Antidepressant Effect of Enzymatic Porcine Placenta Hydrolysate in Repeated Immobilization Stress-Induced Ovariectomized Female Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study “Antidepressant Effect of Enzymatic Porcine Placenta Hydrolysate in Repeated Immobilization Stress–Induced Ovariectomized Female Mice” examines the anti-inflammatory effects of enzymatic porcine placenta 17 hydrolysate (EPPH) on LPS-induced levels of nitric oxide (NO), prostaglandin E2 (PGE2), corticosterone (CORT), and pro-inflammatory cytokine interleukin-1 beta (IL-1β) in RAW 264.7 macrophage cells. Also, the neurite outgrowth of PC12 cells was evaluated to examine the effects of 20 EPPH on neurite growth. A stressed ovariectomized (OVX) female mouse model was used to evaluate the antidepressant effects of EPPH. The obtained results suggested that the high dose of EPPH administration induced the antidepressant-like effect in the ovariectomized mice with repeated stress via downregulating the levels of CORT, IL-1β, and PGE2 in the serum through reducing the expression of c-fos in the paraventricular nucleus (PVN) region.

The textual and illustrative data presented herein are comprehensive, valuable, and engaging.

The entire manuscript is correctly written with a thoroughly described Materials and Methods section and an adequately presented Result section complemented with illustrative data of sufficient quality. The discussion section is appropriate, and a comprehensive and up-to-date reference list accompanies the manuscript.

While there are some minor obstacles that need to be addressed, I am confident that with these corrections, the manuscript will be ready for publication.

These include:

It would be helpful if the authors could provide the catalog numbers of antibodies and ELISA kits used in the study.

The authors should number the figures in the order they appear in the text (Figure 7 appears before Figure 1).

In Figure 2, the authors should designate the columns representing the control. What is defined by the red column in Figure 2C?

 

A minor revision of the manuscript is suggested.

Author Response

Dear Editor;

Attached please find a revised copy of our manuscript cimb-2990931entitled “Antidepressant effect of fermented porcine placenta in repeated restraint stress–induced ovariectomized female mice” which we have modified in response to the critiques of your referees. All changes in this revision are highlighted (yellow text).

We hope the manuscript is now in a form suitable for publication in CIMB.

 

Reviewer 1

The study “Antidepressant Effect of Enzymatic Porcine Placenta Hydrolysate in Repeated Immobilization Stress–Induced Ovariectomized Female Mice” examines the anti-inflammatory effects of enzymatic porcine placenta 17 hydrolysate (EPPH) on LPS-induced levels of nitric oxide (NO), prostaglandin E2 (PGE2), corticosterone (CORT), and pro-inflammatory cytokine interleukin-1 beta (IL-1β) in RAW 264.7 macrophage cells. Also, the neurite outgrowth of PC12 cells was evaluated to examine the effects of 20 EPPH on neurite growth. A stressed ovariectomized (OVX) female mouse model was used to evaluate the antidepressant effects of EPPH. The obtained results suggested that the high dose of EPPH administration induced the antidepressant-like effect in the ovariectomized mice with repeated stress via downregulating the levels of CORT, IL-1β, and PGE2 in the serum through reducing the expression of c-fos in the paraventricular nucleus (PVN) region.

The textual and illustrative data presented herein are comprehensive, valuable, and engaging.

The entire manuscript is correctly written with a thoroughly described Materials and Methods section and an adequately presented Result section complemented with illustrative data of sufficient quality. The discussion section is appropriate, and a comprehensive and up-to-date reference list accompanies the manuscript.

While there are some minor obstacles that need to be addressed, I am confident that with these corrections, the manuscript will be ready for publication.

These include:

It would be helpful if the authors could provide the catalog numbers of antibodies and ELISA kits used in the study.

Response: We have provided the catalog number of antibodies and ELISA kits in the material and methods.

 

The authors should number the figures in the order they appear in the text (Figure 7 appears before Figure 1).

Response: We have modified the figures by numbering them in order.

 

In Figure 2, the authors should designate the columns representing the control. What is defined by the red column in Figure 2C?

Response: We have defined the red column as LPS group and made the corresponding changes to the Figure 2, as shown below (Figure 3 in the revised one).

 

A minor revision of the manuscript is suggested.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript the authors investigated the anti-inflammatory effects of EPPH in vitro and in vivo. The anti-inflammatory property of EPPH has been tested via production of NO, PGE2, CORT and IL-1β by LPS-induced RAW 264.7 cells. Neurite outgrowth of PC12 cells has been used to evaluate EPPH on neurite growth. OVX mouse model was established to exam the antidepressant effects of EPPH. Both in vitro and in vivo results indicated EPPH exhibited anti-inflammation and antidepressant-like effect in a dose-dependent way. There are some concerns need to be addressed before acceptance.

In mice treatment study, the saline was mentioned to be orally delivered. How about the administer way of estradiol and EPPH, still oral administer?

What are the major functional ingredients in EPPH for anti-inflammation and antidepressant effect?

Please correct the ‘2’ of H2O2 with subscript on line 248.

Please correct ‘RAW 265.7’ to ‘RAW 264.7’ on line 275.

The label of LPS and EPPH under the x-axis in figure 2 is not aligned with the columns. Please revise it.

The authors mentioned EPPH significantly decreased the immobility time duration in TST, rather than FST, please explain the possible reason of this phenomenon. What is the difference underlying pathophysiology between these two behavior tests?

Author Response

Dear Editor;

Attached please find a revised copy of our manuscript cimb-2990931entitled “Antidepressant effect of fermented porcine placenta in repeated restraint stress–induced ovariectomized female mice” which we have modified in response to the critiques of your referees. All changes in this revision are highlighted (yellow text).

We hope the manuscript is now in a form suitable for publication in CIMB.

 

Reviewer 2

In this manuscript the authors investigated the anti-inflammatory effects of EPPH in vitro and in vivo. The anti-inflammatory property of EPPH has been tested via production of NO, PGE2, CORT and IL-1β by LPS-induced RAW 264.7 cells. Neurite outgrowth of PC12 cells has been used to evaluate EPPH on neurite growth. OVX mouse model was established to exam the antidepressant effects of EPPH. Both in vitro and in vivo results indicated EPPH exhibited anti-inflammation and antidepressant-like effect in a dose-dependent way. There are some concerns need to be addressed before acceptance.

 

In mice treatment study, the saline was mentioned to be orally delivered. How about the administer way of estradiol and EPPH, still oral administer?

Response: We clearly explained the routes by the drugs were administrated in the material and methods on page 5, as “the ovariectomized, stressed and treated subcutaneously with estradiol 1 μg/kg group (PC, n=6); the ovariectomized, stressed and treated orally with (EPPH) 300 mg/kg group (EPPH 300, n=7); and the stressed and treated orally with (EPPH) 1500 mg/kg group (EPPH 1500, n=7)”.

 

What are the major functional ingredients in EPPH for anti-inflammation and antidepressant effect?

Response: EPPH contains a variety of small peptides and amino acids including arginine, isoleucin and leucine, and most of which have been demonstrated to possess significant anti-inflammatory or anti-depressant properties. [37-39]. It has been reported that decreased levels of leucine, valine and isoleucine were associated with major depression [40], and arginine also has inflammatory properties [41]. Therefore, these major components of EPPH may have been responsible for anti-inflammation and antidepressant effect shown in the present study. Future studies are needed to understand the precise neurochemical and behavioral effects of these components in the OVX mouse model. We include this suggestion in the discussion on page 15.   

 

 

Please correct the ‘2’ of H2O2 with subscript on line 248.

Response: We modified “H2O2” to “H₂O₂”.

 

Please correct ‘RAW 265.7’ to ‘RAW 264.7’ on line 275.

Response: The typo, RAW264.7 has been corrected.

 

The label of LPS and EPPH under the x-axis in figure 2 is not aligned with the columns. Please revise it.

Response: The label in Fig 2 has been modified (Fig. 3 in the revised one).

 

The authors mentioned EPPH significantly decreased the immobility time duration in TST, rather than FST, please explain the possible reason of this phenomenon. What is the difference underlying pathophysiology between these two behavior tests?

Response: Both the Forced Swimming Test (FST) and the Tail Suspension Test (TST) are the most commonly used behavioral tests to assess depressive-like behavior or to screen potential antidepressant drugs. While they share similarities in their aims and applications, and are conceptually similar to each other, they differ in their methodologies and the behaviors they measure. Therefore, they do not show identical sensitivities to pharmacologic agents or to strain differences.

Generally, the TST is considered more reliable and greater sensitivity to antidepressants because the procedures of the two tests differ. In the FST, the degree of immobility in animals may be compounded by the shock of being dropped into water and/or exposed to pharmacologic agents [32]. Consistent with the previous findings [8], it was shown that treatment with estrogen, used as a positive control, reduced immobility time in both tests, with a more pronounced effect observed in the TST than in the FST, suggesting that the TST is more sensitive and reliable and considered to have good predictive validity. However, it should be noted that while a reduction of immobility by EPPH in the FST did not reach statistical significance, its trend is similar to that observed in the TST.

We have included these paragraphs in the discussion section.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This article studied the anti-inflammatory effects of EPPH on animal models of menopause women, and the stimulating effect on dendrite growth on a similar cell model of neurons. This is a beautiful and hard-worked study, which deserves being published after minor details are corrected.

-in the end of Introduction you wrote:” Moreover, the depression-associated inflammatory effects of EPPH on LPS-induced levels of NO, PGE2, CORT, and IL-1ß in RAW 264.7 macrophage cells were analyzed in order to elucidate the mechanism of depressive relief by EPPH.” Why macrophage cells, when talking about menopause and depression? Here it is not clear.

-In Results you clearly wrote” RAW264. 7 cells are widely used as a model for studying anti-inflammatory properties of drugs, therefore, understanding how EPPH treatment can impact these cells proves essential.” Ok. After saying a lot about these macrophages, without explanation. Please move this very good explanation in the beginning, somewhere at the end of Introduction, to make everything clear from the very beginning.

-then you wrote:” 2.2.3. MTT cell viability assay” Please explain what MTT means.

-the second part of randomization of mice is not clear. You wrote:” Randomization was carried out as follows: After 7 days of arrival, animals were assigned to a group and weighed. A total of 30 mice were split into 5 different weight groups (n = 5-7 per group). Each animal was assigned a temporary random number within its respective weight range group. On the basis of their positions on the rack, cages were given a numerical designation. Then, the cages were randomized within the exposure group: sham-operation mice were defined as the SHAM group as control (drug naïve, the operated only abdominal incision and non-stressed group, Sham, n=5); ovariectomized, stressed and saline treatment mice were defined as the OVX+ST group (drug naïve OVX, n=5) (drug naïve OVX, n=5); the ovariectomized, stressed and treated wit estradiol 0.2 mg/kg group (PC, n=6); the ovariectomized, stressed and treated with (EPPH) 300 mg/kg group (EPPH 300, n=7); and the stressed and treated with (EPPH) 1500 mg/kg  group (EPPH 1500, n=7).  Meaning, you placed the mice in cages, according to their weight. Ok. Then you gave some numbers to each mouse in each cage. Then what happened? “cages were given a numerical designation”, meaning that ALL THE CAGE had the same treatment. If this is the case, maybe you could have said something about the influence of weight on the results. If not, please explain clearly from that point on, how the randomization took place.

-in 2.6.1 you wrote:” Briefly, a mouse was placed in which its head was approximately 10 cm above the  floor in each chamber. “ It is not clear. Pease explain/correct “in which its head..”

-in Figure 3B, the length of neurites is highest in NGF group, and is higher in 0.1 compared to 100 mg of EPPH. The stimulating effect of EPPH on neurite length is not convincing in these Figure 3B images. Maybe you could replace one of them. You wrote below:” As shown in Figure 3A, EPPH treatment promoted neurite extension in PC12 cells.”. True. But in Figure 3B, it is not true/visible.

-in References you only have 8 out of 40 titles form 2019 and more recent: 10, 18, 19, 29, 32-35. Please replace some of the older tiles with some more recent ones.

Comments on the Quality of English Language

In a few spots (3-4) two or three words were missing. Otherwise, the English language used is fluent and clear. 

Author Response

Dear Editor;

Attached please find a revised copy of our manuscript cimb-2990931entitled “Antidepressant effect of fermented porcine placenta in repeated restraint stress–induced ovariectomized female mice” which we have modified in response to the critiques of your referees. All changes in this revision are highlighted (yellow text).

We hope the manuscript is now in a form suitable for publication in CIMB.

 

Reviewer 3

This article studied the anti-inflammatory effects of EPPH on animal models of menopause women, and the stimulating effect on dendrite growth on a similar cell model of neurons. This is a beautiful and hard-worked study, which deserves being published after minor details are corrected.

-in the end of Introduction you wrote:” Moreover, the depression-associated inflammatory effects of EPPH on LPS-induced levels of NO, PGE2, CORT, and IL-1ß in RAW 264.7 macrophage cells were analyzed in order to elucidate the mechanism of depressive relief by EPPH.” Why macrophage cells, when talking about menopause and depression? Here it is not clear.

Response: As Reviewer suggested, we have included the rationale of using macrophage cells in introduction as follows; “The activation of macrophages is a general feature for the early stages of pathogens infection. Moreover, anti-inflammatory effects of EPPH on LPS-induced levels of NO, PGE2, CORT, and IL-1β in RAW 264.7 macrophage cells were analyzed in order to elucidate the mechanism of anti-depressive actions by EPPH.”

 

-In Results you clearly wrote” RAW264. 7 cells are widely used as a model for studying anti-inflammatory properties of drugs, therefore, understanding how EPPH treatment can impact these cells proves essential.” Ok. After saying a lot about these macrophages, without explanation. Please move this very good explanation in the beginning, somewhere at the end of Introduction, to make everything clear from the very beginning.

Response: As reviewer suggested, we have reorganized the paragraphs in the introduction section to emphasize the anti-inflammatory properties of EPPG, as follows; “The activation of macrophages is a general feature for the early stages of pathogens infection. Lipopolysaccharide (LPS) is an important activator that binds to macrophages and produces an inflammatory response. RAW264.7 macrophage cell line has been developed as a primary experimental macrophage model for the study of macrophage signaling pathways and the research of macrophage dependent inflammations due to ease of cell propagation, high efficiency for DNA transfection, sensitivity to RNA interference and possession of receptors for many relevant ligands. During an inflammatory-depressive response, pro-inflammatory mediators such as prostaglandin E2 (PGE2) are increased in the cerebrospinal fluid [11] and T-lymphocytes [12]. Similarly, during inflammatory processes, the release of nitric oxide (NO) and pro-inflammatory cytokine interleukin-1 beta (IL-1β) are modulated by the LPS-induced HPA response [13]. Overall, PGE2 and NO are induced by cyclooxygenase (COX-2) and nitric oxide synthase (iNOS) enzymes, respectively. LPS stimulated RAW264.7 cell inflammatory model is widely used as a model for studying anti-inflammatory properties of drugs, therefore, understanding how EPPH treatment can impact these cells proves essential.”

 

-then you wrote:” 2.2.3. MTT cell viability assay” Please explain what MTT means.

Response: MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and is a colorimetric assay for assessing cell viability and proliferation based on metabolic activity. We have provided a detailed explanation of the MTT assay in the Material and Methods section, referred to as “The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a colorimetric assay for assessing cell viability and proliferation based on metabolic activity.”

 

 -the second part of randomization of mice is not clear.

 

You wrote:” Randomization was carried out as follows: After 7 days of arrival, animals were assigned to a group and weighed. A total of 30 mice were split into 5 different weight groups (n = 5-7 per group). Each animal was assigned a temporary random number within its respective weight range group. On the basis of their positions on the rack, cages were given a numerical designation. Then, the cages were randomized within the exposure group: sham-operation mice were defined as the SHAM group as control (drug naïve, the operated only abdominal incision and non-stressed group, Sham, n=5); ovariectomized, stressed and saline treatment mice were defined as the OVX+ST group (drug naïve OVX, n=5) (drug naïve OVX, n=5); the ovariectomized, stressed and treated wit estradiol 0.2 mg/kg group (PC, n=6); the ovariectomized, stressed and treated with (EPPH) 300 mg/kg group (EPPH 300, n=7); and the stressed and treated with (EPPH) 1500 mg/kg  group (EPPH 1500, n=7).  Meaning, you placed the mice in cages, according to their weight. Ok. Then you gave some numbers to each mouse in each cage. Then what happened? “cages were given a numerical designation”, meaning that ALL THE CAGE had the same treatment. If this is the case, maybe you could have said something about the influence of weight on the results. If not, please explain clearly from that point on, how the randomization took place.

Response: Thank you for your comments on the procedures of randomization. Our study employed stratified randomization method in which all animals were divided into subgroups based on weight, and randomization is performed separately within each weight to ensure balance across groups. By creating homogeneous block in which weight variable not under investigation is evenly distributed, evaluation of our drugs can occur without obfuscation.  

In order to avoid confusion, we explained more clearly the procedures of randomization method in the revised manuscript as follows: “After 7 days of arrival, animals were pre-weighed. A total of 30 mice were split into 5 different weight groups (n = 5-7 per group). Each animal was assigned a temporary random number within its respective weight range group, minimizing potential bias of weight.”

 

-in 2.6.1 you wrote:” Briefly, a mouse was placed in which its head was approximately 10 cm above the floor in each chamber. “ It is not clear. Pease explain/correct “in which its head.”

Response: We modified the sentence in 2.6.1 as “Briefly, a mouse was taped approximately 1 cm from the tip of its tail and suspended upside down with its head approximately 10 cm above the floor in each chamber.”

 

-in Figure 3B, the length of neurites is highest in NGF group, and is higher in 0.1 compared to 100 mg of EPPH. The stimulating effect of EPPH on neurite length is not convincing in these Figure 3B images. Maybe you could replace one of them. You wrote below:” As shown in Figure 3A, EPPH treatment promoted neurite extension in PC12 cells.”. True. But in Figure 3B, it is not true/visible.

Response: We have provided improved Figure 3 (Fig. 4 in revised one), as shown below.

 

-in References you only have 8 out of 40 titles form 2019 and more recent: 10, 18, 19, 29, 32-35. Please replace some of the older tiles with some more recent ones.

Response: We have updated the references with more recent ones.

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised version addressed all of my concerns.