Ameliorative Effects of Beetroot Juice Supplementation on Monocrotaline-Induced Pulmonary Hypertension in Rats

Round 1

Reviewer 1 Report (New Reviewer)

Tawa, et al. have reported that beetroot juice (BRJ) exerted the inhibitory action against pulmonary vascular remodeling and right ventricular hypertrophy through reducing oxidative stress in monocrotaline-induced pulmonary hypertension (PH) in rats. This paper provides a new insight into therapeutic strategy against PH, and I think this paper could potentially be suitable for publication in Future Pharmacology. However, there is several comment as below;

１）      The authors described that BRJ reduced TBARS but unchanged nitrite and nitrate in RV of PH rats. How about lung tissue ? Please check and show TBARS and nitrite/nitrate in lung.

２）      It is very strange that low BRJ were more effective than high BRJ against PH. In addition, high BRJ inhibited vascular remodeling in lung although its dose failed to suppress RV hypertrophy. The authors mentioned the above (page 6, line 179-184), it was not enough. You need more consideration and discussion about it.

 

Author Response

We are truly grateful for your careful reading of our manuscript and for your valuable comments. Our changes in the revised manuscript have been denoted in green. We hope our responses have sufficiently clarified the reviewers’ concerns.

 

 

1. The authors described that BRJ reduced TBARS but unchanged nitrite nitrate in RV of PH rats. How about lung tissue? Please check and show TBARS and nitrite/nitrate in lung.

We used lungs for histological evaluation, and no samples remain for measuring TBARS and nitrite/nitrate levels. At the time of experiment, BRJ supplementation did not alter lung weight but decreased RV weight, and therefore we performed biochemical assays on RV. However, as you mentioned, it is of great interest to know whether BRJ supplementation increases nitrate and nitrite in the lung. We would like to take this point to the next issue.

 

2. It is very strange that low BRJ were more effective than high BRJ against PH. In addition, high BRJ inhibited vascular remodeling in lung although its dose failed to suppress RV hypertrophy. The authors mentioned the above (page 6, line 179-184), it was not enough. You need more consideration and discussion about it.

According to your comment, we have added a detailed discussion (see line 195–205). Unfortunately, there are several possible reasons, and we cannot give a definite answer. As PH is a local disease, it is thought that it is not good for NO to increase throughout the body. If blood NO levels are increased, blood vessels may dilate to drop blood pressure. When this happens, the baroreflex mechanism may work and an exacerbating factor for PH may be secreted. For example, noradrenaline has been suggested to induce RV hypertrophy but to have no effect on pulmonary vascular remodeling. We think this is one of the possibilities.

Reviewer 2 Report (New Reviewer)

Manuscript entitled "Ameliorative Effects of Beetroot Juice Supplementation on monocrotaline-Induced Pulmonary Hypertension in Rats" by Tawa et al. is within the scope of Future Pharmacology. The findings of this study show that low-dose beet juice supplementation improves vascular remodeling, heart hypertrophy, and lipid peroxidation (MDA) in the right ventricle of rats with pulmonary arterial hypertension. The manuscript is well written. Results and discussion are logically represented. However, some parts of the article must be addressed prior to acceptance.

1.      Based on the evidence, I suggest that the title specifies the type of pulmonary hypertension that this experimental model represents (pulmonary arterial hypertension, PAH). Likewise, throughout the manuscript the same logic must be followed to standardize the phrase (PAH).

2.      In the abstract the authors state that "these PH symptoms were less severe in rats supplemented with BRJ-L", however, the effect on right ventricular systolic pressure elevation was not significant. These results should be rewritten in a more clear way, and as far as possible showing statistical significance.

3.      Results (Lines 59 and 60): MCT injection reduced body weight and increased lung weight. These two parameters are general characteristics of the model as stated by the authors. However, beetroot juice did not improve these parameters. Could the authors comment on this point?

4.      A major concern are the effects of H-BRJ. Although they were not significant, there is a tendency to negatively affect some parameters such as: BW, HW/BW, LW/BW, right ventricular hypertrophy and RVSP. Could these be signs of early toxicity? How could this be explained? add some information on this point.

5.      Regarding the method of administration of BRJ:

- How many rats were kept per box?

- How did you ensure that all rats ingested a standard dose of BRJ daily?

- Critical point: If MCT generated weight loss throughout the experiment, did this not affect the dosage of BRJ?

There are many questions that will surely be answered in future studies. For example, as stated in the discussion (lines 170,171), an adequate amount of exogenous nitrate can alleviate oxidative stress. So, in the H-BRJ group, in which high levels of nitrates were found, were high levels of NO generated mediated by an increase in the activity or expression of some enzyme? This increase in NO altered the antioxidant/pro-oxidant balance of this molecule as evidenced by an apparently higher level of TBARS?

 

The results indicate that the observed effects are probably independent of the Nitrite-Nitrate-NO pathway, instead, oxidative stress seems to be the mechanism involved. In future studies, it would be interesting to explore the participation of enzymes or key proteins such as eNOS, XOR, CAT, GPx, SOD, among others.

Author Response

We are truly grateful for your careful reading of our manuscript and for your valuable comments. Our changes in the revised manuscript have been denoted in green. We hope our responses have sufficiently clarified the reviewers’ concerns.

 

 

1. Based on the evidence, I suggest that the title specifies the type of pulmonary hypertension that this experimental model represents (pulmonary arterial hypertension, PAH). Likewise, throughout the manuscript the same logic must be followed to standardize the phrase (PAH).

To our knowledge, there are pros and cons to recognizing MCT-treated animals as a PAH model (Chest. 2017;152:1106–1108). The reason for this is because 1) the neointima and plexiform lesions seen in PAH patients are absent in MCT-treated animals, 2) MCT drives pulmonary vascular remodeling not only in arteries but also in veins, and 3) there is a greater involvement of the lung parenchyma in MCT-induced PH. This is why we use the phrase PH not PAH.

 

2. In the abstract the authors state that “these PH symptoms were less severe in rats supplemented with BRJ-L”, however, the effect on right ventricular systolic pressure elevation was not significant. These results should be rewritten in a more clear way, and as far as possible showing statistical significance.

According to your comment, the corresponding parts have been revised (see lines 14–16 and 20). However, as there is a word limit, this is the best we can do.

 

3. Results (Lines 59 and 60): MCT injection reduced body weight and increased lung weight. These two parameters are general characteristics of the model as stated by the authors. However, beetroot juice did not improve these parameters. Could the authors comment on this point?

Body weight was somewhat increased with BRJ-L supplementation, although no significant difference was detected. If PH symptoms had been improved more dramatically, the weight loss might have been lessened. Regarding lung weight, pulmonary vasodilation may enhance pulmonary edema in a MCT model. In fact, many studies have reported that vasodilators fail lung weight reduction in MCT-injected rats. As we judged this issue you commented on is an important one, a related description has been added in the Discussion (see line 153–161).

 

4. A major concern are the effects of H-BRJ. Although they were not significant, there is a tendency to negatively affect some parameters such as: BW, HW/BW, LW/BW, right ventricular hypertrophy and RVSP. Could these be signs of early toxicity? How could this be explained? Add some information on this point.

We do not know if lesser ameliorative effects of BRJ-H supplementation are due to early toxicity. As discussed in the Discussion (see line 195–205), BRJ-H supplementation may trigger the baroreflex, whereby sympathetic output to the heart is increased. As noradrenaline serves as an exacerbating factor for PH, we think this is one of the possibilities that BRJ-H supplementation masked the beneficial effects on PH. In this case, it is not “early toxicity” but “compensatory toxicity”. However, it goes without saying that further research is needed because this is just a hypothesis.

 

5. Regarding the method of administration of BRJ:

• How many rats were kept per box?

Rats were housed at 2 animals per cage. This description has been included in the Materials and Methods (see line 241).

• How did you ensure that all rats ingested a standard dose of BRJ daily?

Rats were allowed ad libitum access to drinking water and no special measures were taken. Nitrate intake was calculated assuming that 2 rats in 1 cage drank the same amount of water.

• Critical point: If MCT generated weight loss throughout the experiment, did this not affect the dosage of BRJ?

Body weight was measured weekly and nitrate intake was calculated weekly. That is, the 3 values (1st week, 2nd week, and 3rd week) were averaged per animal and then per group. The weekly data has been added as a supplementary material (see line 257–261). It is considered that a constant amount was taken throughout the intervention period, although the intake was gradually decreased.

 

There are many questions that will surely be answered in future studies. For example, as stated in the discussion (lines 170, 171), an adequate amount of exogenous nitrate can alleviate oxidative stress. So, in the H-BRJ group, in which high levels of nitrates were found, were high levels of NO generated mediated by an increase in the activity or expression of some enzyme? This increase in NO altered the antioxidant/pro-oxidant balance of this molecule as evidenced by an apparently higher level of TBARS?

As BRJ-H contained more nitrate than BRJ-L (see line 250–252), we believe that this difference in content was reflected in high levels of nitrate in the body. Although we have no data on expression and/or activity of XOR, a key enzyme responsible for NO production from nitrate, our theory is supported by findings that sodium nitrate treatment (300 and 1000 µmol/kg/day) for 16 days did not affect XOR expression in the lung in MCT-injected rats (Basic Clin Pharmacol Toxicol. 2020;126:99–109). In addition, we do not know if higher level of TBARS is due to altered balance between antioxidant and pro-oxidant systems or to higher RVSP. As you mentioned in the next comment, future studies to evaluate alteration of antioxidant/pro-oxidant enzymes are needed (see line 189–193).

 

The results indicate that the observed effects are probably independent of the Nitrate-Nitrite-NO pathway, instead, oxidative stress seems to be the mechanism involved. In future studies, it would be interesting to explore the participation of enzymes or key proteins such as eNOS, XOR, CAT, GPx, SOD, among others.

Whether or not the observed effects are independent of the nitrate-nitrite-NO pathway also remains unclear. We measured nitrate and nitrite levels only at the endpoint of the experiment, but it is possible that the levels may have been increased at some point along the way. In other words, the decrease in oxidative stress at the endpoint may be the result of an increase in NO at intermediate point. This issue should also be addressed in future studies.

Round 2

Reviewer 1 Report (New Reviewer)

There is no further comment. Thank you.

Reviewer 2 Report (New Reviewer)

In the revised manuscript, the authors have properly addressed the reviewer’s concerns.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This study investigated the ameliorative effects of Beetroot juice supplementation on monocrotaline-induced pulmonary hypertension in rats. Interestingly, the same title was published by the authors (Tawa, M., Yano, Y., Yamanaka, M., Sawano, T., Iesaki, K., Murata, Y., Tanaka, R., Nakagawa, K., Ohkita, M., & Matsumura, Y. (2019). Effects of Beet Juice Supplementation on Monocrotaline-Induced Pulmonary Hypertension in Rats. American journal of hypertension, 32(2), 216–222. https://doi.org/10.1093/ajh/hpy144).         

Both papers have a closely similar introduction, objectives, methodology except for the low dose of BRJ, results, and conclusion. In addition, the introduction in the present paper was short and didn’t give significant data about the investigated topic. Also, the novel points in this study should be clearly mentioned. The methodology had serious flaws, and the presentation of the results was inappropriate.

 

Reviewer 2 Report

The present manuscript report the effects of supplementation with beetroot juice in the experimental model of monocrotaline-induced pulmonary hipertension in rats. Although this matter is relevant and results seems to be interesting, the authors have published two articles regarding this effects in the same experimental model, as follows:

Tawa, M.; Yano, Y.; Yamanaka, M.; Sawano, T.; Iesaki, K.; Murata, Y.; Tanaka, R.; Nakagawa, K.; Ohkita, M.; Matsumura Y. 308 Effects of Beet Juice Supplementation on Monocrotaline-Induced Pulmonary Hypertension in Rats. Am. J. Hypertens. 2019, 32, 309 216–222; DOI:10.1093/ajh/hpy144. 310

Tawa, M.; Nagata, R.; Sumi, Y.; Nakagawa, K.; Sawano, T.; Ohkita, M.; Matsumura, Y. Preventive effects of nitrate-rich beetroot 311 juice supplementation on monocrotaline (MCT)-induced pulmonary hypertension in rats. PLoS One 2021, 16, e0249816; 312 DOI:10.1371/journal.pone.0249816.

The reason for the present study was reported in Introduction section by citing the previous studies as "...these studies have not provided evidence that BRJ supplementation is effective for PH, even when the intervention is started with a delay after MCT injection...". In this context, considering Tawa et al. 2019 has supplemented with beetroot juice 7 days before and 14 days after MCT injection with positive results, authors cannot afirm that previous studies there are not provided such evidences. 

Therefore, considering the lack of novelty of the present work. I cannot recommend it for publication, and then my decision is for rejection.