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.
