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Protective Action of Betulinic Acid on Cerebral Ischemia/Reperfusion Injury through Inflammation and Energy Metabolic Homeostasis

Appl. Sci. 2020, 10(7), 2578; https://doi.org/10.3390/app10072578

by Wenjiao Jiang^1,2 and Kun Hao^1,2,*

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Reviewer 3: Anonymous

Appl. Sci. 2020, 10(7), 2578; https://doi.org/10.3390/app10072578

Submission received: 7 March 2020 / Revised: 2 April 2020 / Accepted: 7 April 2020 / Published: 9 April 2020

(This article belongs to the Section Applied Biosciences and Bioengineering)

Round 1

Reviewer 1 Report

In this paper, Jiang and Hao assessed the protective effects of betulinic acid (BA) in a rat model of ischemic stroke through middle cerebral artery occlusion (MCAO), and in oxygen and glucose deprived SHSY5Y cells. They found that treatment with BA resulted in a smaller infarct size and cerebral edema as well as reduced neurological injury. Moreover, a range of neuroinflammatory molecules were downregulated both in vivo and in vitro.

Several major and minor issues need to be addressed prior to publication.

Major:

Introduction:

I’m not sure that clopidogrel is an appropriate positive control as it is used as a preventative for future stroke and to mitigate lifestyle factors (which is not a problem with rodent models as their strokes are induced). Please do not refer to it as an ‘intervention’ in stroke as it is not a treatment. Perhaps referencing previous literature in which clopidogrel has similar effects would help the reader understand. Or removing entirely if you think this is appropriate? I think your results stand for themselves and do not require a positive control, unless you can reference papers that have shown equivalent results.

The methods sections are missing major details that prevent the reader from understanding the paper. For example:

Section 2.3 Timing of the treatment is unclear: it states that BA was given both prior to lesion, and post

“Evaluating animal status after MCAO surgery, we applied two doses of BA (25, 50 mg/kg)”

AND

“animals were given clopidogrel or BA …. once a day for one week prior to the ischemia induction.”

Same for timing of the procedures. Ie. When was serum taken for ELISAs?

Section 2.10. An explanation of how the cells were counted should be provided, as well as your defined region of interest. Ie. Are you looking at the CA1? How many sections/mouse? Is this performed through neurostereology methods? And how is this quantified? Importantly, I’m assuming you are not counting every neuron in the hippocampus, so how is Figure 6 calculated? This is given as a percentage, but what is it compared to?
Section 2.11. please explain how samples were extracted. What kind of extraction buffer (and its contents) were used? Also, describe that this is done for both serum and brain.
Section 2.12. Differences between multiple groups should be tested with a one-way ANOVA with a post hoc test. A students t-test is insufficient.

Results:

In the text, in vitro results are said to be compared to “control treated rats” (same in Fig 4 and 5 legends). I’m assuming this should say “control treated cells”. Cultured cell data should not be compared to animal models.
Figure 4: OGD/R+BA groups should be compared to OGD/R, and not Would be interesting to note – after appropriate statistics have been performed - that 40uM seems to be the most effective dose?

Minor:

Introduction:

Stroke should be introduced as a cause of cerebra ischemia in the first sentence of the paper as this is the cause of cerebral ischemia (and your animal model) that you are investigating.
First time usage of abbreviations should be defined. Ie. AMPK, PPARa
Line 38 “MCAO is an acknowledged animal model of neuroprotection research” should be amended to “MCAO is an acknowledged animal model of cerebral ischemia”
Line 44: References are only for anti-cancer properties. More are needed for its anti-viral/malarial/microbial properties.

Methods sections is missing further (minor) points required for clarification

Section 2.3 although this is titled MCAO, there is no description of the surgery, or animal care e.g. anaesthetic and dose used
Section 2.3, it is unclear what the ‘negative control group’ is. Is this a sham group (surgery but no occlusion)? Or a ‘no surgery’ group? Please explain what that has entailed.
Section 2.5. Details on how the rats were sacrificed are needed (i.e. overdose?)
Section 2.6. what are you referring to by ‘cerebral tissue’? Is this whole brain?
Section 2.7. This sentence makes it sound like you dissolved the cells in bovine serum. Please rephrase. “The cells were incubated with BA at multiple concentrations (10, 20, 40, 80, 160, or 320µM) and dissolved in bovine serum for 1 h.”
Primary antibodies for both immunohistochemical and western blot staining should be listed.

Results:

Referencing individual figure panels (i.e. Fig 2.A) throughout the text would make it easier for the reader to follow.
Line 97: 5x104 should be 5x10⁴
Figure 2: can panel A be enlarged, the writing and images are too small to see. Fig.2C, title of y axis is spelt incorrectly. Legend: remove ‘Schem’
Title of section 3.2 should be changed to include the result (like you have done for 3.1 and 3.3).
In vivo and in vitro should be italicized throughout the text
Doses of 5, 10, 20 and 40 uM are mentioned in the text, but not 80, 160 and 320 uM
Figure 6. Could you orient the pictures in the same direction for better comparison? And the graph will have to be labeled panel ‘G’ and described in the legend.
Figure 7 and 8, blots and graphs should be listed in the same order.
The western blot for IkBa: is the antibody a pan-antibody? Why is it decreased with I/R but the phospho form increased?
The numbers of animals in each group should be indicated in every figure (e.g. in the bars)

Discussion:

Wording: Clopidogrel is not an intervention, it is a preventative. This is noted in the reference you provide.
Line 432: “high doses of BA demonstrated equivalent neuroprotective benefits” is incorrect. Fig 4 shows that 40 uM (middle range) might be more effective (or at least for cell viability assays).
Line 469: “MACO” should be “MCAO”
Line 475-476 “regulates expression of Il-6” but I cannot find this in the papers referenced.
Line 485-487: “our study revealed that brain levels of BA in the control and I/R groups were not detectable until 24 h after BA administration (data not shown).” This data should be included in the figures or else should be removed as we cannot judge it for accuracy. Also, if BA only remains detectable for 24 hours after treatment, it would be important to provide an accurate timeline of when BA was given last, when surgery was performed, when behavior was performed, and how long after this rats were sacrificed.

Author Response

Dear Editors and Reviewers,

Thanks for your comments on our manuscript (applsci-753267, Title: Protective Action of Betulinic Acid on Cerebral Ischemia/Reperfusion Injury through Inflammation and Energy Metabolic Homeostasis). All changes were highlighted in manuscript and responses to reviewers’ comments were included in this cover letter. We revised point to point according to instructions as follows:

Reviewer1

Several major and minor issues need to be addressed prior to publication.

Major:

Introduction:

I’m not sure that clopidogrel is an appropriate positive control as it is used as a preventative for future stroke and to mitigate lifestyle factors (which is not a problem with rodent models as their strokes are induced). Please do not refer to it as an ‘intervention’ in stroke as it is not a treatment. Perhaps referencing previous literature in which clopidogrel has similar effects would help the reader understand. Or removing entirely if you think this is appropriate? I think your results stand for themselves and do not require a positive control, unless you can reference papers that have shown equivalent results.

Answer: Thanks for your comment. As mentioned, it was true that clopidogrel was preventative drug in cerebral ischemia. In clinic, Long-term administration of clopidogrel to patients with atherosclerotic vascular disease is effective in reducing the risk of ischaemic stroke ( Lancet,1996,348(9038): 1329-39.). In MCAO rat, clopidogrel also showed some beneficial effects on cerebral ischemia injury in rats.

Thrombosis Research, 1995, 80(3), 209-216.
Biomed Research International, Volume 2016, Article ID 1859254.
Traditional Chinese Drug Research and Clinical Pharmacology, 2015, 26( 4),451-455. (Influence of Buyang Huanwu Decoction Pretreatment on Expression of Akt Phosphorylation Level in Cerebral Ischemia-reperfusion Rats).

In our study, betulinic acid was also used as a preventative drug. The animals were given clopidogrel or betulinic acid once a day for one week prior to the ischemia induction by MCAO. It was true that there was no definite positive drug for MCAO model in pretreatment regime. Clopidogrel was selected as a comparative drug in our study, so we replaced the word "positive" with " comparative " in text. We also revised ‘intervention’ in text.

The methods sections are missing major details that prevent the reader from understanding the paper. For example:

Section 2.3 Timing of the treatment is unclear: it states that BA was given both prior to lesion,

and post “Evaluating animal status after MCAO surgery, we applied two doses of BA (25, 50 mg/kg)”

AND “animals were given clopidogrel or BA …. once a day for one week prior to the ischemia induction.”

Same for timing of the procedures. Ie. When was serum taken for ELISAs?

Answer: Thanks for your comment. We didn't make timing clearly in the Section 2.3. We employed the pretreatment regime to investigate the effects of betulinic acid (25, 50 mg/kg) on cerebral ischemia/reperfusion. Firstly, the animals were given clopidogrel or betulinic acid (25, 50 mg/kg) once a day for one week prior to the ischemia induction. Secondly, on the 7th day, MCAO was performed 2h after the last drug administration. Thirdly, after 2h occlusions in MCAO, reperfusion was induced by removal of the filament. Finally, after 24 h occlusion (after 22 h reperfusion), the serum and brain were sampled, and then the effect of betulinic acid on ischemic/reperfusion injury were evaluated by different indexes. We have revised in text.

Section 2.10. An explanation of how the cells were counted should be provided, as well as your defined region of interest. Ie. Are you looking at the CA1? How many sections/mouse? Is this performed through neurostereology methods? And how is this quantified? Importantly, I’m assuming you are not counting every neuron in the hippocampus, so how is Figure 6 calculated? This is given as a percentage, but what is it compared to?

Answer: Thanks for your comment. We used neurostereology method to visualize the sections of CA1 (n=5 sections /animal) under light microscope (OlympusBX50, Japan) in a blinded manner. We quantified the HIF- 1α positivity of immunohistochemical staining with the Image J software as it was recommended by instructions. In general, semi-quantitative analysis of immunohistochemical staining was used for positive neurons in hippocampus. Pale yellow to tan cytoplasm dyeing represented the HIF-1 positive neurons in hippocampus. The percentage of Figure 6 was approximately calculated by comparing the staining gray level in different treatment groups (panel B,C,D,E,F) with that in no primary antibody group (panel A). The staining depth of no primary antibody group(panel A) was the background. We have revised in text.

Section 2.11. please explain how samples were extracted. What kind of extraction buffer (and its contents) were used? Also, describe that this is done for both serum and brain.

Answer: Thanks for your comment. The brain samples and SH-SY5Y cells were extracted by Radio Immunoprecipitation Assay (RIPA) buffer. The buffer include 50mM Tris (pH 7.4), 150mM NaCl, 1% NP-40, and 0.5% sodium deoxycholate. First of all, the brain samples and SH-SY5Y cells were lysed in a RIPA buffer (Sigma, St.Louis, MO, USA). After centrifugation at 12,000 rpm for 10 min, the supernatants were harvested for the protein concentration measurement using a bicinchoninic acid assay. Aliquots of protein (40 μg/lane) were separated SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Boston, MA, USA). We have already added in text.

Section 2.12. Differences between multiple groups should be tested with a one-way ANOVA with a post hoc test. A students t-test is insufficient.

Answer: The data were expressed as mean values ± SDs. Data analyses were compared by one-way ANOVA with Tukey multiple comparison test again. We have already revised it in text.

Results:

In the text, in vitro results are said to be compared to “control treated rats” (same in Fig 4 and 5 legends). I’m assuming this should say “control treated cells”. Cultured cell data should not be compared to animal models.

Answer: Thanks for your comment. We have revised it in text.

Figure 4: OGD/R+BA groups should be compared to OGD/R, and not Would be interesting to note – after appropriate statistics have been performed - that 40uM seems to be the most effective dose?

Answer: Thanks for your comment. We did not describe the results in Figure 4 clearly. We selected a relatively wide concentration range for cell viability study. The results showed that cell viability could be reduced seriously in hypoxia, but the decrease of cell viability caused by hypoxia could be alleviated by betulinic acid. However, the cell viability at high concentration of betulinic acid was lower than that at medium concentration of betulinic acid (40uM), which may be due to the cytotoxicity of high concentration betulinic acid itself, which directly led to the decrease of cell viability.Therefore, at the cell level, betulinic acid at 0-40 uM has a certain protective effect on OGD/R cells, while the protective effects were decreased at high concentrations. So the concentration range of betulinic acid (10-40uM) was selected in following cell studies. Additionally, the data of OGD/R+BA groups was compared to that of OGD/R. We have added it in text.

Minor:

Introduction:

Stroke should be introduced as a cause of cerebra ischemia in the first sentence of the paper as this is the cause of cerebral ischemia (and your animal model) that you are investigating.

Answer: The pathological background for stroke may either be ischemic or hemorrhagic disturbances of the cerebral blood circulation. We have already added in text.

First time usage of abbreviations should be defined. Ie. AMPK, PPARa

Answer: We have already added in text.

Line 38 “MCAO is an acknowledged animal model of neuroprotection research” should be amended to “MCAO is an acknowledged animal model of cerebral ischemia”

Answer: We have already revised in text.

Line 44: References are only for anti-cancer properties. More are needed for its anti-viral/malarial/microbial properties.

Answer: We have already added in text.

Methods sections is missing further (minor) points required for clarification

Section 2.3 although this is titled MCAO, there is no description of the surgery, or animal care e.g. anaesthetic and dose used

Answer: Thanks for your comment. 2 h after the last drug treatment, the rats were intraperitoneally anesthetized and subjected to a protocol of MCAO. Briefly, the internal carotid artery (ICA), right common carotid artery, and external carotid artery (ECA) of individual rats were surgically exposed. A monofilament nylon suture with silicon coated tip (Beijing Sunbio Biotech, Beijing, China) was inserted into the ICA through the ECA stump and gently advancing to the middle cerebral artery (MCA) to occlude the origin of middle carotid artery (MCA). After 2h occlusion, reperfusion was induced by removal of the filament and then the skin was sutured. Simultaneously, the control animals received a sham surgery. The core body temperature of each rat was maintained at 37±0.5 °C during the whole experiment with a thermostatically controlled infrared lamp. Sham-operated rats received the same surgical exposure procedures without occlusion of MCA. We have already added description in text.

Section 2.3, it is unclear what the ‘negative control group’ is. Is this a sham group (surgery but no occlusion)? Or a ‘no surgery’ group? Please explain what that has entailed.

Answer: Negative control group is sham group. We have already revised it in text.

Section 2.5. Details on how the rats were sacrificed are needed (i.e. overdose?)

Answer: Animals were sacrificed by cervical dislocation. We have already added it in text.

Section 2.6. what are you referring to by ‘cerebral tissue’? Is this whole brain?

Answer: Cerebral tissue is whole brain. We have already revised in text.

Section 2.7. This sentence makes it sound like you dissolved the cells in bovine serum. Please rephrase. “The cells were incubated with BA at multiple concentrations (10, 20, 40, 80, 160, or 320µM) and dissolved in bovine serum for 1 h.”

Answer: Thanks for your comment. The cells were incubated with BA at multiple concentrations (5,10, 20, 40, 80, 160, and 320μM) and dissolved in culture medium for 1 h. We have already revised in text.

Primary antibodies for both immunohistochemical and western blot should be listed.

Answer: The primary antibody against HIF-1α was used in immunohistochemical staining. The primary antibodies against HIF-1α, AMPK, PPARα, PGC-1α, IκBα, P-IκBα, NF-kBP65, and P-NF-kBP65 were used in western blot. All antibodies were provided by Cell Signaling Technology (Danvers, USA). We have already added in text.

Results:

Referencing individual figure panels (i.e. Fig 2.A) throughout the text would make it easier for the reader to follow.

Answer: We have already revised in text.

Line 97: 5x104 should be 5x10⁴

Answer: We have already revised in text.

Figure 2: can panel A be enlarged, the writing and images are too small to see.

Fig.2C, title of y axis is spelt incorrectly. Legend: remove ‘Schem’

Answer: We have already revised Figure 2.

Title of section 3.2 should be changed to include the result (like you have done for 3.1 and 3.3).

Answer: We have already revised it in text.

In vivo and in vitro should be italicized throughout the text

Answer: We have already revised in text.

Doses of 5, 10, 20 and 40 uM are mentioned in the text, but not 80, 160 and 320 uM

Answer: We have already added some information in text. We selected a relatively wide concentration range for cell viability study. The results showed that cell viability could be reduced seriously in hypoxia, but the decrease of cell viability caused by hypoxia could be alleviated by betulinic acid. However, the cell viability at high concentration of betulinic acid (80, 160 and 320 uM) was lower than that at medium concentration of betulinic acid, which might be due to the cytotoxicity at high concentration betulinic acid itself, which directly led to the decrease of cell viability. We have already added in text.

Figure 6. Could you orient the pictures in the same direction for better comparison? And the graph will have to be labeled panel ‘G’ and described in the legend.

Answer: We have already revised Figure 6 in text.

Figure 7 and 8, blots and graphs should be listed in the same order.

Answer: We have already revised Figure 7 and 8.

The western blot for IkBa: is the antibody a pan-antibody? Why is it decreased with I/R but the phospho form increased?

Answer: IkBa antibody is a pan-antibody. IκBα is an inhibitor of NF-κB-p65. In normal state (inactive), both IkBa and NF-κB were nonphosphorylated in the form of NF-κB-IkBa complex. IkBa inhibited the phosphorylation of NF-κB, so the expression of IkBa and NF-κB was high, while the expression of phosphorylated IkBa and phosphorylated NF-κB was low. But in I/R injury state (active), IkBa in the NF-κB-IkBa complex was phosphorylated by upstream signal factors, and then the free NF-κB was also phosphorylated, which led to the decreased expression of IkBa and NF-κB and the increased expression of phosphorylated IkBa and phosphorylated NF-κB.

The numbers of animals in each group should be indicated in every figure (e.g. in the bars)

Answer: We have already added it in Figure legends.

Discussion:

Wording: Clopidogrel is not an intervention, it is a preventative. This is noted in the reference you provide.

Answer: We have already revised it in text.

Line 432: “high doses of BA demonstrated equivalent neuroprotective benefits” is incorrect. Fig 4 shows that 40 uM (middle range) might be more effective (or at least for cell viability assays).

Answer: We have already revised it in text.

Line 469: “MACO” should be “MCAO”

Answer: We have already revised it in text.

Line 475-476 “regulates expression of Il-6” but I cannot find this in the papers referenced.

Answer: We have already revised the sentence in text.

Line 485-487: “our study revealed that brain levels of BA in the control and I/R groups were not detectable until 24 h after BA administration (data not shown).” This data should be included in the figures or else should be removed as we cannot judge it for accuracy. Also, if BA only remains detectable for 24 hours after treatment, it would be important to provide an accurate timeline of when BA was given last, when surgery was performed, when behavior was performed, and how long after this rats were sacrificed.

Answer: We have already revised in text. We revealed that brain level of BA (50 mg/kg) was 20.5 ng/(g brain tissue) at 24 h in MCAO rats, while the plasma concentration of BA (50 mg/kg) was 29.33 ng/ml in previous pre-experiments.

Author Response File: Author Response.docx

Reviewer 2 Report

Stroke is a world problem and we need as much research as possible to improve the outcome. The authors have tested the neuroprotective properties of betulinic acid. The study has potential and I will like it, however some problems with the manuscripts can not be overlooked.

My major concern is that some experimental data is missing:

I was not able to find the number of animals used (in total and per each tested group), there age and gender.
When the authors talk about the MCAO they do not say which variant they used, eg: what was the time of ischemia 30 minutes, 60, 120?
I assume the serum was obtained at 24 h after MCAO, right? there is not mention of this data point.
IHC description is not sufficient in the Materials and method section. Name of the antibody, concentration and provider should be provided.
please show the data you are referring in line 486, one should not just say there data is different form existing studies without showing it.

Some minor problems:

I was not able to find the numbers referred in the first paragraph in the cited articles, I would suggest the authors use the WHO data (https://www.who.int/healthinfo/statistics/bod_cerebrovasculardiseasestroke.pdf). the phrase at the 38 line "Middle cerebral ...." is confusing. MCAO is a stroke model and can be also use to asses neuroprotection.

Suggestions:

To improve the quality of the manuscript I will think the authors need to take a step back and reorganize it. E.g. section 2.5 and 2.6 could be one, 3.2 is extremely confusing please rephrase. The panels in Figure 1 have different markings, please use the same, for the same subgroup. Also there infarct size has not letter given to it and the SHAM is missing (it will be 0 but if you added it in panel B one should add it everywhere).

Please also compare results from BA with Clopidogrel group.

Author Response

Dear Editors and Reviewers,

Reviewer2

I was not able to find the number of animals used (in total and per each tested group), there age and gender.

Answer: Male adult Sprague-Dawley rats were assigned into five groups (n = 12 per group). We have already added it in text.

When the authors talk about the MCAO they do not say which variant they used, eg: what was the time of ischemia 30 minutes, 60, 120?

Answer: We have already added these information in text. MACO was operated as follows: Briefly, the internal carotid artery (ICA), right common carotid artery, and external carotid artery (ECA) of individual rats were surgically exposed. A monofilament nylon suture with silicon coated tip (Beijing Sunbio Biotech, Beijing, China) was tinserted into the ICA through the ECA stump and gently advancing to the middle cerebral artery (MCA) to occlude the origin of middle carotid artery (MCA). After 2h occlusion, reperfusion was induced by removal of the filament and then the skin was sutured. Simultaneously, the control animals received a sham surgery. The core body temperature of each rat was maintained at 37 ± 0.5 °C during the whole experiment with a thermostatically controlled infrared lamp. Sham-operated rats received the same surgical exposure procedures without occlusion of MCA.

I assume the serum was obtained at 24 h after MCAO, right? there is not mention of this data point.

Answer: The serum was obtained at 24 h after MCAO. We have already added it in text.

IHC description is not sufficient in the Materials and method section. Name of the antibody, concentration and provider should be provided.

Answer: The primary antibody against HIF-1α was used in immunohistochemical staining. All antibodies were provided by Cell Signaling Technology (Danvers, USA). HIF-1α antibody (1mg/ml) were diluted by 1:100. We have already added it in text.

please show the data you are referring in line 486, one should not just say there data is different form existing studies without showing it.

Answer: We revealed that brain level of BA (50 mg/kg) was 20.5 ng/(g brain tissue) at 24 h in MCAO rats, while the plasma concentration of BA (50 mg/kg) was 29.33 ng/ml in previous pre-experiments. We have already added it in text.

Some minor problems:

Answer: We have already revised the sentences and cited the WHO data in text.

the phrase at the 38 line "Middle cerebral ...." is confusing. MCAO is a stroke model and can be also use to asses neuroprotection.

Answer: Thanks for your comment. We have already revised it in text.

Suggestions:

Answer: Thanks for your comment. We have already revised section 2.5 , 2.6 and 3.2 in text. We revised Figure1 and Figure2 in text. The structure of betulinic acid has different molecular conformations, so there are different dotted lines and solid lines in Fig. 1. There are photos of brain infarction (Figure2A), so we deleted the infarction size computed by software.

Please also compare results from BA with Clopidogrel group.

Answer: Thanks for your comment. We have already added the comparison results from BA with Clopidogrel group in text.

Author Response File: Author Response.docx

Reviewer 3 Report

In section 2.3 it is unclear whether the middle cerebral artery occlusion is unilateral or bilateral. Unilateral may have offered an opportunity to compare sides of the brain. The duration of the occlusion and whether it was single or multiple is also not described.

It is unclear why the researchers looked at the effects of clopidogrel and different doses of BA only as independent interventions and not as a combined intervention too. Was the reperfusion in room air or 100% oxygen.

It is unclear why the cell culture OGPD/R part of the study was performed from the introduction. Was this to provide samples for IL-6, IL-8 and TNF and signalling etc? If so this should be clearly stated How do we interpret the cell culture model with respect to the rat model? Why should they be seen as similar?

Lines 439-443 are a little redundant and clumsy by making such a long sentence. Clarity would be improved by breaking this into a few sentences.

There is are typos in lines 444 and 490

Author Response

Dear Editors and Reviewers,

Reviewer3

Answer: Thanks for your comment. In our study, middle cerebral artery occlusion was unilateral. MCAO surgery was complex and had a lot of trauma, so it was difficult to compare the left and right brain injuries in the same animal at the same time. The duration of the occlusion is 2h.

Answer: Thanks for your comment. This study was to investigate the protective effect of BA on cerebral ischemia injury. Clopidogrel was only used as a comparative drug of BA for comparative study. The combination of the two drugs has not been considered in this study. After 2h occlusion, reperfusion was induced by removal of the filament and then the blood flow was recovered.

Answer: Thanks for your comment. This OGD/R cell culture known as in vitro ischemia model can simulate cerebral ischemia model in vivo. The cells were transferred to an anaerobic chamber supplemented with 1% O₂, 5% CO₂, and 94% N₂ for 4 h under glucose deprivation. We have already added it in introduction.

Lines 439-443 are a little redundant and clumsy by making such a long sentence. Clarity would be improved by breaking this into a few sentences.

Answer: We have already revised the sentences.

There is are typos in lines 444 and 490

Answer: We have already revised it in text.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Major English revisions are required before publication.

Antibody concentrations should be included (as has been given for HIF-1 in the IHC section)

Fig 6 needs a scale bar. Also I still don’t understand: HIF-1 positive neurons are expressed as a percentage of what? Total neurons in the CA1 region? There is no description of how the total number of neurons are stained (and therefore counted). At the moment only HIF-1 neurons are stained and counted. So you can't calculate a %.

Fig 4 currently shows me control vs. OGD/R +/-BA. Your statistics and graph do not show me the effect of BA on OGD/R induced death (despite this being said in the text): “Upon OGD/R induction, SHSY5Y cell proliferation was considerably reduced. Intervention with BA (5, 10, 20, and 40 µM) presented effective cell viability improvement under OGD/R in vitro (Figure 4).” - This is not what your figure shows

Also, 80, 160 and 320 show same viability as 10uM. So I don’t understand this sentence: “However, the cell viability at high concentration of BA (80, 160, and 320 µM) is lower than that at medium concentration of BA (40 µM), which may be due to the cytotoxicity of high concentration BA itself, which directly leads to the decrease of cell viability.”

Fig 7. What is @@P<0.01 in the legend? This is not in the figures

Name of anesthetic used and dose (per g bodyweight) are needed.

In vivo and in vitro should be italicized throughout the text.

Abstract needs correcting, you have: “in vivo and in vivo”

I asked for these corrections last time but they were not addressed:

Figure 6. Could you orient the pictures in the same direction for better comparison? And the graph will have to be labeled panel ‘G’ and described in the legend.

Figure 7 and 8, blots and graphs should be listed in the same order.

The numbers of animals in each group should be indicated in every figure (e.g. in the bars)

Line 455: “our study revealed that brain levels of BA in the control and I/R groups were not detectable until 24 h after BA administration (data not shown).” This data should be included in the figures or results (not in the discussion) or else should be removed as we cannot judge it for accuracy

Author Response

Dear Editors and Reviewers,

Thanks for your comments on our manuscript (applsci-753267, Title: Protective Action of Betulinic Acid on Cerebral Ischemia/Reperfusion Injury through Inflammation and Energy Metabolic Homeostasis). Now, all changes were highlighted with blue in manuscript and responses to reviewers’ comments were included in this cover letter. We revised point to point according to instructions as follows:

Major English revisions are required before publication.

Antibody concentrations should be included (as has been given for HIF-1 in the IHC section)

Answer: We have added antibody concentrations in text.

Answer: We have added a scale bar in Figure 6 and some related descriptions in text. The HIF-1 positive cells were stained as pale yellow to tan cytoplasm. The numbers of positive cells in different groups were obtained by counting the cells in six randomly selected microscopic fields, respectively. The percentage of positive cells in each field was calculated and the average percentage in all six fields was obtained. No primary antibody group was only background to verify the reliability of staining method. We deleted no primary antibody group in Figure 6 and revised the Figure6 again.

Answer: The data of OGD/R+BA groups was compared to that of OGD/R in figure4. We have revised it in figure4. We selected a relatively wide concentration range for cell viability study. Figure 4 showed that cell viability could be reduced in hypoxia, but the decrease of cell viability caused by hypoxia could be alleviated by BA at the concentrations (5, 10, 20, and 40 µM). Intervention with BA (80, 160 and 320 uM) show same viability as 10uM, lower than that with 20 uM and 40 uM. This may be due to BA’s cytotoxicity at high concentration (80, 160, and 320 µM). If the data (80, 160 and 320 µM) made readers confused, we can remove the data at high concentration (80, 160, and 320 µM). We removed this sentence: “However, the cell viability at high concentration of BA (80, 160, and 320 µM) is lower than that at medium concentration of BA (40 µM), which may be due to the cytotoxicity of high concentration BA itself, which directly leads to the decrease of cell viability.”

Fig 7. What is @@P<0.01 in the legend? This is not in the figures

Answer: The original Figure 7 was replaced by updated Figure 7.

Name of anesthetic used and dose (per g bodyweight) are needed.

Answer: Rats were anesthetized by an intraperitoneal injection of 10% chloral hydrate (3.5 mL/kg).

In vivo and in vitro should be italicized throughout the text.

Answer: We have revised it in text.

Abstract needs correcting, you have: “in vivo and in vivo”

Answer: We have revised it in text.

I asked for these corrections last time but they were not addressed:

Answer: In the latest version of the manuscript for revision, we uploaded our correct revision in online submission system, but we don't know why Figure 1, Figure 3, Figure 4, Figure 6, Figure 7 and Figure 8 were not updated in the process of system transformation. We have updated these figures in this time again.

Figure 6. Could you orient the pictures in the same direction for better comparison? And the graph will have to be labeled panel ‘G’ and described in the legend.

Answer: We have revised Figure 6 in text.

Figure 7 and 8, blots and graphs should be listed in the same order.

Answer: We have updated the Figure 7 and 8 in text.

The numbers of animals in each group should be indicated in every figure (e.g. in the bars)

Answer: We have added it in figures.

Answer: We have removed the sentence in text.

Author Response File: Author Response.pdf

Reviewer 2 Report

Just some minor points:

-line 134-135: "; Then The sections ..." should be reviewed

-line 178: "**P<0.01).." should be reviewed

-line 218 the authors added "(@P<0.05)." but no @ can be found in the figure, the same for line 342.

Figure 6, please add a scale bar

and some suggestion:

If the authors want, they could also use the summary of the WHO report (https://www.thelancet.com/action/showPdf?pii=S1474-4422%2819%2930034-1).

Less than 20% of all ischemic strokes are reperfused. In my opinion stroke research should be done on both permanent and reperfused animals, as the results will greatly vary.

Author Response

Dear Editors and Reviewers,

Thanks for your comments on our manuscript (applsci-753267, Title: Protective Action of Betulinic Acid on Cerebral Ischemia/Reperfusion Injury through Inflammation and Energy Metabolic Homeostasis) Now, all changes were highlighted with blue in manuscript and responses to reviewers’ comments were included in this cover letter. We revised point to point according to instructions as follows:

Just some minor points:

-line 134-135: "; Then The sections ..." should be reviewed

Answer: We have revised it in text.

-line 178: "**P<0.01).." should be reviewed

Answer: We have revised it in text.

-line 218 the authors added "(@P<0.05)." but no @ can be found in the figure, the same for line 342.

Answer: The original figure was replaced by updated figure.

Figure 6, please add a scale bar

Answer: We have added a scale bar in text.

and some suggestion:

If the authors want, they could also use the summary of the WHO report (https://www.thelancet.com/action/showPdf?pii=S1474-4422%2819%2930034-1).

Answer: We have added some summary of the WHO reports in discussion.

Less than 20% of all ischemic strokes are reperfused. In my opinion stroke research should be done on both permanent and reperfused animals, as the results will greatly vary.

Answer: Thanks for your suggestion on our experiment ideal. Our study on stroke will be focused on permanent animals in future.

Author Response File: Author Response.pdf

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Protective Action of Betulinic Acid on Cerebral Ischemia/Reperfusion Injury through Inflammation and Energy Metabolic Homeostasis

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