Trehalose Attenuates Oxidative Stress and Endoplasmic Reticulum Stress-Mediated Apoptosis in IPEC-J2 Cells Subjected to Heat Stress

Round 1

Reviewer 1 Report

This manuscript provides evidence that trehalose is able to attenuate IPEC-J2 cells injury caused by heat exposure. The present study showed that supplementation with 10 mM trehalose can relieve oxidative stress and cellular apoptosis in heat-stressed IPEC-J2 cells. The mechanism is associated with Tre attenuating apoptosis by inhibiting pro-apoptotic arm of UPR.

Although in general the manuscript is of good quality, there are several minor points that need attention.

Comments for author File: Comments.pdf

Author Response

This manuscript provides evidence that trehalose is able to attenuate IPEC-J2 cells

injury caused by heat exposure. The present study showed that supplementation with

10 mM trehalose can relieve oxidative stress and cellular apoptosis in heat-stressed

IPEC-J2 cells. The mechanism is associated with Tre attenuating apoptosis by

inhibiting pro-apoptotic arm of UPR.

Although in general the manuscript is of good quality, there are several minor points

that need attention.

Response: Thank you for your comments.

Line 21 "allevate" should be "alleviate".

Response: We have corrected the error in revised version (line 21).

Line 66 "in vitro " need to be italic.

Response: We have revised it (line 70).

Line 112 10μL should be 10 μL.

Response: We have revised it (line 121).

Line 122 "themanufacturer " should be "the manufacturer "

Response: We have revised it (line 130).

Line 125 please simplify the methods on ROS detection.

Response: We have simplify the methods on ROS detection, as described at line 139-145 in revised version of the manuscript.

Cellular ROS levels were determined according to the instructions of ROS detection (kitBeyotime, Shanghai, China). Briefly, the DCFH-DA probe was added to the mediumat a ratio of 1:1000, and then incubated at 37°C for 1 h. The cells were washed twice with serum-free culture medium and observed with a fluorescence microscope (NTX-N3, Ni-kon Corp.,Tokyo, Japan) Intracellular ROS levels were quantified using the mean fluorescence intensity per cell by Image J software (National Institute of Health, Bethesda, MD, USA).

Line 128 please delete the " and dilute"

Response: We have revised it (line 140).

Line 138 please delete the " within the cells"

Response: We have revised it (line 147).

Line 140 "themanufacturer " should be "the manufacturer "

Response: We have revised it (line 149).

Line 146 "by " should be "with"

Response: We have revised it (line 153).

Line 151 Please delete the "being"

Response: We have revised it (line 159).

Line 157 Please add the reference.

Response: We have added the reference 26 (line 166).

Cui, Y.; Qi, S.; Zhang, W.; Mao, J.; Tang, R.; Wang, C.; Liu, J.; Luo, X.M.; Wang, H. Lactobacillus reuteri zj617 culture supernatant attenuates acute liver injury induced in mice by lipopolysaccharide. The Journal of nutrition 2019, 149, 2046-2055.

Line 240" expression" should be "expressions"

Response: We have revised it (line 254).

Line 249-251 delete duplicates.

Response: We have deleted them (line 264-265).

Line 265 “in vitro” need to be italic.

Response: We have revised it (line 281).

Author Response File: Author Response.docx

Reviewer 2 Report

The paper evaluates the potential of trehalose to ameliorate IPEC-J2 cells injuries induced by heat stress. The authors found that trehalose has protective effects in allevating oxidative stress and endoplasmic reticulum stress-mediated apoptosis. The manuscript is well written, data presented of good quality. However, there was still some small error or problems. 

Line 42 Use “hallmarks” instead of “hall marks”.

Line 46 Use“cytochrome c” instead of “cytochrome C”.

Line 70 “Both heat exposure or ROS…” should be as the beginning of the next paragraph.

Line 131-134 are repeated, pleease clarify determination and quantificationt of the ROS.

Line 151 delate “being”.

Line 157 Please supplement the reference.

Line 237 Use “indeed” instead of “In deed”.

Line 265 “in vitro” need to be italic.

Please supplement the abbreviations involved in this manuscript as supplementary materials.

Author Response

The paper evaluates the potential of trehalose to ameliorate IPEC-J2 cells injuries induced by heat stress. The authors found that trehalose has protective effects in alleviating oxidative stress and endoplasmic reticulum stress-mediated apoptosis. The manuscript is well written, data presented of good quality. However, there was still some small error or problems.

Response: Thank you for your comments.

Line 42 Use “hallmarks” instead of “hall marks”.

Response: We have revised it (line 45).

Line 46 Use“cytochrome c” instead of “cytochrome C”.

Response: We have revised it (line 48).

Line 70 “Both heat exposure or ROS…” should be as the beginning of the next paragraph.

Response: We have revised it (line 75).

Line 131-134 are repeated, please clarify determination and quantification of the ROS.

Response: We have revised the methods on ROS detection, as described at line 139-145 in revised version of the manuscript.

Cellular ROS levels were determined according to the instructions of ROS detection (kitBeyotime, Shanghai, China). Briefly, the DCFH-DA probe was added to the mediumat a ratio of 1:1000, and then incubated at 37°C for 1 h. The cells were washed twice with serum-free culture medium and observed with a fluorescence microscope (NTX-N3, Ni-kon Corp., Tokyo, Japan) Intracellular ROS levels were quantified using the mean fluorescence intensity per cell by Image J software (National Institute of Health, Bethesda, MD, USA).

Line 151 delate “being”.

Response: We have revised it (line 159).

Line 157 Please supplement the reference.

Response: We have added the reference 26 (line 166).

Cui, Y.; Qi, S.; Zhang, W.; Mao, J.; Tang, R.; Wang, C.; Liu, J.; Luo, X.M.; Wang, H. Lactobacillus reuteri zj617 culture supernatant attenuates acute liver injury induced in mice by lipopolysaccharide. The Journal of nutrition 2019, 149, 2046-2055.

Line 237 Use “indeed” instead of “In deed”.

Response: We have revised it (line 251).

Line 265 “in vitro” need to be italic.

Response: We have revised it (line 281).

Please supplement the abbreviations involved in this manuscript as supplementary materials.

Response: We have supplemented the abbreviations involved in this manuscript as supplementary materials.

Author Response File: Author Response.docx

Reviewer 3 Report

This study investigated the beneficial role of Trehalose in attenuating heat stress induced oxidative stress, endoplasmic reticulum stress and apoptosis in porcine IPEC-J2 cells. While the data are interesting and add to the existing knowledge, the manuscript would benefit from revisions.

To improve this manuscript, I suggest the authors to clearly state the experimental design, experimental unit and the replicates especially in the method section. Also, please include more details on chemicals and experimental protocols used.

In general, the interpretation and conclusions were sound and well supported by their citations. However, the authors need to discuss the major findings and implications of the current study before heavily discussing others’ works. The readers would be more interested in interpretation of current findings. Please revise accordingly.

Abstract:

Line 32: please clarify the temperature information for CON and HS groups within the parentheses

Line 33, please include the pre-treated trehalose concentration again here for the TRE+HS group.

Line 40: please clarify if it is mRNA expression.

Introduction:

Line 55, HS - please do not use an abbreviation to start a sentence. Please check it throughout the manuscript.

Materials and Methods

General information: you need to clearly state the experimental design. There are a couple of general comments:

1.     You might do no need to repeat the cell culture and the treatment in each assay paragraph as long as you clearly state the experimental design in the earlier paragraphs.

2.      Please clearly state the experimental design at the beginning of the methods following the cell culture procedures. You mentioned this well in the abstract. So, please expand it and clarify clearly here.

3.       What is the biological replicate in the experiment?

4.    Please clarify if you have repeated each of the experiment. If so, for how many times?

5.  To me, it seems that both non-treated and trehalose treated cells were subjected to either 37C or 43C. However, based on the Abstract and Results sections, only trehalose treated cells, but the CON cells, were subjected to 43C; and the non-treated cells were only under 37C. Again, please clarify the experimental design of each experiment clearly.

6.      I suggest that you use CON vs. HS only to refer temperature treatments (37C vs. 43C). It might be confused for readers if you use ‘CON’ for ‘DMEM’ only as you also have temperature differences, unless you say it is ‘DMEM+37C’ for CON.

7.   Please define the trehalose (0.1, 1,5…) as ‘Tre+0.1’, ‘Tre+1’…in the methods section, although you have mentioned this in Table 1.

Line 101: provide the source information of the IPEC-J2 cells please.

Line 105, Can you provide the volume of the trehalose solutions administrated?

Line 109: pre-incubated with DMEM or DEM/F12? Please be consistent. Please remove the’ CON’ in the parentheses and put it wherever relevant to the temperature (37C).

Line 107: It looks like that trehalose treat cells were also subjected to 37C? Please clarify.

Line 110-114: Again, I would assume that both non-trehalose treated and trehalose treated cells were subjected to either 37C or 43C. Please clarify without reading the results section. Again, please clearly state the trehalose treatment/temperature groups.

Line 112: Please clarify – it sounds like that the CCK-8 solution was added only to the HS treatment group?

Line 114: Did you measure the OD and all other assay in duplicates within the microplate? How about the intra-plate variation, if possible?

Line 119, Again, please clarity whether the supernatant of the medium was collected from only the HS group or both the HS group and 37C?

Line 121: Please provide the LDH kit’ supplier info.

Line 125: You might need one or two sentences to remind the readers (and the reasons) of only using 10 mM for the remainder of the experiments.

Line 146: Please provide the pH values of the PBS solution, if possible.

Line 152: What was the incubation temperature?

Line 160-163: Please provide the host species for all the primary antibodies or the category numbers of the antibodies. Please provide the manufacture info of the secondary antibodies.

Statistical Analysis

General comments: Did you check the normality of your data before conducting the ANOVA?

Line 168: I suggest that you go back to the Methods section and clearly state that all experiments were repeated three times. Also, was each experiment conducted based at similar experimental protocols (e.g., incubation time & temperature, antibody dilution, etc.).

Results

General comments:  Again, you need to well define what the CON and HS groups were (in the method section). Otherwise, the readers would be confused about the results and comparisons reported here. Also, I suggest reformatting the table 1. The readers would be interested to know the actual P value for the main effect of temperature and treatment concentrations. I suggest that you put the three parameters in rows in Table. Also, the error term needs to be one additional significant figure to the average.

Line 174: maybe change the subtitle from ‘IPEC-J2 cells’ to ‘heat-treated IPEC-J2 cells’

Line 175: how about the 37C and non-Tre treated groups? Is this your CON group? As mentioned earlier, for CON, you might clarify that it was “non-TRE treated + 37C’.

Line 177: again, I am confused with what the CON and HS groups were without referring to the Table. Please clarify.

Line 180: Should not it be ‘compared to non-Tre treated at 43C’? I don’t think you can simply say it as ‘HS group’ as all Tre-treated cells were exposed to HS as well. See my comments above.

Line 191: please revise the captions accordingly.

Line 193: Why n=6 here whereas n=3 for other assays?

Line 203-204: no discussion in the Methods section please.

Discussion and Conclusion

General comments: Please first discus your (principal) findings and implications before showing results from others’ or your previous studies. The readers would be more interested to know the findings from the current work. Again, it is not appropriate to start a sentence with an abbreviation.

Line 279: The whole paragraph was only talking about the background information and previous findings from others, which were unnecessary, at least before discussion of your own data. Again, you need to discuss your results first. I suggest condensing and combining this paragraph with the following one.

Line 301: inhibition or activation of the eif2α-CHOP signal pathway? Please clarify.

Line 331: Please conclude the study using one paragraph.

Author Response

This study investigated the beneficial role of Trehalose in attenuating heat stress induced oxidative stress, endoplasmic reticulum stress and apoptosis in porcine IPEC-J2 cells. While the data are interesting and add to the existing knowledge, the manuscript would benefit from revisions.

To improve this manuscript, I suggest the authors to clearly state the experimental design, experimental unit and the replicates especially in the method section. Also, please include more details on chemicals and experimental protocols used.

In general, the interpretation and conclusions were sound and well supported by their citations. However, the authors need to discuss the major findings and implications of the current study before heavily discussing others’ works. The readers would be more interested in interpretation of current findings. Please revise accordingly.

Response: Thank you for your comments and suggestions. According to the suggestions, we have clarified the experimental design in the method section, and discussed the major findings.

Abstract:

Line 32: please clarify the temperature information for CON and HS groups within the parentheses

Response: We have revised it (line 33-35).

IPEC-J2 cells were divided into three groups: cultured at 37°C until the end of the experiment (control, CON); exposed to heat stress for 2 h (43°C HS) ; or pretreated with 10 mM trehalose for 4 h at 37°C prior to heat stress treatments (Tre+HS) for 2 h.

Line 33, please include the pre-treated trehalose concentration again here for the TRE+HS group.

Response: We have revised it (line 33-35).

Line 40: please clarify if it is mRNA expression.

Response: It is protein expression (line 42)

Introduction:

Line 55, HS - please do not use an abbreviation to start a sentence. Please check it throughout the manuscript.

Response: We have revised them and carefully checked them throughout the manuscript.

Materials and Methods

General information: you need to clearly state the experimental design. There are a couple of general comments:

1. You might do no need to repeat the cell culture and the treatment in each assay paragraph as long as you clearly state the experimental design in the earlier paragraphs.

Response: We have clarify the cell culture and the treatment in the Materials and Methods section (Line 106-115) and deleted the repeated parts in each assay paragraph. 

The IPEC-J2 cells was a gift from Prof. Haifeng Wang at Zhejiang university. The cells were seeded in DMEM/F12 culture medium (KeyGEN, Shanghai, China) containing 10% fetal bovine serum (Gibco, Waltham, MA, USA) and incubated in a 5% CO2 humidified in-cubator at 37 °C. The media was renewed every day. The well-grown cells in log phase were cultured at 37°C for 24 h in 96-well plates after resuscitation. Thereafter, IPEC-J2 cells were cultured at 37°C until the end of the experiment (control, CON); exposed to heat stress for 2 h (43°C, HS); or pretreated with 0.1, 1, 5, 10, and 15 mM trehalose (Sigma-Aldrich, St. Louis, Missouri, USA) at 37°C for 4 h prior to heat stress treatments for 2 h. All cell samples were used to determine the cell viability, lactate dehydrogenase (LDH) release to supernatant, and malondialdehyde (MDA) level. All experiments were repeated 3 times.

2. Please clearly state the experimental design at the beginning of the methods following the cell culture procedures. You mentioned this well in the abstract. So, please expand it and clarify clearly here.

Response: We have clarify clearly the experimental design at the beginning of the methods (Line 106-115)

The IPEC-J2 cells was a gift from Prof. Haifeng Wang at Zhejiang university. The cells were seeded in DMEM/F12 culture medium (KeyGEN, Shanghai, China) containing 10% fetal bovine serum (Gibco, Waltham, MA, USA) and incubated in a 5% CO2 humidified in-cubator at 37 °C. The media was renewed every day. The well-grown cells in log phase were cultured at 37°C for 24 h in 96-well plates after resuscitation. Thereafter, IPEC-J2 cells were cultured at 37°C until the end of the experiment (control, CON); exposed to heat stress for 2 h (43°C, HS); or pretreated with 0.1, 1, 5, 10, and 15 mM trehalose (Sigma-Aldrich, St. Louis, Missouri, USA) at 37°C for 4 h prior to heat stress treatments for 2 h. All cell samples were used to determine the cell viability, lactate dehydrogenase (LDH) release to supernatant, and malondialdehyde (MDA) level. All experiments were repeated 3 times.

3. What is the biological replicate in the experiment?

Response: All experiments were repeated 3 times.

4. Please clarify if you have repeated each of the experiment. If so, for how many times?

Response: All experiments were repeated 3 times.

5. To me, it seems that both non-treated and trehalose treated cells were subjected to either 37C or 43C. However, based on the Abstract and Results sections, only trehalose treated cells, but the CON cells, were subjected to 43C; and the non-treated cells were only under 37C. Again, please clarify the experimental design of each experiment clearly.

Response: The cells were seeded in DMEM/F12 culture medium (KeyGEN, Shanghai, China) containing 10% fetal bovine serum (Gibco, Waltham, MA, USA) and incubated in a 5% CO2 humidified in-cubator at 37 °C. The media was renewed every day. The well-grown cells in log phase were cultured at 37°C for 24 h in 96-well plates after resuscitation. Thereafter, IPEC-J2 cells were cultured at 37°C until the end of the experiment (control, CON); exposed to heat stress for 2 h (43°C, HS); or pretreated with 0.1, 1, 5, 10, and 15 mM trehalose (Sigma-Aldrich, St. Louis, Missouri, USA) at 37°C for 4 h prior to heat stress treatments for 2 h. All cell samples were used to determine the cell viability, lactate dehydrogenase (LDH) release to supernatant, and malondialdehyde (MDA) level. All experiments were repeated 3 times.

The optimum level of trehalose to protecting against HS-induced cell injuries was determined to be 10 mM, as evidenced by the highest cellular viability, lowest MDA content and lactate dehydrogenase (LDH) activity. Thus, pretreatment with 10mM Tre was selected for the following experiments. Based on this, IPEC-J2 cells were divided into three groups: cultured at 37°C until the end of the experiment (Control, CON); exposed to heat stress for 2 h (43°C HS) ; or pretreated with 10 mM trehalose for 4 h at 37°C prior to heat stress treatments for 2 h (Tre+HS). All cell samples were used to determine cellular ROS level, SOD activity, protein expression level, mitochondrial membrane potential, and cell apoptosis. All experiments were repeated 3 times.

6. I suggest that you use CON vs. HS only to refer temperature treatments (37C vs. 43C). It might be confused for readers if you use ‘CON’ for ‘DMEM’ only as you also have temperature differences, unless you say it is ‘DMEM+37C’ for CON.

Response: We have revised it in the in the Materials and Methods section (Line 106-115)

7. Please define the trehalose (0.1, 1,5…) as ‘Tre+0.1’, ‘Tre+1’…in the methods section, although you have mentioned this in Table 1.

Response: We have changed the Table 1 as Figure 1. 

Line 101: provide the source information of the IPEC-J2 cells please.

Response: The IPEC-J2 cells was a gift from Prof. Haifeng Wang at Zhejiang university (Line 106).

Line 105, Can you provide the volume of the trehalose solutions administrated?

Response: 200 μL

Line 109: pre-incubated with DMEM or DEM/F12? Please be consistent. Please remove the’ CON’ in the parentheses and put it wherever relevant to the temperature (37C).

Response: We have revised it (Line 118-123)

Line 107: It looks like that trehalose treat cells were also subjected to 37C? Please clarify.

Response: We have clarify clearly the experimental design at Cell Culture and treatments section (Line 106-115)

Line 110-114: Again, I would assume that both non-trehalose treated and trehalose treated cells were subjected to either 37C or 43C. Please clarify without reading the results section. Again, please clearly state the trehalose treatment/temperature groups.

Response: We have clarify clearly the experimental design at Cell Culture and treatments section (Line 106-115)

Line 112: Please clarify – it sounds like that the CCK-8 solution was added only to the HS treatment group?

Response: We have clarify clearly it in the Cell viability assay section (Line 118-123)

Line 114: Did you measure the OD and all other assay in duplicates within the microplate? How about the intraplate variation, if possible?

Response: We measured the OD in triplicate within the microplate and the intraplate variation is less than 1%.

Line 119, Again, please clarity whether the supernatant of the medium was collected from only the HS group or both the HS group and 37C?

Response: IPEC-J2 cells were cultured at 37°C until the end of the experiment (control, CON); exposed to heat stress for 2 h (43°C, HS); or pretreated with 0.1, 1, 5, 10, and 15 mM trehalose at 37°C for 4 h prior to heat stress treatments for 2 h. Then the supernatant of the medium from all groups was collected for lactate dehydrogenase (LDH, cat. no. A020) assay.

Line 121: Please provide the LDH kit’ supplier info.

Response: lactate dehydrogenase (LDH, cat. no. A020)

Line 125: You might need one or two sentences to remind the readers (and the reasons) of only using 10 mM for the remainder of the experiments.

Response: Thank your for your suggestions. We have supplemented it (Line 132-138).

Pretreatment with 10 mM Tre has best effects in relieving HS-induced cell injuries and thus was selected for the following experiments. Based on this, IPEC-J2 cells were di-vided into three groups: cultured at 37°C until the end of the experiment (Control, CON); exposed to heat stress for 2 h (43°C HS) ; or pretreated with 10 mM trehalose for 4 h at 37°C prior to heat stress treatments for 2 h (Tre+HS). All cell samples were used to determine cellular ROS level, SOD activity, protein expression level, mitochondrial membrane potential, and cell apoptosis. All experiments were repeated 3 times.

Line 146: Please provide the pH values of the PBS solution, if possible.

Response: pH = 7.2

Line 152: What was the incubation temperature?

Response: at 25°C

Line 160-163: Please provide the host species for all the primary antibodies or the category numbers of the antibodies. Please provide the manufacture info of the secondary antibodies.

Response: We have supplemented them (Line 163-173)

Statistical Analysis

General comments: Did you check the normality of your data before conducting the ANOVA?

Response: Yes. The data is suitable for normal distribution.  

Line 168: I suggest that you go back to the Methods section and clearly state that all experiments were repeated three times. Also, was each experiment conducted based at similar experimental protocols (e.g., incubation time & temperature, antibody dilution, etc.).

Response: We have clarified them in the Methods section.

Results

General comments:  Again, you need to well define what the CON and HS groups were (in the method section). Otherwise, the readers would be confused about the results and comparisons reported here. Also, I suggest reformatting the table 1. The readers would be interested to know the actual P value for the main effect of temperature and treatment concentrations. I suggest that you put the three parameters in rows in Table. Also, the error term needs to be one additional significant figure to the average.

Response: We have changed the Table 1 as Figure 1.

Line 174: maybe change the subtitle from ‘IPEC-J2 cells’ to ‘heat-treated IPEC-J2 cells’

Response: We have revised it (line 185).

Line 175: how about the 37C and non-Tre treated groups? Is this your CON group? As mentioned earlier, for CON, you might clarify that it was “non-TRE treated + 37C’.

Response: We have clarified them in the Methods section (Line 106-116).

Line 177: again, I am confused with what the CON and HS groups were without referring to the Table. Please clarify.

Response: We have clarified them in the Methods section (Line 106-116) and figure captions.

Line 180: Should not it be ‘compared to non-Tre treated at 43C’? I don’t think you can simply say it as ‘HS group’ as all Tre-treated cells were exposed to HS as well. See my comments above.

Response: We have clarified them in the Methods section (Line 106-116).

Line 191: please revise the captions accordingly.

Response: We have clarified them in the Methods section (Line 106-116) and figure captions.

Line 193: Why n=6 here whereas n=3 for other assays?

Response: I am sorry for this mistake. n =3 for all assays. We have revised it.

Line 203-204: no discussion in the Methods section please.

Response: We have deleted it.

Discussion and Conclusion

General comments: Please first discus your (principal) findings and implications before showing results from others’ or your previous studies. The readers would be more interested to know the findings from the current work. Again, it is not appropriate to start a sentence with an abbreviation.

Response: Thank your for your suggestions. We have revised the discussion and conclusion.

Line 279: The whole paragraph was only talking about the background information and previous findings from others, which were unnecessary, at least before discussion of your own data. Again, you need to discuss your results first. I suggest condensing and combining this paragraph with the following one.

Response: Thank your for your suggestions. According to your suggestions, We have condensed and combined this paragraph with the following one.

Line 301: inhibition or activation of the eif2α-CHOP signal pathway? Please clarify.

Response: inhibition of the eif2α-CHOP signal pathway (line 306)

Line 331: Please conclude the study using one paragraph.

Response: We have revised it (line 336-343).

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

I appreciate the authors' revisions. 

Author Response

Thank you for your comments.