Review Reports - Puerarin Alleviates Blood Pressure via Inhibition of ROS/TLR4/NLRP3 Inflammasome Signaling Pathway in the Hypothalamic Paraventricular Nucleus of Salt-Induced Prehypertensive Rats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

To:

Editorial Board

Nutrients

Title: “Puerarin Alleviates Blood Pressure via Inhibition of ROS/TLR4/NLRP3 Inflammasome Signaling Pathway in Hypothalamic Paraventricular Nucleus of Salt-Induced Prehypertensive Rats”

Dear Editor,

I read this paper and I think that:

- Introduction section is too long. Please be more concise and focused on the aims of the study.

- The English of the paper should be revised by a native English speaker.

- The impact of Puerarin on RAAS and atherosclerosis should also be discussed.

Comments on the Quality of English Language

see comments

Author Response

Response to Reviewer 1 Comments

Manuscript ID: nutrients-3112906

July 24, 2024

Dear Ms. Jolene Lv and Reviewer:

We are submitting our revised manuscript entitled “Puerarin alleviates blood pressure via inhibition of ROS/TLR4/NLRP3 inflammasome signaling pathway in hypothalamic paraventricular nucleus of salt-induced prehypertensive rats”. (Manuscript ID: nutrients-3112906).

Thank you very much for your interest in our manuscript, for your time and patient during this review. We thank the Reviewers for the constructive suggestions, and these suggestions have helped us tremendously in revising the manuscript. Those comments are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to make us think more deeply about our research.

We have carefully considered those comments and have made correction accordingly and changes are highlighted in yellow in the revised manuscript. We have revised the manuscript following the Reviewers' suggestions. We believe we have addressed all of the issues raised by the Reviewers, and we hope these changes will be considered favorable by you and the Reviewers. The main correction in the manuscript and the point by point responses to the reviewer’s comments are as following.

We greatly appreciate your kindness and help. We hope you will find this revision acceptable for publication on Nutrients.

Best regards,

Xiao-Jing Yu, on behalf of all authors

Point by point responses to reviewers

Reviewer 1

Comments and Suggestions for Authors

- Introduction section is too long. Please be more concise and focused on the aims of the study.

Authors’ response: We appreciate all your supports and suggestions. These constructive suggestions helped us tremendously. We have modified Introduction section in a more concise way according to your suggestion. Please see the revised manuscript.

-The English of the paper should be revised by a native English speaker.

Authors’ response: We thank the reviewer for pointing this out. We have checked and revised the grammatical errors in the manuscript. To improve the readability and quality of our manuscript, we asked three native English speakers to help us to revise the English expression. All changes in the revised version are highlighted in yellow color. Please see the revised manuscript.

- The impact of Puerarin on RAAS and atherosclerosis should also be discussed.

Authors’ response: Thank you so much for these suggestions. We have discussed the impact of puerarin on RAAS and atherosclerosis into the Discussion section. Please see the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Puerarin Alleviates Blood Pressure via Inhibition of ROS/TLR/NLRP3 Inflammasome Signaling Pathway in Hypothalamic Paraventricular Nucleus of Salt-Induced Prehypertensive Rats” by Hong-Li Gao et al examined the effect of administration of isoflavone derivative puerarin on the blood pressure, signal transduction pathway, expression of inflammatory genes and antioxidant enzymes.

It is true that the authors carried out many experiments, however, the experimental procedures in general are not interesting. The authors used the normal procedures using various kit and instruments. The explanation of the usage of puerarin is not clear, as there are many metabolites in kudzu roots of leguminous plants, the clear description for the usage of puerarin must be necessary.

There are some general points which should be revised.

1, There are some commercially available spontaneous hypertensive rats.

However, the authors used the salt-sensitive rats in their study, the reason for the usage of salt-sensitive rat is not clear.

2, The methodology is heavily dependent on the immunofluorescence staining studies (Fig.2-Fig.7), the image for the readers is good, however, the explanation is not enough for all these figures.

3, The English usage should be revised. For example, line 333-336, what is the subject of this sentence? The authors use pre hypertensive (line 360), in other cases, they use prehypertensive. In line 394, the authors did not capitalize the first letter, is there any meaning?

Basically, the authors should pay attention before submitting the manuscript, small grammatical mistakes might be easily be found by the computer soft (MS word).

4, It is not clear to use the advantage of puerarin. In other words, there are many candidates to carry out same studies.

5, How many rats did authors use in these experiments? The authors divided rats into four groups, did all rats survive until the end of the experiment? It is not clear these points.

Figures

In Fig. 1, the figure is not clear in Figure 1(A), it is quite difficult to check the figure. This figure should be revised. In Figure 1 (B-D), the authors wanted to describe the administration effect of peurarin in HS (No. 4). The authors pointed out the significant difference (0.05) was observed in the cases of No. 3 (HS vehicle) and No. 4 (HS + puerarin) in every three cases (B-D). The administration of puerarin to NS (No. 2) decreased the HR, NR, and NF-kB showed almost same level as those of NS (No. 1 vehicle). Is there any significant difference observed in cases of No. 2 and No. 4? In the case of Fig. 1C, there is not so big difference between No. 2 and No. 4, however, there is a big difference in the case of Fig. 1D. Is this difference important or not? All these effects are dose-dependent or not?

In Figures 2-8, the similar observations were described. The administration of peurarin to HS rat improved the state to the control level. The ratio is different, but the trend is the same. Basically, the authors wanted to describe the difference between No. 3 and No. 4. Precisely examining the effect of puerarin, the moderate effects were observed in the cases of Figure 2-7, however, in the case of Figure 8, the significant effects were observed in all these cytokines (almost same level as control No. 2(in some case No.1)). Concerning for this point, the authors made some explanation for the antioxidant activity of puerarin (line 361-362), if so, isoflavone derivatives might show the similar effect. Do the authors carry out the experiments to use other isoflavonoids in their system?

Comments on the Quality of English Language

The English usage must be improved. It is very careful to prepare the manuscript before submission. Some grammatical mistakes are found in the manuscript. The reviewer can’t understand several sentences. In this meaning, the advice and revision of highly specialized person must be necessary.

Author Response

Response to Reviewer 2 Comments

Manuscript ID: nutrients-3112906

July 24, 2024

Dear Ms. Jolene Lv and Reviewer:

We greatly appreciate your kindness and help. We hope you will find this revision acceptable for publication on Nutrients.

Best regards,

Xiao-Jing Yu, on behalf of all authors

Point by point responses to reviewers

Reviewer 2

Comments and Suggestions for Authors

There are some general points which should be revised.

1, There are some commercially available spontaneous hypertensive rats.

However, the authors used the salt-sensitive rats in their study, the reason for the usage of salt-sensitive rat is not clear.

Authors’ response: We appreciate for all your effort to make our manuscript better and would try our best to answer your questions. Hypertension is a common chronic disease worldwide, seriously affecting human health. Establishing an animal model for hypertension is a crucial step in exploring the pathogenesis, prevention, and treatment measures of hypertension. Hypertension animal models are important tools for studying hypertension and have wide applications in medical research and drug screening. Through in-depth research on these models, we can better understand the pathogenesis of hypertension, search for effective therapeutic drugs, and provide more scientific theoretical basis for clinical practice. Currently, number of models for hypertension have been validated and available (commercially, for the case of SHR). Each model typically addresses some contributing factors or causes believed to promote hypertension. Salt-induced hypertension is one model validated that simulate secondary hypertension observed among people with high salt intake and its mechanism is different from other hypertensive models. Further studies may assess this drug in other model so as to cover the panoply of believed pathogenesis of hypertension. This work aimed to clarify the important role of puerarin in the prehypertensive phase, that is, will it improve blood pressure and prevent it from developing into high blood pressure.

2, The methodology is heavily dependent on the immunofluorescence staining studies (Fig.2-Fig.7), the image for the readers is good, however, the explanation is not enough for all these figures.

Authors’ response: We thank the reviewer for pointing this out. The explanation for all these figures has been reformulated. Please see the revised manuscript.

Authors’ response: We appreciate all your supports and suggestions. We have checked and revised all the inappropriate description and grammatical errors in the manuscript. To improve the readability and quality of our manuscript, we asked three native English speakers to help us to revise the English expression. All changes in the revised version are highlighted in yellow color. Please see the revised manuscript.

Basically, the authors should pay attention before submitting the manuscript, small grammatical mistakes might be easily be found by the computer soft (MS word).

Authors’ response: To improve the readability and quality of our manuscript, we asked three native English speakers to help us to revise the English expression. All changes in the revised version are highlighted in yellow color. Please see the revised manuscript.

4, It is not clear to use the advantage of puerarin. In other words, there are many candidates to carry out same studies.

Authors’ response: Thank you so much for your careful review. These constructive suggestions helped us tremendously. Traditional Chinese medicine offers several advantages in treating chronic metabolic diseases, including its natural acquisition, affordability, and fewer side effects. Owing to theirpleiotropic biological effects, low biotoxicity, and easy access, natural medicinal molecules have become the preferred treatment option for most patients with early-stage chronic diseases. Pueraria is the dried root of leguminous plant Pueraria lobata (Willd.) Ohwi, which is mainly found in Eastern Asia and Liaoning, Hebei, and Henan in China. Pueraria, which is rich in starch, dietary fiber, flavonoids and other nutrients, has been an important edible natural resource in China for thousands of years and was recommended as the sixth food crop by Food and Agriculture Organization of the United Nations in 2005[1]. Puerarin, as a natural isoflavonoid phytoestrogen, puerarin has recently attracted attention owing to its promising pharmacological activities, such as anti-inflammatory, cardioprotective, neuroprotective, antioxidant, and insulin resistance propertiess[2, 3]. Puerari, as a main active ingredient in pueraria, has been widely used as a nutritious food and an herb medicine to alleviate diabetes, cardiovascular and cerebrovascular diseases with a long history[4].

As a phytoestrogen, puerarin has weak estrogen-like effect and less toxic and side effects than estrogen, which has been confirmed in previous study[1]. Therefore, the research on puerarin in the prevention and treatment of cardiovascular diseases has become a hot spot in recent years. However, the precise mechanisms underlying its therapeutic effects remain incompletely understood. The present study, therefore, our goal was to uncover the underlying mechanisms of puerarin in improving blood pressure.

5, How many rats did authors use in these experiments? The authors divided rats into four groups, did all rats survive until the end of the experiment? It is not clear these points.

Authors’ response: We appreciate for your thorough review. Due to the success rate (64%) of bilateral PVN cannulation the actual number of rats used in this study was 64.

Figures

In Fig. 1, the figure is not clear in Figure 1(A), it is quite difficult to check the figure. This figure should be revised. In Figure 1 (B-D), the authors wanted to describe the administration effect of puerarin in HS (No. 4). The authors pointed out the significant difference (0.05) was observed in the cases of No. 3 (HS vehicle) and No. 4 (HS + puerarin) in every three cases (B-D). The administration of puerarin to NS (No. 2) decreased the HR, NR, and NF-κB showed almost same level as those of NS (No. 1 vehicle). Is there any significant difference observed in cases of No. 2 and No. 4? In the case of Fig. 1C, there is not so big difference between No. 2 and No. 4, however, there is a big difference in the case of Fig. 1D. Is this difference important or not? All these effects are dose-dependent or not?

Authors’ response: We appreciate for your thorough review. In this study, we found that there is no difference between NS+PVN (No. 1) vehicle and NS+PVN puerarin (No. 2) . There is a significant difference between HS+PVN vehicle (No. 3) and HS+PVN puerarin (No. 4) . The purpose of this study is to explore the effect and mechanism of puerarin in improving blood pressure. Therefore, as long as the model is successful, the statistical significance of blood pressure and other molecular biological indicators in the puerarin treatment group (No. 4) compared to the model group (No. 3) is the focus of our attention.

In addition, we set the dose according to preliminary experiments and published articles (Cardiovasc Res. 2009 Jun 1;82(3):503-12. doi: 10.1093/cvr/cvp073. Cardiovasc Toxicol. 2016 Jul;16(3):276-85. doi: 10.1007/s12012-015-9335-x.). In our pilot study, three different doses of 10, 20 and 50 μg/h were used on the rats. The 20 μg/h dose was found to be optimal and used in the subsequent studies to evaluate the effects of puerarin in Dahl salt-sensitive rats, which produced significant blood pressure lowering effects, whereas the 50 μg/h dose caused mortality, and 10 μg/h dose did not produce a significant effect. Therefore, 20 μg/h dosage is the optimal.

In Figures 2-8, the similar observations were described. The administration of puerarin to HS rat improved the state to the control level. The ratio is different, but the trend is the same. Basically, the authors wanted to describe the difference between No. 3 and No. 4. Precisely examining the effect of puerarin, the moderate effects were observed in the cases of Figure 2-7, however, in the case of Figure 8, the significant effects were observed in all these cytokines (almost same level as control No. 2(in some case No.1)). Concerning for this point, the authors made some explanation for the antioxidant activity of puerarin (line 361-362), if so, isoflavone derivatives might show the similar effect. Do the authors carry out the experiments to use other isoflavonoids in their system?

Authors’ response: We are very grateful for your suggestions. Puerarin, an isoflavone glycoside derived from Pueraria lobata (Willd.) Ohwi, has been identified as a pharmacologically active component with diverse benefits. Increasing evidence indicates that puerarin has anti-hypertensive effect in a variety of animal models of hypertension. Our results elucidated that PVN pretreatment puerarin could alleviate inflammatory response and Nrf2-mediated intracellular ROS, which mediate sympathetic excitability and elevated arterial pressure. We first revealed that the central mechanism of early prevention of high salt induced hypertension by puerarin. Puerarin alleviates blood pressure via inhibition of ROS/TLR4/NLRP3 inflammasome signaling pathway in the PVN of salt-induced prehypertensive rats.

Previous experimental studies have suggested that isoflavonoids have the blood pressure-lowering effect, such as green tea, grape seeds, genistein and resveratrol. Genistein is an isoflavone derived from soy-rich products and accounts for nearly 60% of total isoflavones in soybeans. In our previous research, we found that PVN infusion of genistein could inhibit the progression of hypertension by activating the Sirt1/Nrf2 pathway[5]. Resveratrol within the PVN attenuates hypertension via the SIRT1/NF-κB pathway to decrease ROS and restore the balance of excitatory and inhibitory neurotransmitters[6]. Additionally, we have demonstrated that treatment with Epigallocatechin-3-O-gallate (EGCG) infusion in the PVN attenuates the sympathetic activity and hypertensive response by restoring the balance between the excitatory and inhibitory neurotransmitters and the balance between pro-inflammatory cytokines and anti-inflammatory cytokines[7].

Comments on the Quality of English Language

References

Li B, Wang Y, Gong S, Yao W, Gao H, Liu M, Wei M: Puerarin improves OVX-induced osteoporosis by regulating phospholipid metabolism and biosynthesis of unsaturated fatty acids based on serum metabolomics.Phytomedicine 2022, 102:154198.
Zhou W, He H, Wei Q, Che L, Zhao X, Liu W, Yan Y, Hu L, Du Y, Yin Z, et al: Puerarin protects against acetaminophen-induced oxidative damage in liver through activation of the Keap1/Nrf2 signaling pathway.Food Sci Nutr 2023, 11:6604-6615.
Liu Y, Hu Z, Wang J, Liao Y, Shu L: Puerarin alleviates depressive-like behaviors in high-fat diet-induced diabetic mice via modulating hippocampal GLP-1R/BDNF/TrkB signaling.Nutr Neurosci 2023, 26:997-1010.
Chen R, Wu P, Cai Z, Fang Y, Zhou H, Lasanajak Y, Tang L, Ye L, Hou C, Zhao J: Puerariae Lobatae Radix with chuanxiong Rhizoma for treatment of cerebral ischemic stroke by remodeling gut microbiota to regulate the brain-gut barriers.J Nutr Biochem 2019, 65:101-114.
Niu LG, Sun N, Liu KL, Su Q, Qi J, Fu LY, Xin GR, Kang YM: Genistein Alleviates Oxidative Stress and Inflammation in the Hypothalamic Paraventricular Nucleus by Activating the Sirt1/Nrf2 Pathway in High Salt-Induced Hypertension.Cardiovasc Toxicol 2022, 22:898-909.
Qi J, Fu LY, Liu KL, Li RJ, Qiao JA, Yu XJ, Yu JY, Li Y, Feng ZP, Yi QY, et al: Resveratrol in the Hypothalamic Paraventricular Nucleus Attenuates Hypertension by Regulation of ROS and Neurotransmitters.Nutrients 2022, 14.
Yi QY, Qi J, Yu XJ, Li HB, Zhang Y, Su Q, Shi T, Zhang DM, Guo J, Feng ZP, et al: Paraventricular Nucleus Infusion of Epigallocatechin-3-O-Gallate Improves Renovascular Hypertension.Cardiovasc Toxicol 2016, 16:276-285.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised version of the manuscript entitled “Puerarin Alleviates Blood Pressure via Inhibition of ROS/TLR/NLRP3 Inflammasome Signaling Pathway in the Hypothalamic Paraventricular Nucleus of Salt-Induced Prehypertensive Rats” by Hong-Li Gao et al examined the effect of administration of isoflavone derivative puerarin on the blood pressure, signal transduction pathway, expression of inflammatory genes and antioxidant enzymes.

The authors revised the manuscript according to the suggestions and comments indicated by the reviewers. Although some points were still remaining.

The number of the sacrificed animals should be reduced as much as possible. For this purpose, well-designed (organized) experimental procedure must be planned and performed.

As pointed out in the previous comments, there are many flavonoids isoflavonoids (including aglycon and glycoside), unfortunately, there are no comments (studies) regarding these topics.

So, in the future study, 1) the authors should pay much attention on the number of sacrificed animals, 2) the authors carry out several in vitro experiments before animal experiments, 3) the authors pay attention on the behavior change of animals dosed vs non-dosed.

Comments on the Quality of English Language

The authors asked three native English speakers to brush up the manuscript. Some editorial revision might be necessary.