparticles act through activation of Nrf2 in protecting gastric mucosa against alcohol toxicity could be well conducted in commercially available Nrf2-deficient mice.

    Thank you very much for your suggestion and the comment is very constructive for us to improve this paper. We purchased Nrf2-deficient mice (Cat. NO. NM-KO-190433) and performed experiments, and the results showed that knockout mice with Nrf2 were more sensitive to EtOH and the effects of CAP were partially eliminated (Figure 9), which further validated the role of Nrf2-related signaling pathway in EtOH-induced gastric mucosal injury and the therapeutic effect of CAP.

    Reviewer #1 (Recommendations For The Authors):

    Minor concerns include proofreading the paper. Actinomycin is not an inhibitor of translation.

    Thank you for your comment. We have revised “Actinomycin” to “Cycloheximide”.

    Reviewer #2 (Recommendations For The Authors):

    - Please have a careful look at your conclusions: just because two effects happen at the same time and may be plausible explanations for each other, it does not mean that they are really in a causative relationship in your given test system (unless unambiguously proven by additional experiments).

    Your suggestions are very constructive for us to improve this paper.

    We further discussed the role of capsaicin with TRPV1, DPP3 and Nrf2deficient mice, hoping to make our conclusions more credible to some extent.

    - You may want to frankly discuss other targets of capsaicin (e.g. the TrpV1 receptor) that possibly could also account for your observations, and that binding to Keap1 not only releases Nrf2 from proteasomal degradation.

    Thank you for your comment. As a result, we further explored the TRPV1 and DPP3 to detect the potential off-target effects of CAP respectively. Capsazepine (CAPZ), which is TRPV1 receptor antagonist does not affect the protection of CAP against GES-1 (Fig S4f and S4g). DPP3 with an ETGE motif was detected by BLI, and we found that the KD between CAP and DPP3 was 1.653 mM, which may indicate the potential off-target effect of CAP is low (Fig S4h and S4i). At the same time, the activation of NRF2 by non-classical pathways such as CAP regulation of DPP3 or other proteins also deserves more discussion and experimental verification.

    - For Figure 1G it does not become entirely clear what has been done (and thus deduction of conclusions is hampered).

    Thank you for your comment. Network targets analysis (Figure 1g) was performed to obtain the potential mechanism of effects of CAP on ROS. Biological effect profile of CAP was predicted based our previous networkbased algorithm：drug CIPHER. Enrichment analysis was conducted based on R package ClusterProfiler v4.9.1 and pathways or biological processes enriched with significant P value less than 0.05 (Benjamini-Hochberg adjustment) were remained for further studies. Then pathways or biological processes related to ROS and significantly enriched were filtered and classified into three modules, including ROS, inflammation and immune expression. Network targets of CAP against ROS were constructed based on above analyses, and finally we combined proteomics to determine the research idea of this paper

    - Figure 1L: is there a reason/explanation why UC.MSC needs a comparably very high concentration of capsaicin.

    Thank you for your comment. Because the experimental results of 8 μM and 32 μM on this cell were more stable, and the activation effect of NRF2 downstream was more obvious.

    - Figure 2C: it is surprising that naïve (unstressed /untreated cells) already show a rather high nuclear abundance of Nrf2 (shouldn´t Nrf2 be continuously tagged for degradation by Keap1).

    Thank you for your comment. This is a real experimental result, and we have found in many experiments that the untreated group can also show NRF2 when immunoblotting. We think that this phenomenon may be related to the cell state at that time.

    - Figure 2E: the claim of synergy between CAP and the proteasome inhibitor is not justified with this single figure.

    Thank you for your comment. Multiple experiments have shown that CAP significantly up-regulates NRF2 in the presence of additional stimuli such as EtOH (Figure 1i), H2O2 (Figure 1l), PS-341 (Figure 2e) and DTT (Figure 4d), which pattern is consistent with our understanding of allosteric regulation and as expected. However, this synergy does warrant more research.

    - CHX is cycloheximide (in the main text it is referred to as actinomycin).

    Thank you very much for your comment. We have revised “Actinomycin” to “Cycloheximide”.

    - Figures 2G-H: why switch to rather high concentrations? Is it due to the overexpression of Keap1?

    Thank you for your comment. At the time of this part of the experiment, we had obtained in vitro data on the interaction of CAP and the Kelch domain of KEAP1 (about 32 μM). To keep the results uniform and valid, we chose a relatively higher concentration.

    - Figure 2I: in the pics of mitochondria the control mitochondria look way more punctuated (likely fissed) than the ones treated with EtOH or EtOH + CAP. Wouldn´t one expect that EtOH leads to mitochondrial fission and CAP can prevent it?

    Thank you for your comment. MitoTracker® Red CMXRos (M9940, Solarbio, China) is a cell-permeable X-rosamine derivative containing weakly sulfhydryl reactive chloromethyl functional groups that label mitochondria. This product is an oxidized red fluorescent stain (Ex=579 nm, Em=599 nm) that simply incubates the cell and can be passively transported across the cell membrane and directly aggregated on the active mitochondria. Therefore, red does not represent broken mitochondria, but active mitochondria. Quantitative analysis of the mean branch length of mitochondria was calculated using MiNA software (https://github.com/ScienceToolkit/MiNA) developed by ImageJ.

    - Figure 3C: figure legend is somewhat poor.

    Thank you for your comment. We have revised: “KEAP1-NRF2 interaction was detected with Surface plasmon resonance (SPR) in vitro.”

    - Figure 3E: given that CAP disrupts Nrf2/Keap1- PPI, why is there no Nrf2 stabilization seen in the fourth lane (input/lysate)?

    Thank you for your comment. The fourth lane may promote the degradation of NRF2 due to overexpression of KEAP1.

    - Figure 3H: high basal Nrf2 levels in unstressed/untreated HEK WT cells, why?

    Thank you for your comment. This is a real experimental result, and we have found in many experiments that the untreated group can also show NRF2 when immunoblotting in 293T cells. We think that this phenomenon may be related to the cell state at that time.

    - Figure 3G/I: this data suggests to me that the alcohol-mediated toxicity is Keap1-dependent (rather than the protection by CAP), doesn´t it?

    Thank you for your comment. We can see that KEAP1-KO cells had a high expression of NRF2, which was also in line with our expectations, and EtOH-induced GES-1 damage may be closely related to oxidative stress.

    - Figure 4a: the inclusion of an additional Keap1 binding protein (one with an ETGE motif) would have been desirable (to get information on specificity/risks of off-target (unwanted) effects of CAP).

    Thank you for your comment. DPP3 with an ETGE motif was detected by BLI, and we found that the KD between CAP and DPP3 was 1.653 mM, which may indicate the potential off-target effect of CAP is low (Fig S4h and S4i).

    - Figure 4D: why is there no stabilization of Nrf2 by CAP in lane 2 ? How can the DTT-mediated boost on Nrf2 levels be explained?

    Thank you for your comment. Multiple experiments have shown that CAP significantly up-regulates NRF2 in the presence of additional stimuli such as EtOH (Figure 1i), H2O2 (Figure 1l), PS-341 (Figure 2e) and DTT (Figure 4d), which pattern is consistent with our understanding of allosteric regulation and as expected. However, this synergy does warrant more research.

    - Figure 4f: 5% DMSO is a rather high solvent concentration, why so high (the solvent alone seems to have quite marked effects).

    Thank you for your comment. Because our maximum concentration was set relatively high, we have also recognized relevant problems and resupplemented the more critical Pull-down experiment (Figure 4g). The current DMSO of 0.2% had no effect on the experimental results.

    - Figure 5: it should be described in the figure legend which mutant is used. Based on the previous data, I would expect an investigation of mutants carrying amino acid exchanges at the newly identified allosteric site.

    Thank you for your comment. The mutated version involved substitutions at residues Y334A, R380A, N382A, N414A, R415A, Y572A, and S602A (the orthostatic site), which are residues reported to engage NRF2 and classic Keap1 inhibitors. The exploration of newly discovered allosteric sites is worthy of further study.

    - Figure 6/7: I am not expert enough to judge formulations and histology scores. However, the benefit of the encapsulated capsaicin does not become entirely clear to me, as CAP and IRHSA@CAP mostly do not significantly differ in their elicited response.

    Thank you for your comment. On the one hand, nanomedicine improves the safety of administration: it helps to reduce the intense spicy irritation of CAP itself when administered in the stomach; On the other hand, the dosage of drugs is reduced to a certain extent to achieve better therapeutic effect.

    - Figure 7: rebamipide was introduced as positive control in the text with an activating effect on Nrf2, but there is no induction of hmox and nqo in Figure 7f, why?

    Thank you for your comment. The effect of addition of positive control drug (Rebamipide) on NRF2 activation is not the focus of this paper. We speculate that the transcription and translation of related genes may not be completely synchronized when Rebamipide was taken at the same time.

    - Figure 8: the CAP effect on inflammation is visible, however, a clear causal connection between ROS/Nrf2/KEap1 is not given in the presented experiments.

    Thank you for your comment. The simple mechanics of this paper are illustrated in the Graphic diagram. The activation of NRF2 exerts both antiinflammatory and antioxidant functions, which has been reported in many articles, but the causal relationship is still open to exploration.

    Points related to presentation:

    - The data with the encapsulated CAP appear a little as a sidearm that does not bolster your main message (maybe take out and elaborate on this topic more extensively in another manuscript).

    - Revise the introduction on the Nrf2 signaling pathway as it is written at the moment, someone outside the Nrf2 field might have trouble understanding it.

    - The use of language requires proofreading and revision.

    Thank you for your comment. We rearranged and proofread it.

    Reviewer #3 (Recommendations For The Authors):

    Overall, the manuscript is well-written and the results are presented in a concise and comprehensible manner.

    Some recommendations on the experimental evidence and further suggestions:

    • The authors should state how they assessed the distribution of the data. Description of data with mean and standard deviation as well as comparisons between different groups with t-test assumes that the underlying data is normally distributed.

    Your suggestions are very constructive for us to improve the paper. The differences in the mean values between the two groups were analyzed using the student’s t-test, while the differences among multiple groups were analyzed using a one-way ANOVA test in the GraphPad Prism software.

    Therefore, we checked and proofread the statistical analysis.

    • Additional experiments further characterising and validating the activation of CAP via direct KELCH1-binding could include parallel experiments with similar agonists like dimethyl fumarate. It would be interesting to know how CAP activation compares to DMF activation.

    Thank you very much for your comment. We believe that the activation of NRF2 by DMF has been widely reported and well-studied, so we did not purchase this drug for comparative study here. If it can be promoted clinically in the future, we may consider comparing with DMF.

    • Also, the knock-down of NRF2 would be a suggested experiment to do because it rules out that the benefit of CAP is independent of KEAP1-NRF2 binding and activation.

    Thank you very much for your suggestions. We purchased Nrf2-deficient mice and performed experiments, and the results showed that knockout mice with Nrf2 were more sensitive to ethanol and the effects of CAP were partially eliminated (Figure 9), which further validated the role of Nrf2-related signaling pathway in alcohol-induced gastric mucosal injury and the therapeutic effect of CAP.

    Some corrections on text and figures:

    • Figure 1b: incorrect spelling of DNA stain. Should be Hoechst33324.

    Thank you very much for your comment. We have revised.

    • Figure 1c: don't put the label inside the plot.

    Thank you very much for your comment. We have revised.

    • Figure 1d: choose less verbose axes titles (this also applies to other figures).

    Thank you very much for your comment. We have revised.

    • Figures 1e and 1f: please state the units.

    Thank you very much for your comment. The enzyme activity of SOD and the content of MDA were compared with that of the control group.

    • Heading 2.2: NRF2-ARE instead of NRF-ARE.

    Thank you very much for your comment. We have revised.

    • Line 118: missing expression after immune.

    Thank you very much for your comment. We have revised.

    • Figure 1g: names of proteins are not readable.

    Thank you very much for your comment. We have revised.

    • Line 120: You performed transcriptomic analyses to identify differentially expressed GENES not proteomic.

    Thank you very much for your comment. This part of the work we do is proteomics.

    • Line 122: Fold change should be stated in both directions, i.e. absolute FC like |FC| > 1. Or did you select only upregulated DEGs? Is it not log2 FC?

    Thank you very much for your comment. We have revised.

    • Figure 1h (and Supplementary Figure 1a): Missing heatmap legend for FC.

    What do the colors show? Sample (column) description missing.

    Thank you very much for your comment. We used red to indicate up-regulation, blue to indicate down-regulation, and the vertical coordinate on the right side were antioxidant genes such as GSS and SOD1, respectively, and the proportion between the treatment group and the model group (CAP + EtOH/EtOH) had been calculated and labeled.

    • Line 145: A Western blot is not a proteomic analysis.

    Thank you very much for your comment. We have revised: “Concurrently, the elevated expression levels of GSS and Trx proteins, which were also downstream targets of NRF2, further validated by western blotting (Figure 1j).”

    • Supplementary Figure 2e-j: expression fold change is not the right quantity. The signal of the actual protein was quantified. And what are you comparing to with the statistics? The stars on one bar are not clear.

    Thank you very much for your comment. The expression level of this part was normalized compared with that of the control group. The significance differentiation analysis is compared with the model group.

    • What was the concentration of H2O2 used?

    Thank you very much for your comment. 200 μM H2O2 was used.

    • Figure 2d: use a more precise y-axis label.

    Thank you very much for your comment. We do want to compare the amount of NRF2 entering the nucleus, so the relative expression is compared to the internal reference

    • Figure 2g: missing molecular weight markers.

    Thank you very much for your comment. Since the ubiquitination modification is a whole membrane, and only marking the size of HA and GAPDH is not beautiful enough here.

    • Line 221: lactate is the endproduct of the anaerobic glycolytic pathway.

    Thank you very much for your comment. We have revised.

    • Supplementary Figure 3d: should it be PKM2 (instead of PKM) and LDHA (instead of LDH). Should fit with the text in the manuscript.

    Thank you very much for your comment. We have revised.

    • Supplementary Figures 3 e-f: brackets in y-axis labels are too bold.

    Thank you very much for your comment. We have revised.

    • Figures 3a and b. Brackets should only be used if two conditions are being compared statistically. Remove the one line with ns as it could imply that you have compared the first with the last condition only.

    Thank you very much for your comment. We have revised.

    • Consistent labeling of kDa in figures (no capital K in KDa).

    Thank you very much for your comment. We have revised.

    • Figure 4a. Move kDa on top of 70.

    Thank you very much for your comment. We have revised.

    • Figure 3 g-h: Why 2% EtOH. Used 5% previously?

    Thank you very much for your comment. Because here we changed the 293T cell line, 5% EtOH concentration is too high on this cell.

    • Supplementary Figure b-e: correct typo in y-axis label: expression.

    Thank you very much for your comment. We have revised.

    • Figure 4a: correct x-axis label for temperature unit. Too bold. Not readable.

    Add a clear label and unit for y-axis.

    Thank you very much for your comment. We have revised.

    • Figure 4 b-c: should have a legend explaining colors.

    Thank you very much for your comment. Our Figure legend already contains the meaning of colors: “(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 CAPbinding pocket with KEAP1-NRF2 interface. The KEAP1-NRF2 interaction interface is represented in purple.”

    • Supplementary Figure 5a. Add axis units.

    Thank you very much for your comment. We have revised.

    • Figure 4e: Missing b ions value for number 19.

    Thank you very much for your comment. This part is not missing, but corresponds to 19 of y ions.

    • Figure 7f: adjust brackets - they are too bold.

    Thank you very much for your comment. We have revised.

    • Supplementary Figure 8b-i: labels not readable. c should be spleen.

    Thank you very much for your comment. We have revised.

    • Line 787: specify BH adjustment to Benjamini-Hochberg.

    Thank you very much for your comment. We have revised.

    • Check spelling of µl throughout the Methods section e.g. line 854 - shouldn't be "ul".

    Thank you very much for your comment. We have revised.

    • Line 974: correct spelling of species names: E. coli should be in italics.

    Thank you very much for your comment. We have revised all of these corrections on text and figures. For me, the writing of papers will be more rigorous and careful in the future.

  11. Version published to 10.7554/elife.97632.2 on eLife
  12. Version published to 10.7554/elife.97632.1 on eLife
  13. eLife

    eLife assessment

    This important study suggests that capsaicin nanoparticle administration in rats activates the transcription factor Nrf2 by directly binding to its repressor KEAP1, leading to cytoprotective gene induction, and preventing alcohol-induced gastric damage, an avenue to treat alcoholism-related gastric disorders. The evidence is currently incomplete as there is no experimental proof that capsaicin exerts its cytoprotective effects via Nrf2, and not via any of its multiple known pharmacological effects. In particular, Nrf2-deficient mice should be used to show that Nrf2 is causal to the cytoprotective effect, and better controls should be provided for the direct KEAP2-capsaicin interaction, given the high concentrations used.

  14. eLife

    Reviewer #1 (Public Review):

    Summary:

    This paper by Gao et al. describes the effect of capsaicin on the NRF2/KEAP1 pathway. The authors carried out a set of in vitro experiments that addressed the mechanisms of the protective effect of capsaicin on ethanol-induced cytotoxicity. They also conducted in vivo studies in rats focusing on ethanol-induced gastric mucosal oxidative damage. The authors conclude that capsaicin activates NRF2, which leads to the induction of cytoprotective genes, preventing oxidative damage. This effect has already been shown, and it is well established that capsaicin activates NRF2, but what can be novel in the paper is the demonstration that capsaicin may directly bind to KEAP1 and that it is a noncovalent modification of the Kelch domain. The authors also designed new albumin-coated capsaicin nanoparticles, which were tested for the therapeutic effect in vivo. Apart from novelty concerns, the manuscript may be potentially interesting, but in my opinion, it is not fully technically sound, which weakens the strength of the evidence.

    Major concerns:

    For studies investigating capsaicin binding to KEAP1, the authors used capsaicin concentrations that are toxic to cells (Figures S1D and 4F, G). In vivo studies were performed only in 3 rats per group. The T-test was used for the comparison of more than two groups. Given the well-known issues with the specificity of the NRF2 antibody, the authors should provide appropriate controls, especially for IF and IHC staining.

  15. eLife

    Reviewer #2 (Public Review):

    Summary:

    In this paper, the authors wanted to show that capsaicin can disrupt the interaction between Keap1 and Nrf2 by directly binding to Keap1 at an allosteric site. The resulting stabilization of Nrf2 would protect CAP-treated gastric cells from alcohol-induced redox stress and damage as well as inflammation (both in vitro and in vivo).

    Strengths:

    One major strength of the study is the use of multiple methods (CoIP, SPR, BLI, deuterium exchange MS, CETSA, MS simulations, target gene expression) that consistently show for the first time that capsaicin can disrupt the Nrf2/Keap1 interaction at an allosteric site and lead to stabilization and nuclear translocation of Nrf2.

    Weaknesses:

    One major weakness of the study is that plausibility is taken as proof for causality. The finding that capsaicin directly binds to Keap1 and releases Nrf2 from its fate of degradation (in vitro) is taken for granted as the sole explanation for the observed improved gastric health upon alcohol exposure (in vivo). There is no consideration or exclusion of any potential unrelated off-target effect of capsaicin, or proteins other than Nrf2 that are also controlled by Keap1.

    Another point that hampers full appreciation of the capsaicin effect in cells is that capsaicin is not investigated alone, but mostly in combination with alcohol only.

    Bottom Line:

    Overall, the authors are convincing that capsaicin (although weakly) binds to Keap1 and releases Nrf2 from degradation. With this, the authors provide a significant finding with marked relevance for the redox/Nrf2 as well as natural products /hit discovery communities. Moreover, the employed toolbox of different complementary methodologies can set the bar for future PPI inhibitor studies. The translation of this novel finding in a biological setting (alcohol-stressed gastric cells) still leaves some open questions and concerns. These concerns mainly arise from lacking control experiments and/or somewhat biased conclusions from the obtained data sets.

  16. eLife

    Reviewer #3 (Public Review):

    Summary:

    The paper by Gao et al. describes that capsaicin (CAP) might act as a novel NRF2 agonist capable of suppressing ethanol (EtOH)-induced oxidative damage in the gastric mucosa by disrupting the KEAP1-NRF2 interaction. Initially, the authors established and validated a cell model for EtOH-induced oxidative stress which they used to experiment with different CAP concentrations and to determine changes in a variety of parameters such as cell morphology, ROS production, status of redox balance to mitochondrial function, amongst others.

    The proposed mechanism by which CAP activates NRF2 and mitigates oxidative stress is thought to be via non-covalent binding to the Kelch domain of KEAP1. A variety of assays such as BLI, CETSA, Pull-down, Co-IP, and HDX-MS were employed to investigate the KEAP1 binding behavior of CAP both in vitro and in GES1 cells. Consequently, the authors developed in vivo nanoparticles harboring CAP and tested those in a rat model. They found that pretreatment with the CAP-nanoparticles led to significant upregulation of NRF2 and subsequent effects on pro- (suppression of IL-1β, TNF-α, IL-6, and CXCL1) and anti-inflammatory (activation of IL-10) cytokines pointing to a resolved state of inflammation and oxidative stress.

    Strengths:

    The work comprises a comprehensive approach with a variety of in vitro assays as well as cell culture experiments to investigate CAP binding behaviour to KEAP1. In addition, the authors employ in vivo validation by establishing an ethanol-induced acute gastric mucosal damage rat model and providing evidence of the potential therapeutic effect of CAP.

    The study further provides novel insights into the mode of CAP action by elucidating the mechanism by which CAP promotes NRF2 expression and downstream antioxidant target gene activation.

    The design of IR-Dye800 modified albumin-coated CAP nanoparticles for enhanced drug solubility and delivery efficiency demonstrates a valuable practical application of the research findings.

    In summary, the study's findings suggest that CAP could be a safe and novel NRF2 agonist with implications for the development of lead drugs for oxidative stress-related diseases. Collectively, the data support the significance and potential impact of CAP as a therapeutic agent for oxidative stress-related conditions.

    Weaknesses:

    While the study provides valuable insights into the molecular mechanisms and in vivo effects of CAP, further clinical studies are needed to validate its efficacy and safety in human subjects. The study primarily focuses on the acute effects of CAP on ethanol-induced gastric mucosa damage. Long-term studies are necessary to assess the sustained therapeutic effects and potential side effects of CAP treatment.

    Furthermore, the study primarily focuses on the interaction between CAP and the KEAP1-NRF2 axis in the context of ethanol-induced gastric mucosa damage. It may be beneficial to explore the broader effects of CAP on other pathways or conditions related to oxidative stress. CAP has been known for its interaction with the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel and subsequent NRF2 signaling pathway activation. Those receptors are also expressed within the gastric mucosa and could potentially cross-react with CAP leading to the observed outcome. Including experiments to investigate this route of activation could strengthen the present study.

    While the design of CAP nanoparticles is innovative, further research is needed to optimize the nanoparticle formulation for enhanced efficacy and targeted delivery to specific tissues.

    Addressing these weaknesses through additional research and clinical trials can strengthen the validity and applicability of CAP as a therapeutic agent for oxidative stress-related conditions.

  17. Version published to 10.1101/2024.01.06.574490 on bioRxiv