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

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.

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.

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.