The Effects of Royal Jelly Acid, 10-Hydroxy-trans-2-decenoic Acid, on Neuroinflammation and Oxidative Stress in Astrocytes Stimulated with Lipopolysaccharide and Hydrogen Peroxide

Round 1

Reviewer 1 Report

Although the results obtained in this study were a bit negative, the reviewer has no objection to accept it . 

Here are the major comments:

(1) What is the level of 10-hydroxy-trans-2-decenoic acid in brain? Are the concentrations used in this study physiologically relevant?

(2) Why 10-hydroxy-trans-2-decenoic acid appeared to be pro-inflammatory at about 3 micromolar but such effect was not observed at higher concentration (Fig 1 a)? 

(3) Any safety data of 10-hydroxy-trans-2-decenoic acid reported in literatrue?

Author Response

Manuscript ID: immuno-1278934

Title: The effects of royal jelly acid, 10-hydroxy-trans-2-decenoic acid, on neuroinflammation and oxidative stress in astrocytes stimulated with lipopolysaccharide and hydrogen peroxide

Reviewer #1

Thank you so much for the careful and insightful comments. The reviewer has raised questions, which would probably be asked by many readers. Given that MDPI publishes the review reports along with the manuscript, we reply to these queries while quoting and reporting on aspects of the literature that hit the points raised by the reviewer and common readers. Authors’ responses come underneath following the comments in red. When relevant, the location of the modification in the text is reported for the reviewer’s reference.

Here are the major comments:

• What is the level of 10-hydroxy-trans-2-decenoic acid in brain? Are the concentrations used in this study physiologically relevant?

Unfortunately, we could not find studies reporting on the level of 10-hydroxy-trans-2-decenoic acid in brain. In fact, we used doses within the range of 100 μM based on a previous study reporting stimulation of neurogenesis by 1, 10, and 100 μM [1] (reported in line 142). Reports show that injected intraperitoneally injected RJ (250 mg/kg/day) is more effective for alleviating depressive symptoms in stressed mice than higher doses (2.5 g/kg/day) administered through gavage feeding [2]. Orally consumed Low-dose (3 mg/kg) 10H2DA group (10HDA-L) had no effect on the development of induced encephalomyelitis in mice compared with high-dose (6 mg/kg) 10H2DA group (10HDA-H). However, both doses exhibited anti-inflammatory activity, albeit the effect of low dose was less than the high dose [3]. Thus, we could assume that direct treatment of cultured cells with the reported concentrations might be physiologically relevant. However, in microglia concentrations of 4mM inhibited LPS-induce inflammation. Therefore, there is a possibility that the concentrations of10H2DA in the current study may not be physiologically relevant. We have reported on this in this revised version of the manuscript (line 273-278).

• Why 10-hydroxy-trans-2-decenoic acid appeared to be pro-inflammatory at about 3 micromolar but such effect was not observed at higher concentration (Fig 1 a)? 

It is well-known that cytokines induce glial cell activation and advance neurodegeneration. However, slight levels of cytokines facilitate normal physiological functions e.g., during ovulation [4, 5]. Slight levels of free radicals (cytokines in parallel) may exert preconditioning effects, which may lower the occurrence of damage following exposure to severer stimulations [6]. The following quote from a detailed review note that cytokines may elicit potent neuroprotective and regenerative responses from astrocytes.

 “However, there is a wealth of data indicating that cytokines ‘activate’ astrocytes, and that cytokine-stimulated astrocytes can promote the recovery of CNS function. Supporting evidence demonstrates that cytokine-activated astrocytes produce energy substrates and trophic factors for neurons and oligodendrocytes, act as free radical and excess glutamate scavengers, actively restore the blood–brain barrier, promote neovascularization, restore CNS ionic homeostasis, promote remyelination and also stimulate neurogenesis from neural stem cells. Accordingly, a re-assessment of cytokine-activated astrocytes is necessary. Here, we review studies that promote the thesis that cytokines elicit potent neuroprotective and regenerative responses from astrocytes.” [7]

The literature spots several instances in which bioactive natural compounds, with documented pharmacological activities, express pro-inflammatory properties. For example, quercetin decreased the risk for cardiovascular injury but moderately increased TNFα levels (P = 0.024) in men with apolipoprotein E (APOE) genotype [8]. Given this literature, it might be expected that the proinflammatory activity of 0-hydroxy-trans-2-decenoic acid at about 3 micromolar might induce positive effects in astrocytes under normal conditions (i.e., in the absence of LPS). This notion highlighted in line 246 “Astrocytes maintain homeostasis under normal physiological conditions by producing low levels of cytokines in response to byproducts of metabolic processes [9].”

(3) Any safety data of 10-hydroxy-trans-2-decenoic acid reported in literatrue?

Royal jelly is considerably safe, and allergic reactions that might affect some individuals following the consumption of royal jelly are associated with its protein fraction [10, 11]. For this revision, all studies retrieved in an ad hoc search in Google Scholar “search term: safety of 10-hydroxy-trans-2-decenoic acid” reported no adverse effects, and most results were positive.

In final, we thank Reviewer 1 for the time, effort, and help provided. We hope that the comments were properly handled and that the revised version will be suitable for publication.

 Best regards,

Corresponding author

References

1. Hattori, N., et al., Royal jelly and its unique fatty acid, 10-hydroxy-trans-2-decenoic acid, promote neurogenesis by neural stem/progenitor cells in vitro. Biomedical Research, 2007. 28(5): p. 261-266.
2. Ito, S., et al., Antidepressant-Like Activity of 10-Hydroxy-Trans-2-Decenoic Acid, a Unique Unsaturated Fatty Acid of Royal Jelly, in Stress-Inducible Depression-Like Mouse Model. Evidence-based Complementary and Alternative Medicine : eCAM, 2012. 2012: p. 139140.
3. Shahla, J., et al., Comparative immunomodulatory effects of jelly royal and 10-H2DA on experimental autoimmune encephalomyelitis. Gene Reports, 2021. 24: p. 101217.
4. Poulsen, L.l.C., et al., Human granulosa cells function as innate immune cells executing an inflammatory reaction during ovulation: a microarray analysis. Molecular and Cellular Endocrinology, 2019. 486: p. 34-46.
5. Molyneaux, K.A., K. Schaible, and C. Wylie, GP130, the shared receptor for the LIF/IL6 cytokine family in the mouse, is not required for early germ cell differentiation, but is required cell-autonomously in oocytes for ovulation. Development, 2003. 130(18): p. 4287-94.
6. Inoue, Y., et al., 4-Hydroperoxy-2-decenoic acid ethyl ester protects against 6-hydroxydopamine-induced cell death via activation of Nrf2-ARE and eIF2α-ATF4 pathways. Neurochemistry International, 2018. 112: p. 288-296.
7. Liberto, C.M., et al., Pro-regenerative properties of cytokine-activated astrocytes. J Neurochem, 2004. 89(5): p. 1092-100.
8. Pfeuffer, M., et al., Effect of quercetin on traits of the metabolic syndrome, endothelial function and inflammation in men with different APOE isoforms. Nutr Metab Cardiovasc Dis, 2013. 23(5): p. 403-9.
9. Casedas, G., et al., Polyphenol-associated oxidative stress and inflammation in a model of LPS-induced inflammation in glial cells: do we know enough for responsible compounding? Inflammopharmacology, 2019. 27(1): p. 189-197.
10. Gu, H., et al., Antioxidant Activity of Royal Jelly Hydrolysates Obtained by Enzymatic Treatment. Korean journal for food science of animal resources, 2018. 38(1): p. 135-142.
11. Moriyama, T., et al., Hypoallergenicity and Immunological Characterization of Enzyme-Treated Royal Jelly from Apis mellifera. Bioscience, Biotechnology, and Biochemistry, 2013. 77(4): p. 789-795.

Author Response File: Author Response.pdf

Reviewer 2 Report

Ali et al. wrote a very interesting research article describing “The effects of royal jelly acid, 10-hydroxy-trans-2-decenoic acid, on neuroinflammation and oxidative stress in astrocytes stimulated with lipopolysaccharide and hydrogen peroxide”. The manuscript represents an interesting way to discover new scenarios for neuroinflammation mechanisms and diagnosis. However, I suggest only minor revisions needed to improve the reliability of the paper:

• “Methods”: were all experiments performed at least in triplicated?
• “Cell Culture” section: this section should be more detailed.
• Finally, manuscript requires English revisions and typos correction.

Author Response

Manuscript ID: immuno-1278934

Title: The effects of royal jelly acid, 10-hydroxy-trans-2-decenoic acid, on neuroinflammation and oxidative stress in astrocytes stimulated with lipopolysaccharide and hydrogen peroxide

Reviewer #2

We thank Reviewer 2 for his/her productive and insightful comments. The comments are addressed line-by-line as shown below. Replies come underneath in red.

I suggest only minor revisions needed to improve the reliability of the paper:

• “Methods”: were all experiments performed at least in triplicated?

Yes, my mentor and I doubted the results at first. So, all experiments were performed several times for confirmation. Those involving the anti-inflammatory effects of 10-H2DA were performed at least in triplicates.

• “Cell Culture” section: this section should be more detailed.

As per this comment and similar comments from other reviewers, we have added more details to the Methods and other sections of the manuscript.

• Finally, manuscript requires English revisions and typos correction.

Yes, we have revised the whole manuscript taking into account the correction of typos.

In final, we thank Reviewer 2 for the time, effort, and help provided. We hope that the comments were properly handled and that the revised version will be suitable for publication.

 Best regards,

Corresponding author

Reviewer 3 Report

Neuroinflammation is an area of high relevance and importance for numerous neurological disorders. These authors investigate the potential role of a natural compound found in royal jelly, 10-H2DA, in regulating astrocyte inflammation and cytokine expression. The authors found no effect of 10-H2DA on astrocytes. The overall results, while simple, are interpreted correctly. The scope of the study and overall novelty is a bit limited.

Specific concerns:

1. Abstract: The abstract ends with this statement: “However, it is possible that 10-H2DA might prevent neuronal loss during neuroinflammation by reducing the probability of developing a late pro-inflammatory response and by enhancing ketogenesis secondary to IGF-1 inhibition” which is not appropriate for the abstract. This point can be included in the discussion, not in the abstract.

2. Since the focus of the manuscript is on neuroinflammation, it would be interesting if the authors had explored any effect of RJ on microglia.

3. Authors measure mRNA levels and should thus describe cytokine expression instead of production when talking about their results.

4. Please put figure labels (A-E) above and to the left of the graph, not underneath.

5. Can the authors confirm that the 0uM treatment of 10-H2DA is represented by a vehicle and not just media. The 10-H2DA was dissolved in ethanol and thus this vehicle needs to be represented in the control condition.

6. Figure 2- remove boxes around graphs and fix x-axis label on bottom panel.

Author Response

Manuscript ID: immuno-1278934

Title: The effects of royal jelly acid, 10-hydroxy-trans-2-decenoic acid, on neuroinflammation and oxidative stress in astrocytes stimulated with lipopolysaccharide and hydrogen peroxide

Reviewer #3

We thank the Reviewer for his/her productive and insightful comments. The comments are addressed line-by-line as shown below. Replies come underneath in red.

1. Abstract: The abstract ends with this statement: “However, it is possible that 10-H2DA might prevent neuronal loss during neuroinflammation by reducing the probability of developing a late pro-inflammatory response and by enhancing ketogenesis secondary to IGF-1 inhibition” which is not appropriate for the abstract. This point can be included in the discussion, not in the abstract.

 Yes, we have removed this sentence from the abstract.

1. Since the focus of the manuscript is on neuroinflammation, it would be interesting if the authors had explored any effect of RJ on microglia.

 Thank you very much. Yes, of course microglia play a key role in neuroinflammation. We have located a recent study examining the effect of 10H2DA on neuroinflammation in vivo and in LPS-challenged microglia. The used concentration of 10H2DA was exceptionally high (4 mM) [12] compared with the dose used in the current study. Based on this comment, along with a similar comment by Reviewer #1, we have noted in the discussion (line 273-278) that lack of effects may be because the used concentrations of 10H2DA might not be physiologically relevant.

1. Authors measure mRNA levels and should thus describe cytokine expression instead of production when talking about their results.

 Yes, thank you very much. We have replaced “production” by “expression” in the text within the relevant contexts.

1. Please put figure labels (A-E) above and to the left of the graph, not underneath.

 Yes, we have moved figure labels (A-E) from underneath the graphs to be above and to the left.

1. Can the authors confirm that the 0uM treatment of 10-H2DA is represented by a vehicle and not just media. The 10-H2DA was dissolved in ethanol and thus this vehicle needs to be represented in the control condition.

Yes, we agree with the reviewer; we should have used ethanol as vehicle in the control cells. Unfortunately, it was just media. Ethanol was not used in control cells. We noted that in the text in this version (line 143).

1. Figure 2- remove boxes around graphs and fix x-axis label on bottom panel.

Yes, we have modified Figure 2 accordingly.

In final, we thank Reviewer 3 for the time, effort, and help provided. We hope that the comments were properly handled and that the revised version will be suitable for publication.

 Best regards,

Corresponding author

Author Response File: Author Response.pdf