Probiotics-Derived Extracellular Vesicles Protect Oxidative Stress against H₂O₂ Induction in Placental Cells

Round 1

Reviewer 1 Report

Wang et al found that the probiotics-derived extracellular vesicles protect senescence against H2O2-induced oxidative stress in placental cells. During the review, several major problems need to be concerned.

In Line 156, In Figure 1, the author found that H2O2 (500 μM) induction can reduce the cell survival in 3A-sub-E placental cells. However, whether this effect was significant did not show in Figure 1.

In line 158, the author declared that “These results indicated that oxidative stress could result in cell degeration.”  “Cell degeration” did you mean cell degeneration?

This declaration is not proper. First, the author did not show results (such as ROS) which confirmed the oxidative stress induced by H2O2. Second, reduced cell survival was not enough to evidence cell degeneration.

The legend of Figure 3A and 3B was not correct. In addition, the morphology of L. crispatus- derived EVs by transmission electron microscope was not marked in Figure 3A. I can not tell which is L. crispatus and which is L. crispatus- derived EVs from Figure 3A.

In Figure 2, a significant effect of H2O2 on pAKT was shown, however, in Figure 6, the effect of H2O2 on pAKT was not significant. This effect was inconsistent. 

Author Response

Wang et al found that the probiotics-derived extracellular vesicles protect senescence against H2O2-induced oxidative stress in placental cells. During the review, several major problems need to be concerned.

In Line 156, In Figure 1, the author found that H2O2 (500 μM) induction can reduce the cell survival in 3A-sub-E placental cells. However, whether this effect was significant did not show in Figure 1.

ANS: We have rewritten the sentence in lines 168-173.

In line 158, the author declared that “These results indicated that oxidative stress could result in cell degeration.”  “Cell degeration” did you mean cell degeneration?

ANS: We have corrected it in line 173.

This declaration is not proper. First, the author did not show results (such as ROS) which confirmed the oxidative stress induced by H2O2. Second, reduced cell survival was not enough to evidence cell degeneration.

ANS: We have added some description about placental degeneration in line 168-173.

The legend of Figure 3A and 3B was not correct. In addition, the morphology of L. crispatus- derived EVs by transmission electron microscope was not marked in Figure 3A. I can not tell which is L. crispatus and which is L. crispatus- derived EVs from Figure 3A.

ANS: We have corrected it.

Reviewer 2 Report

- The introductory part on the effects of IVs and host-organism relationships needs to be modified to make it more concise and clear.
- The effects of the treatment with H2O2 500uM can be relevant on the cellular redox balance, the authors should test not only the effect in fluorescence but also the functional alteration of the enzymes involved such as GPX.
- The discussion of the results is extremely concise, a results section before the discussion would make the description more linear.
- The blots of the presented WBs should be accompanied by reference markers and pm of the band.
- The text is to be revised due to the presence of numerous typing errors and absent punctuation.

Author Response

- The introductory part on the effects of IVs and host-organism relationships needs to be modified to make it more concise and clear.

ANS: We have introduced function of EVs regulated host in lines 75-82.

- The effects of the treatment with H2O2 500uM can be relevant on the cellular redox balance, the authors should test not only the effect in fluorescence but also the functional alteration of the enzymes involved such as GPX.

ANS: Thanks for reviewers comment. The role of EVs regulate mitochondrial function and oxidative stress is major specific aim in this article.

- The discussion of the results is extremely concise, a results section before the discussion would make the description more linear.

ANS: We have re-written in full article.

- The blots of the presented WBs should be accompanied by reference markers and pm of the band.

ANS: Thanks for reviewers comment.

- The text is to be revised due to the presence of numerous typing errors and absent punctuation.

ANS: We have corrected errors in full article.

Reviewer 3 Report

It has long been known that probiotics (especially lactic acid bacteria) have a beneficial effect on the female reproductive system. However, little is known about the mechanisms by which bacteria interact with human cells. Recent studies indicate that membrane vesicles secreted by bacterial cells into the environment, known as EVs, may make a significant contribution to these intercellular interactions. Therefore, it is worth studying it in detail. In this manuscript, the authors follow this trend. However, I have many reservations about the presented study, which are listed below.

1. In line 106, "PKH25" should not be changed to "PKH26"?
2. Regarding subchapter 2.3. There is a lack of technical description of the EVs` concentration determination. Is it possible to calculate the EVs` concentration using DLS? Maybe NTA analysis would be more appropriate? Furthermore in lines 185-186 authors wrote “The physicochemical characterization of these EVs was performed using a nanoparticle tracking analysis technique”. So, was DLS or NTA applied?Were the DNA, RNA and proteins somehow released or extracted from EVs prior the determination of its concentration in EVs? Please clarify this in the manuscript.
3. In the description of Western blot (point 2.6), there should be information about: How many times were primary antibodies diluted?Were membranes incubated at RT or other temperature? Where were the HRP-linked secondary antibody purchased from, and how many times diluted? Besides, there is no information how were calculated values presented in Fig. 2
4. In line 150, there should be "PBC" or rather "PBS"?
5. In lines 151-152, there should be a technical description of confocal microscope and parameters set up during observations.
6. The title of the subchapter 3.1. "Senescence induction by oxidative stress in 3A-sub-E placental cells treated with H2O2 " is not precise, because authors did not studied the senescence induction but the oxidative stress caused by H2O2
7. Authors should give a description of transmission electron microscopy (TEM) methodology including how were the samples prepared.
8. Fig. 3A is unclear, the EVs and the scale bar value are not well visible.
9. In Figures 5 - 6, there are given EVs concentrations. It is not clear, whether these values mean the final concentrations of EVs in cell cultures or the concentrations of EVs in the samples added to the cell cultures?
10. In Fig. 5 the scale bar value is invisible
11. In lines 189-190 authors wrote “We further stained EVs with PKH26 (red fluorescent stain) and observed them entering the cells with confocal microscopy. As presented in Figure 5”. The photo does not show whether the EVs have entered the cells or not.
12. In lines 199-200 authors wrote “The mitochondrial fission caused by H2O2 induction was attenuated by treatment with crispatus-derived EVs (10^10 particles/mL) for 24 h (Figure 8).” The photos do not show that.

Author Response

1. In line 106, "PKH25" should not be changed to "PKH26"?

ANS: We have corrected it.

1. Regarding subchapter 2.3. There is a lack of technical description of the EVs` concentration determination. Is it possible to calculate the EVs` concentration using DLS? Maybe NTA analysis would be more appropriate? Furthermore in lines 185-186 authors wrote “The physicochemical characterization of these EVs was performed using a nanoparticle tracking analysis technique”. So, was DLS or NTA applied?Were the DNA, RNA and proteins somehow released or extracted from EVs prior the determination of its concentration in EVs? Please clarify this in the manuscript.

ANS: We have corrected it.

1. In the description of Western blot (point 2.6), there should be information about: How many times were primary antibodies diluted?Were membranes incubated at RT or other temperature? Where were the HRP-linked secondary antibody purchased from, and how many times diluted? Besides, there is no information how were calculated values presented in Fig. 2

ANS: We have corrected it.

1. In line 150, there should be "PBC" or rather "PBS"?

ANS: We have corrected it.

1. In lines 151-152, there should be a technical description of confocal microscope and parameters set up during observations.

ANS: We have corrected it.

1. The title of the subchapter 3.1. "Senescence induction by oxidative stress in 3A-sub-E placental cells treated with H2O2 " is not precise, because authors did not studied the senescence induction but the oxidative stress caused by H2O2

ANS: We have corrected it.

1. Authors should give a description of transmission electron microscopy (TEM) methodology including how were the samples prepared.

ANS: We have described it in section 2.9.

1. Fig. 3A is unclear, the EVs and the scale bar value are not well visible.

ANS: Thank for reviewer’s comment.

1. In Figures 5 - 6, there are given EVs concentrations. It is not clear, whether these values mean the final concentrations of EVs in cell cultures or the concentrations of EVs in the samples added to the cell cultures?

ANS: It is final concentrations. We have improved it.

1. In Fig. 5 the scale bar value is invisible

ANS: Thank for reviewer’s comment.

1. In lines 189-190 authors wrote “We further stained EVs with PKH26 (red fluorescent stain) and observed them entering the cells with confocal microscopy. As presented in Figure 5”. The photo does not show whether the EVs have entered the cells or not.

ANS: We have improved it.

1. In lines 199-200 authors wrote “The mitochondrial fission caused by H2O2 induction was attenuated by treatment with crispatus-derived EVs (10^10 particles/mL) for 24 h (Figure 8).” The photos do not show that.

ANS: It is clearly induced mitochondrial fission by H2O2. And attenuation of mitochondrial fission was markedly inhibited by EVs treatment.

Round 2

Reviewer 3 Report

The revised manuscript does not include all comments, including those listed below.

1. In the description of Western blot (point 2.6), there should be information about: Were membranes incubated at RT or other temperature?  Besides, there is no information how were calculated values presented in Fig. 2
2. There should be a technical description of confocal microscope and parameters set up during observations.
3. Fig. 3A and Fig. 5  the scale bars` values are not well visible. Besides in Fig. 8, there is no scale bar
4.  Figure 5 does not show whether the EVs have entered the cells or not. Authors should have taken photos under higher magnification, the same as in Fig. 8, and it would be more clear.

The title of manuscript: "Probiotics-derived extracellular vesicles protect senescence against H2O2-induced oxidative stress in placental cells" is not precise, because authors did not studied the senescence it self but the oxidative stress caused by H2O2

Author Response

The revised manuscript does not include all comments, including those listed below.

1. In the description of Western blot (point 2.6), there should be information about: Were membranes incubated at RT or other temperature?  Besides, there is no information how were calculated values presented in Fig. 2

ANS: We have supplied information in 2.6 section.

1. There should be a technical description of confocal microscope and parameters set up during observations.

ANS: We have shown the excition in 2.8 section.

1. Fig. 3A and Fig. 5 the scale bars` values are not well visible. Besides in Fig. 8, there is no scale bar

ANS: We have showed the scale bars in Fig. 3, Fig. 5, and Fig. 8.

1.  Figure 5 does not show whether the EVs have entered the cells or not. Authors should have taken photos under higher magnification, the same as in Fig. 8, and it would be more clear.

ANS: We have improved the photo quality.

The title of manuscript: "Probiotics-derived extracellular vesicles protect senescence against H2O2-induced oxidative stress in placental cells" is not precise, because authors did not studied the senescence it self but the oxidative stress caused by H2O2

ANS: We have rewritten the title.

Round 3

Reviewer 3 Report

I have no further comments.