Effect of Bombyx mori on the Liver Protection of Non-Alcoholic Fatty Liver Disease Based on In Vitro and In Vivo Models
Round 1
Reviewer 1 Report
The manuscript by Park et al. demonstrates that Bombyx mori (silkworm; SW) extract protects against diet-induced NAFLD through the modulation of hepatic AMPK and ACC-mediated lipid metabolism. In general, the study is well prepared. However, following points arose during the revision of this manuscript:
Major comments:
- In general, representative blots presented are overexposed, so authors have to change some of them in order to better show the difference between conditions (FAS, PPARa and CPT1 in Figure 2, CPT1 in Figure 6). Moreover, blot of pACC in Figure 7 should be improved.
- Did SW modify the food intake of mice?
- In Figure 5, a quantification of adipose size from WAT of all mice will help to notice the difference between animal groups. Moreover, the quantification of Oil Red O staining will be appreciated.
- Did SW modify any fibrogenic markers in the in vivo model?
- As authors mentioned in the discussion “An increasing expression level of hepatic CD36 has been observed in NAFLD, and appears to mediate enhanced intake of free fatty acids derived from the plasma through de novo lipogenesis”. Are CD36 levels regulated by SW in the in vitro and/or in vivo model?
- Authors stated that “These results provide a novel therapeutic agent of hepatoprotective effects of SW for non-alcoholic hepatic steatosis that targets hepatic AMPK and ACC-mediated lipid metabolism”. In order to support this conclusion, experiments focused on the inhibition of AMPK or ACC in the presence of FFA ± SW should be performed.
Minor comments:
- Why did the authors use this positive control in the in vivo study? A brief explanation will be appreciated.
- Primer sequences are in Supplement Table 3, not 1.
- The reference and dilution of primary antibodies are missing.
- In line 267, authors defined NASH as non-alcoholic fatty hepatitis, but it is non-alcoholic fatty steatohepatitis.
Author Response
Reviewer 1
The manuscript by Park et al. demonstrates that Bombyx mori (silkworm; SW) extract protects against diet-induced NAFLD through the modulation of hepatic AMPK and ACC-mediated lipid metabolism. In general, the study is well prepared. However, following points arose during the revision of this manuscript:
Major comments:
- In general, representative blots presented are overexposed, so authors have to change some of them in order to better show the difference between conditions (FAS, PPARa and CPT1 in Figure 2, CPT1 in Figure 6). Moreover, blot of pACC in Figure 7 should be improved.
à We changed western blot of PPARα in Figure 2 and improved the intensity of figures (FAS and CPT1 in figure 1, CPT1 in Figure 6, and pACC in Figure 7).
- Did SW modify the food intake of mice?
à We didn’t alter the SW, and the SW treatment (in PBS) was orally supplemented.
- In Figure 5, a quantification of adipose size from WAT of all mice will help to notice the difference between animal groups. Moreover, the quantification of Oil Red O staining will be appreciated.
à In Figure 5, we added the graph about the quantification of adipocyte area.
- Did SW modify any fibrogenic markers in the in vivo model?
à We didn’t check the several markers about fibrosis, but in figure 4, we measured AST, which is one of the blood markers (Cales et al.) of liver fibrosis, and SW treatment significantly improved liver function.
Cales P, Oberti F, Michalak S, et al. A novel panel of blood markers to assess the degree of liver fibrosis. Hepatology. 2005 Dec;42(6):1373–81.
- As authors mentioned in the discussion “An increasing expression level of hepatic CD36 has been observed in NAFLD, and appears to mediate enhanced intake of free fatty acids derived from the plasma through de novo lipogenesis”. Are CD36 levels regulated by SW in the in vitro and/or in vivo model?
à Thank you for your query. It was cited to demonstrate that an increased amount of CD36 has been linked to NAFLD in previous research in our discussion.
Miquilena-Colina, M.E.; Lima-Cabello, E.; Sánchez-Campos, S.; García-Mediavilla, M.V.; Fernández-Bermejo, M.; Lozano-Rodríguez, T.; Vargas-Castrillón, J.; Buqué, X.; Ochoa, B.; Aspichueta, P., et al. Hepatic fatty acid translocase CD36 upregulation is associated with insulin resistance, hyperinsulinaemia and increased steatosis in non-alcoholic steatohepatitis and chronic hepatitis C. Gut 2011, 60, 1394-1402, doi:10.1136/gut.2010.222844.
Koonen, D.P.; Jacobs, R.L.; Febbraio, M.; Young, M.E.; Soltys, C.L.; Ong, H.; Vance, D.E.; Dyck, J.R. Increased hepatic CD36 expression contributes to dyslipidemia associated with diet-induced obesity. Diabetes 2007, 56, 2863-2871, doi:10.2337/db07-0907.
- Authors stated that “These results provide a novel therapeutic agent of hepatoprotective effects of SW for non-alcoholic hepatic steatosis that targets hepatic AMPK and ACC-mediated lipid metabolism”. In order to support this conclusion, experiments focused on the inhibition of AMPK or ACC in the presence of FFA ± SW should be performed.
à Thank you for your valuable comment. We used both models to understand the novel modulating effect of SW on NAFLD. As mentioned in the title, we concentrated on in vivo tests. As a result, we did not use an AMPK inhibitor (Compound C or siRNA).
As far as we know, inhibitors have mainly been used in studies focusing solely on in vitro experiments. For your reference, kindly find below the manuscripts justifying our viewpoint.
At that time, we did not have enough grant to conduct experiment with inhibitors and our project was finished last year. We totally agree with the reviewer’s opinion. We would like to incorporate inhibitor aspects in our further study (In vitro/in vivo).
- Water Extract of Curcuma longa L. Ameliorates Non-Alcoholic Fatty Liver Disease.
Mun J, Kim S, Yoon HG, You Y, Kim OK, Choi KC, Lee YH, Lee J, Park J, Jun W.
Nutrients. 2019 Oct 21;11(10):2536.
- Lonicera caerulea Extract Attenuates Non-Alcoholic Fatty Liver Disease in Free Fatty Acid-Induced HepG2 Hepatocytes and in High Fat Diet-Fed Mice.
Park M, Yoo JH, Lee YS, Lee HJ.
Nutrients. 2019 Feb 26;11(3):494.
- Peroxiredoxin 5 ameliorates obesity-induced non-alcoholic fatty liver disease through the regulation of oxidative stress and AMP-activated protein kinase signaling.
Kim MH, Seong JB, Huh JW, Bae YC, Lee HS, Lee DS.
Redox Biol. 2020 Jan;28:101315.
- Coenzyme Q10 attenuates high-fat diet-induced non-alcoholic fatty liver disease through activation of the AMPK pathway.
Chen K , Chen X , Xue H , Zhang P , Fang W , Chen X , Ling W .
Food Funct. 2019 Feb 20;10(2):814-823.
Minor comments:
- Why did the authors use this positive control in the in vivo study? A brief explanation will be appreciated.
à Line 399~400, we added a brief explanation about silymarin.
- Primer sequences are in Supplement Table 3, not 1.
à Line 432, we changed the number of supplement Table 1 to 3.
- The reference and dilution of primary antibodies are missing.
à Line 440~442, we added the dilution of primary antibodies.
- In line 267, authors defined NASH as non-alcoholic fatty hepatitis, but it is non-alcoholic fatty steatohepatitis.
à We are so sorry about that. Line 264, we changed ‘non-alcoholic fatty hepatitis’ to ‘Non-alcoholic fatty steatohepatitis’
Reviewer 2 Report
In this study Park et al. investigated protective effects of silk worm supplementation on progression on non alcoholic fatty liver disease. Authors showed silk worm supplementation lead to reductions in serum triglycerides and total cholesterol increased fatty acid oxidation in livers and beneficial metabolic effects appear to be mediated by AMPK. Although the study is well designed and experiments were performed well. There are few things need to be addressed to publish this study.
Most of the beneficial effects of silk worm supplementation on progression on non alcoholic fatty liver disease appear to be mediated thorough AMPK. This should be tested by treating with AMPK inhibitors in HepG2 cells.
In figure 2D quantification of PPARalpha does not match band intensity in western blots. Authors need to address this.
Author Response
Reviewer 2
In this study Park et al. investigated protective effects of silk worm supplementation on progression on non alcoholic fatty liver disease. Authors showed silk worm supplementation lead to reductions in serum triglycerides and total cholesterol increased fatty acid oxidation in livers and beneficial metabolic effects appear to be mediated by AMPK. Although the study is well designed and experiments were performed well. There are few things need to be addressed to publish this study.
Most of the beneficial effects of silk worm supplementation on progression on non alcoholic fatty liver disease appear to be mediated thorough AMPK. This should be tested by treating with AMPK inhibitors in HepG2 cells.
à Thank you for your valuable comment. We used both models to understand the novel modulating effect of SW on NAFLD. As mentioned in the title, we concentrated on in vivo tests. As a result, we did not use an AMPK inhibitor (Compound C or siRNA).
As far as we know, inhibitors have mainly been used in studies focusing solely on in vitro experiments. For your reference, kindly find below the manuscripts justifying our viewpoint.
At that time, we did not have enough grant to conduct experiment with inhibitors and our project was finished last year. We totally agree with the reviewer’s opinion. We would like to incorporate inhibitor aspects in our further study (In vitro/in vivo).
- Water Extract of Curcuma longa L. Ameliorates Non-Alcoholic Fatty Liver Disease.
Mun J, Kim S, Yoon HG, You Y, Kim OK, Choi KC, Lee YH, Lee J, Park J, Jun W.
Nutrients. 2019 Oct 21;11(10):2536.
- Lonicera caerulea Extract Attenuates Non-Alcoholic Fatty Liver Disease in Free Fatty Acid-Induced HepG2 Hepatocytes and in High Fat Diet-Fed Mice.
Park M, Yoo JH, Lee YS, Lee HJ.
Nutrients. 2019 Feb 26;11(3):494.
- Peroxiredoxin 5 ameliorates obesity-induced non-alcoholic fatty liver disease through the regulation of oxidative stress and AMP-activated protein kinase signaling.
Kim MH, Seong JB, Huh JW, Bae YC, Lee HS, Lee DS.
Redox Biol. 2020 Jan;28:101315.
- Coenzyme Q10 attenuates high-fat diet-induced non-alcoholic fatty liver disease through activation of the AMPK pathway.
Chen K , Chen X , Xue H , Zhang P , Fang W , Chen X , Ling W .
Food Funct. 2019 Feb 20;10(2):814-823.
In figure 2D quantification of PPARalpha does not match band intensity in western blots. Authors need to address this.
à We changed western blot of PPARα in Figure 2.
Round 2
Reviewer 1 Report
The responses are proper, and I understand that authors cannot conduct any experimental work since the project was finished last year. However, at least they should solve the following items:
-Did SW treatment modify the food intake of mice? It means, did mice treated with SW eat the same amount of food compared with untreated mice?
-The quantification of Oil Red O staining will be appreciated
Author Response
-Did SW treatment modify the food intake of mice? It means, did mice treated with SW eat the same amount of food compared with untreated mice?
Thank you for your understanding and comment. We calculated the cumulative mean between HFHF and HFHF + S100 and HFHF + S200 from start to end of SW supplementation. The cumulative average food intakes of the S100, S200, and HFHF groups for 8 weeks were 2.10 ± 0.5 g/day/mouse, 2.32 ±0.4 g/day/mouse, and 2.35 ± 0.6 g/day/mouse, respectively. There was no significance between groups. Also, there was not significant between HF-treated group and HF+SW treated groups at 16week before scarifying mice. SW treatment did not modify the food intake of mice.
-The quantification of Oil Red O staining will be appreciated
Thank you for your kind advice. The quantification of Oil Red O staining in HepG2 cells was shown in Figure 1c. Oil Red O optical density (%) in the liver was measured by Image-pro (ver.10) (Media cybernetics Inc., Rockville, USA). It is added in Figure 5a.
Author Response File: 
Author Response.pdf
