Treatment with Curcumin Delays the Development of Type 1 Diabetes Mellitus by Decreasing Proinflammatory Cytokines in Non-Obese Diabetic Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Line 50: “años”is in Spanish

Line 61: beta cells, but line 66 B cells when should be beta cells or β-cells

Lines 92-93: This is should be explained in the introduction section, not in materials and methods

Lines 97-98: When you say “Twenty-four-week-old female mice” Specifiy the exact number of animals (I guess is 24) and that they are NOD. Why only female animals? CD1 mice are also female? Text only mentions same age, include that.

Line 105: Regarding the research project number, where can externally be validated?

Line 105: 6 animals per cage is okey for CD1 animals but not for diabetic animals like NOD. Both groups were housed in the same conditions?

Lines 117-118: “To explore whether curcumin altered the development of DM1, we administered this compound in a mix with olive oil because turmeric curcumin (C1386, Sigma-Aldrich™) had previously been described as dose-dependent”. Could you explain this in more detail? Why the fact of being dose-dependent would justify the use of olive oil? You should mention that curcumin cannot be properly solved in water because curcumin is a highly lipophilic compound. Which type of olive oil?

Lines 124-125: Could you explain the therapeutic guidelines used? How many doses per day? Was a daily administration?

Line 127: While the study appropriately includes a vehicle control group receiving olive oil, it should be noted that extra virgin olive oil possesses biologically active compounds with documented antioxidant and anti-inflammatory properties. [PMID 29141574; 40749711; 22000808] Therefore, without a naïve group (no vehicle), it is difficult to fully separate the effects of the experimental compound from potential effects of the vehicle itself. Including a group without olive oil administration would strengthen the study design and clarify the specific contribution of curcumin. Why naïve groups were not included?

Lines 133-137: Glucose levels were measured during the same time slots each week?

 

Lines 190-192. Which was the criteria used to use mean+SD or median+IQR?

Line 193: You should include a justification for the use of Kruskal-Wallis instead of ANOVA. It is “Kruskal” not “Kruskall”

Line 195: Regarding Krukal-Wallis test and Wilcoxon Rank sum test, did you include a correction for multiple comparisons (Bonferroni, Tukey, etc.)?

Lines 202-206: If you have 5 experimental groups why are you only showing 4 in Figure 1?

Lines 208-211: The vehicle group here are NOD mice or CD1 animals? Include both. I think it would be more interesting to include the measurements during all the weeks in a fashion similar to Figure 1A to observe the evolution of body weight instead of the final results. Also that would help to see if all the groups were similar in terms of body weight before the beginning of the treatment.

Lines 216-218: I recommend to change “Box” for “Figure”.

Line 226: Nod should be changed to NOD. Be consistent with the terminology

Lines 233–235: The claim that higher doses of curcumin resulted in islets with a score of 0 should be softened, as supplementary data indicate residual inflammatory infiltrate in some animals.

Lines 239–244: Conclusions regarding VDR involvement are speculative and based only on qualitative immunostaining. Please rephrase to indicate an association rather than a mechanistic link.

Lines 266–275: Given the variability observed in individual cytokine values, the conclusions regarding anti-inflammatory effects should be more cautious.

Line 286: Discussion is extremely long for the small amount fo results. It must be reduced.

Lines 286–295: The data support a delay in hyperglycemia development rather than prevention. Please revise wording accordingly.

Lines 309–316: Statements suggesting VDR-mediated genetic regulation go beyond the data presented and should be toned down.

Lines 424–430: The limitations section should more clearly emphasize the small sample size, lack of naïve controls, and variability observed in the supplementary data.

Lines 439–446: Conclusions should be moderated to reflect the lack of dose dependency and the exploratory nature of the mechanistic interpretation.

Author Response

We sincerely appreciate the attention and time dedicated to our manuscript; we have addressed each of the observations as detailed below:

 

Line 50: “años”is in Spanish

Answer: We appreciate the observation; the mistake has been corrected, as shown in the new version of our manuscript.

Line 61: beta cells, but line 66 B cells when should be beta cells or β-cells}.

Answer: The term has been replaced in the new version of our work.

Lines 92-93: This is should be explained in the introduction section, not in materials and methods

Answer: Following the recommendation, the information mentioned was removed from the materials and methods section and relocated to the introduction.

Lines 97-98: When you say “Twenty-four-week-old female mice” Specifiy the exact number of animals (I guess is 24) and that they are NOD. Why only female animals? CD1 mice are also female? Text only mentions same age, include that.

Answer: Information regarding the exact number of animals used in the protocol was added to the Materials and Methods section of the revised version of our manuscript. The reason for selecting only females for the experiment is that the animals coexist in chronic situations; selecting only females eliminates the factors of fighting and aggression associated with hierarchies. All groups consisted solely of female animals.

Line 105: Regarding the research project number, where can externally be validated?

Answer: The research and ethics committees of the Faculty of Medicine, National Autonomous University of Mexico, meet monthly to evaluate proposed research projects. Once evaluated by both committees, each project is assigned an approval number for identification and follow-up. If necessary, anyone can contact the committees, which maintain records of the sessions and observations made on each project. This process allows for external verification of any project of interest.

Line 105: 6 animals per cage is okey for CD1 animals but not for diabetic animals like NOD. Both groups were housed in the same conditions?

Answer: To resolve this condition, the size of the box housing each strain of animals was varied, always respecting the Mexican national standard NOM-062-ZOO-1999 regarding the vital space required for each animal based on its weight, and this has been reflected in the new version of our manuscript.

Lines 117-118: “To explore whether curcumin altered the development of DM1, we administered this compound in a mix with olive oil because turmeric curcumin (C1386, Sigma-Aldrich™) had previously been described as dose-dependent”. Could you explain this in more detail? Why the fact of being dose-dependent would justify the use of olive oil? You should mention that curcumin cannot be properly solved in water because curcumin is a highly lipophilic compound. Which type of olive oil?

 

Answer: Several studies describe an anti-inflammatory effect from curcumin administration. One of the clearest examples is: "The Dose-Dependent Effect of Curcumin Supplementation on Inflammatory Response and Gut Microbiota Profile in High-Fat Fed C57BL/6 Mice Mol Nutr Food Res 2023, 67, e2300378, doi:10.1002/mnfr.202300378." In this study, the authors demonstrate a dose-dependent effect on the expression of two cytokines: IL-10, an anti-inflammatory cytokine, which shows a dose-dependent increase at 50, 250, and 500 mg/kg; and TNF-alpha, which shows a dose-dependent reduction in mice. The authors also present histological, biochemical, and molecular findings. Furthermore, we fully agree with the reviewer that curcumin is highly lipophilic, and for this reason, olive oil was selected as the carrier. Olive oil is a non-toxic ingredient with significant amounts of monounsaturated fats and antioxidants, the effects of which can be mitigated by including a control group that receives the oil without curcumin. The olive oil used was commercially available and of extra virgin quality.

Lines 124-125: Could you explain the therapeutic guidelines used? How many doses per day? Was a daily administration?

Answer: According to the question, the frequency of administration of the curcumin + olive oil mixture was added to the methodology section.

Curcumin was administered daily once a day for 6 weeks as follows:  Group A 50 mg/kg body weight (bw)[25], group B 100 mg/kg bw [27], and group C 200 mg/kg bw[28].

The mixture was prepared before each administration and administered by intragastric gavage, introducing the substance directly into the stomach with precise doses and consistent timing.

Line 127: While the study appropriately includes a vehicle control group receiving olive oil, it should be noted that extra virgin olive oil possesses biologically active compounds with documented antioxidant and anti-inflammatory properties. [PMID 29141574; 40749711; 22000808] Therefore, without a naïve group (no vehicle), it is difficult to fully separate the effects of the experimental compound from potential effects of the vehicle itself. Including a group without olive oil administration would strengthen the study design and clarify the specific contribution of curcumin. Why naïve groups were not included?

Answer: We fully agree with the reviewer that extra virgin olive oil contains multiple compounds that have been documented as antioxidants and anti-inflammatories; however, the addition of a control group that received this compound did not at any time show any effect that could be interpreted as an improvement, and that would warrant the inclusion of a group without the oil to discount said effect.

Lines 133-137: Glucose levels were measured during the same time slots each week?

Answer: Indeed, glucose levels in all animals were measured weekly at the same time periods for each animal in each group, establishing a routine during the development of the experiment.

Lines 190-192. Which was the criteria used to use mean+SD or median+IQR?

Answer: The criterion for using mean+SD or median+IQR was the distribution of each variable analyzed during the protocol.

Line 193: You should include a justification for the use of Kruskal-Wallis instead of ANOVA. It is “Kruskal” not “Kruskall”

Answer: We used the Kruskal-Wallis test for our analysis because the data did not meet the assumptions for an ANOVA, especially normality. This is common for proinflammatory cytokines. On the other hand, thank you for the correction. In the new version of our manuscript, we have changed the highlighted word.

Line 195: Regarding Krukal-Wallis test and Wilcoxon Rank sum test, did you include a correction for multiple comparisons (Bonferroni, Tukey, etc.)?

Answer: The statistical test was used only when differences were found with the Kruskal-Wallis test. This procedure is described in the statistics section of the revised version of our manuscript.

Lines 202-206: If you have 5 experimental groups why are you only showing 4 in Figure 1?

Answer: The reason for only showing the first 4 groups (Comprised of NOD animals) is that the CD1 mouse group was only used to show the appearance of a pancreas without alteration from the development of the autoimmune response causing T1DM, which is impossible using only NOD mice, because these will always develop changes derived from this autoimmune process, and since they would not show any change in serum glucose levels or in pro-inflammatory cytokines, they are not included in the figure.

Lines 208-211: The vehicle group here are NOD mice or CD1 animals? Include both. I think it would be more interesting to include the measurements during all the weeks in a fashion similar to Figure 1A to observe the evolution of body weight instead of the final results. Also that would help to see if all the groups were similar in terms of body weight before the beginning of the treatment.

Answer: The kind of animals in the control group was clarified in the new version of our manuscript:

The body weight of the curcumin-treated mice was significantly greater than that of the control group of NOD mice without curcumin after six weeks. This finding indicated that curcumin treatment at any of the three doses prevented the weight loss accompanying the characteristic hyperglycemia in T1DM (Figure 1B).

Lines 216-218: I recommend to change “Box” for “Figure”.

Answer: Regarding the recommendation, we changed the word in the paragraph:

Figure 1. Kinetics of glycemia, weights, and insulitis in non-obese diabetic mice treated with three different doses of curcumin administered orally. Figure 1A shows the kinetics of glycemia throughout the experimental process. The gray area shows hyperglycemia; Figure 1 B shows the weight of the mice after six weeks of treatment; and Figure 1 C shows the insulitis in the pancreas determined at the end of the experimental process.

Line 226: Nod should be changed to NOD. Be consistent with the terminology

Answer: Thank you for the observation; the terminology has been standardized in the new version of our manuscript.

Lines 233–235: The claim that higher doses of curcumin resulted in islets with a score of 0 should be softened, as supplementary data indicate residual inflammatory infiltrate in some animals.

Answer: The information was rephrased to highlight the presence of residual inflammation.

Lines 239–244: Conclusions regarding VDR involvement are speculative and based only on qualitative immunostaining. Please rephrase to indicate an association rather than a mechanistic link.

Answer: In accordance with the reviewer's recommendation, the conclusion related to VDR was rephrased in the new version of our manuscript.

Lines 266–275: Given the variability observed in individual cytokine values, the conclusions regarding anti-inflammatory effects should be more cautious.

Answer: We fully agree with the reviewer regarding the variability of cytokines; for this reason, we have added a paragraph clarifying that the observed changes in serum cytokine concentrations reflect only the specific conditions of the treatment.

The anti-inflammatory effect was statistically significant under these treatment conditions, as reflected by lower serum cytokine concentrations in animals treated with three different doses.

Line 286: Discussion is extremely long for the small amount fo results. It must be reduced.

Answer: We appreciate the observation; in the new version of our manuscript, we present a shorter discussion section than in the previous version.

Lines 286–295: The data support a delay in hyperglycemia development rather than prevention. Please revise wording accordingly.

Thank you for the recommendation. We have carefully reviewed the text and ensured that our proposal is a delay in the development of the inflammatory phenomenon.

Lines 309–316: Statements suggesting VDR-mediated genetic regulation go beyond the data presented and should be toned down.

Answer: In accordance with the recommendation, we have removed the phrase to avoid interpretations beyond our results.

Lines 424–430: The limitations section should more clearly emphasize the small sample size, lack of naïve controls, and variability observed in the supplementary data.

Answer: In accordance with the recommendation, we have modified the limitations paragraph of our new manuscript.

Lines 439–446: Conclusions should be moderated to reflect the lack of dose dependency and the exploratory nature of the mechanistic interpretation.

Answer: In accordance with the recommendation, we have modified the conclusions of our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors The manuscript is clearly written and presents a straightforward multi-dose oral curcumin intervention with longitudinal glucose monitoring, pancreatic histology, and serum cytokine measurements. The results are directionally consistent across these readouts and support the main message at a descriptive level.  Suggested minor points :

1. Please standardize the terminology for “control” groups throughout the text.

2. Please ensure the insulitis scoring definitions match the reported scoring scale.

3. Please double-check Table 1 for potential typographical inconsistencies (e.g., TNF-α summary statistics) and align Figure/Table statements where needed.

Author Response

We deeply appreciate the attention and time dedicated to our manuscript; we have addressed each of the observations as detailed below

The manuscript is clearly written and presents a straightforward multi-dose oral curcumin intervention with longitudinal glucose monitoring, pancreatic histology, and serum cytokine measurements. The results are directionally consistent across these readouts and support the main message at a descriptive level.  Suggested minor points:

1. Please standardize the terminology for “control” groups throughout the text.

Answer: Thank you for the observation. We have reviewed the text to standardize the term mentioned.

1. Please ensure the insulitis scoring definitions match the reported scoring scale.

Answer: We have reviewed this aspect and modified the text to emphasize that an inflammatory infiltrate is present in all NOD mice.

1. Please double-check Table 1 for potential typographical inconsistencies (e.g., TNF-α summary statistics) and align Figure/Table statements where needed.

Answer: Thank you for the observation. We have modified the table according to your recommendation.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for the revised version of the manuscript and for addressing my previous comments. Before I can recommend acceptance, I have three remaining minor points:

1. Figure 1 should include all five experimental groups. Figure 1B should be updated in the same way as Figure 1A, showing the progression of body weight over the three-week period.

2. The Discussion section remains too long in relation to the amount of results presented and would benefit from further condensation.

3. Please clarify whether the ethics approval (project number 105-2014) covered the entire data collection period, or whether renewals were obtained if the study extended beyond the initial approval.

Comments on the Quality of English Language

The manuscript is generally clear, but minor English language editing would improve fluency and readability.

Author Response

We again thank you for your attention and the time dedicated to our work. In this second round of corrections, we have addressed the observations as detailed below:

1. Figure 1 should include all five experimental groups. Figure 1B should be updated in the same way as Figure 1A, showing the progression of body weight over the three-week period.

Answer: In accordance with the reviewer's recommendation, we have modified the aforementioned graphs. In the new version of our manuscript, the data for the CD1 mice used as controls in the experiments are presented.

2. The Discussion section remains too long in relation to the amount of results presented and would benefit from further condensation.

Answer: In accordance with the reviewer's recommendation, we have further shortened the discussion, keeping only the minimum content necessary to convey the idea.

3. Please clarify whether the ethics approval (project number 105-2014) covered the entire data collection period, or whether renewals were obtained if the study extended beyond the initial approval.

Answer: Regarding the approval period allowed for developing the research project, the local committees of the Faculty of Medicine of the National Autonomous University of Mexico approved it for three years, which was sufficient time to develop the presented project, so it was not necessary to request a renewal of the approval provided.