Reviewer ’s report  
Title: Geranylgeranylacetone attenuates fibrogenic activity an d induces apoptosis in cultured 
human hepatic stellate cells and reduce s liver fibrosis in carbon tetrachloride -treated mice  
 
Version: 0 Date: 07 Jun 2017  
 
Reviewer: Kinji Asahina  
 
Reviewer's report:  
The present study examined the effect of Geranylgeranylacetone (GGA) on activation of hepatic 
stellate cells (HSC) and liver fibros is. The authors showed GGA induces cell death and reduces 
fibrogenic marker expression in a human HSC line in culture. GGA treatment also reduced 
CCl4 -induced fibrosis in mice.  
 
Although the data suggest beneficial effects of GGA on liver fibrosis, many w eaknesses reduce 
my enthusiasm on this manuscript. The effect of GGA on CCl4 -induced liver fibrosis was 
already published by other group (ref 16). The authors only used the human HSC line for their 
analysis and it remains unclear the effects of GGA in prim ary HSCs. The authors failed to clarify 
the mechanism underlying GGA -induced cell death in HSCs.  
 
1. It will be better to include the in vivo study in the title.  
2. Page 3, Background. "However, the mechanisms underlying the development of liver 
fibrosis r emain largely unexplored". This sentence is inaccurate. Indeed, the mechanisms 
have been extensively studied.  
3. Page 9. The method of CCl4 -induced model is less clear. Did the authors mix 400 ml of 
CCl4 and 600 ml of salad oil and inject 0.5 ul of this s olution per gram body weight?  
4. The authors need to switch Supplemental Figure A and B.  
5. Figure 1. GGA may reduce the cell proliferation.  
6. Figure 2. Only merged pictures are sufficient.  
7. The authors should confirm the effects of GGA in primary mouse  HSCs.  
8. It remains unclear whether GGA modulates ER stress in HSCs and how GGA participates 
cell death.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
No 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
No 
Are the conclusions  drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
No 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statis tical review is recommended, please speci fy what aspects require further 
assessment in your comments to the editors.  
Not relevant to this manuscript  
Quality of written English  
Please indicate the quality of language in the manuscript:  
Acceptable  
Declaratio n of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose f inancial ly from the publication of this 
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
3. Do  you hold or are you currently applying for any patents  relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
No competing interests.  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand t hat any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
Title: Geranylgeranylacetone attenuates fibrogenic activity an d induces apoptosis in cultured 
human hepatic stellate cells and reduce s liver fibrosis in carbon tetrachloride -treated mice  
 
Version: 0 Date: 07 Jun 2017  
 
Reviewer: Kinji Asahina  
 
Reviewer's report:  
The present study examined the effect of Geranylgeranylacetone (GGA) on activation of hepatic 
stellate cells (HSC) and liver fibros is. The authors showed GGA induces cell death and reduces 
fibrogenic marker expression in a human HSC line in culture. GGA treatment also reduced 
CCl4 -induced fibrosis in mice.  
 
Although the data suggest beneficial effects of GGA on liver fibrosis, many w eaknesses reduce 
my enthusiasm on this manuscript. The effect of GGA on CCl4 -induced liver fibrosis was 
already published by other group (ref 16). The authors only used the human HSC line for their 
analysis and it remains unclear the effects of GGA in prim ary HSCs. The authors failed to clarify 
the mechanism underlying GGA -induced cell death in HSCs.  
 
1. It will be better to include the in vivo study in the title.  
2. Page 3, Background. "However, the mechanisms underlying the development of liver 
fibrosis r emain largely unexplored". This sentence is inaccurate. Indeed, the mechanisms 
have been extensively studied.  
3. Page 9. The method of CCl4 -induced model is less clear. Did the authors mix 400 ml of 
CCl4 and 600 ml of salad oil and inject 0.5 ul of this s olution per gram body weight?  
4. The authors need to switch Supplemental Figure A and B.  
5. Figure 1. GGA may reduce the cell proliferation.  
6. Figure 2. Only merged pictures are sufficient.  
7. The authors should confirm the effects of GGA in primary mouse  HSCs.  
8. It remains unclear whether GGA modulates ER stress in HSCs and how GGA participates 
cell death.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
No 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
No 
Are the conclusions  drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
No 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statis tical review is recommended, please speci fy what aspects require further 
assessment in your comments to the editors.  
Not relevant to this manuscript  
Quality of written English  
Please indicate the quality of language in the manuscript:  
Acceptable  
Declaratio n of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose f inancial ly from the publication of this 
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
3. Do  you hold or are you currently applying for any patents  relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
No competing interests.  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand t hat any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
Title: Geranylgeranylacetone attenuates fibrogenic activity an d induces apoptosis in cultured 
human hepatic stellate cells and reduce s liver fibrosis in carbon tetrachloride -treated mice  
 
Version: 0 Date: 07 Jun 2017  
 
Reviewer: Kinji Asahina  
 
Reviewer's report:  
The present study examined the effect of Geranylgeranylacetone (GGA) on activation of hepatic 
stellate cells (HSC) and liver fibros is. The authors showed GGA induces cell death and reduces 
fibrogenic marker expression in a human HSC line in culture. GGA treatment also reduced 
CCl4 -induced fibrosis in mice.  
 
Although the data suggest beneficial effects of GGA on liver fibrosis, many w eaknesses reduce 
my enthusiasm on this manuscript. The effect of GGA on CCl4 -induced liver fibrosis was 
already published by other group (ref 16). The authors only used the human HSC line for their 
analysis and it remains unclear the effects of GGA in prim ary HSCs. The authors failed to clarify 
the mechanism underlying GGA -induced cell death in HSCs.  
 
1. It will be better to include the in vivo study in the title.  
2. Page 3, Background. "However, the mechanisms underlying the development of liver 
fibrosis r emain largely unexplored". This sentence is inaccurate. Indeed, the mechanisms 
have been extensively studied.  
3. Page 9. The method of CCl4 -induced model is less clear. Did the authors mix 400 ml of 
CCl4 and 600 ml of salad oil and inject 0.5 ul of this s olution per gram body weight?  
4. The authors need to switch Supplemental Figure A and B.  
5. Figure 1. GGA may reduce the cell proliferation.  
6. Figure 2. Only merged pictures are sufficient.  
7. The authors should confirm the effects of GGA in primary mouse  HSCs.  
8. It remains unclear whether GGA modulates ER stress in HSCs and how GGA participates 
cell death.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
No 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
No 
Are the conclusions  drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
No 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statis tical review is recommended, please speci fy what aspects require further 
assessment in your comments to the editors.  
Not relevant to this manuscript  
Quality of written English  
Please indicate the quality of language in the manuscript:  
Acceptable  
Declaratio n of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose f inancial ly from the publication of this 
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
3. Do  you hold or are you currently applying for any patents  relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
No competing interests.  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand t hat any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant