Title:Fulvic acid attenuates homocysteine-induced cyclooxygenase-2 expression in human monocytes

Transcription

1 Author's response to reviews Title:Fulvic acid attenuates homocysteine-induced cyclooxygenase-2 expression Authors: Shao-Ju Chien Te-Chuan Chen Hsing-Chun Kuo Cheng-Nan Chen Shun-Fu Chang Version:3Date:5 December 2014 Author's response to reviews: see over

2 Dear Editors: Thank you for your letter of Nov. 17, We have made major revision of the manuscript by Shao-Ju Chien et al. on Fulvic acid attenuates homocysteine-induced cyclooxygenase-2 expression in human monocytes in accordance with the reviewers and your valuable comments. Additional experiments have been added and the text has been modified accordingly in this revised manuscript to respond to the reviewers and your comments. All co-authors have read and approved the submission of this revised manuscript. The responses to the reviewers comments, including the point-by-point description of the changes made, have been appended to next page. On the website please find our revised manuscript ( ). We wish to thank you and the reviewers for your constructive critiques, which have greatly improved the paper. Your editorial consideration of this revised manuscript would be much appreciated. Sincerely, Shun-Fu Chang, Ph.D. Biophotonics and Molecular Imaging Research Center National Yang-Ming University No. 155, Sec. 2, Linong St. Beitou District, Taipei, Taiwan, ROC. Phone: Fax:

3 MS NO.: MS TITLE: Fulvic acid attenuates homocysteine-induced cyclooxygenase-2 expression AUTHORS: Shao-Ju Chien, Te-Chuan Chen, Hsing-Chun Kuo, Cheng-Nan Chen and Shun-Fu Chang Reply to the Reviewer--Zongbing You: Major Comment (1): Please provide data from primary monocytes in the experiments of Figure 4, Figure 5, and Figure 6. Response: It is much appreciated that the Reviewer made this valuable comment. Pursuant to the reviewer s comment, in the revised manuscript we have performed additional experiments by using human primary monocytes in the Figure 4~6. We have also added these new data and statements regarding these results in the revised manuscript (New Figure 4A, 4B, 5A, 5B, and 6A; Results: page11, line 203; page 12, lines ; Discussion: page 15, lines ; Figure legends: page 24, lines 462, 466, 476, and 478). Major Comment (2): What is the relationship between activation of ERK/JNK and activation of NF-kB? In Figures 4C and 4D, please provide data showing changes in p-ikb and/or nuclear localization of p65, and does inhibitors of ERK/JNK affect activation of NF-kB? Response: It is much appreciated that the Reviewer made this valuable comment. Pursuant to the reviewer s comment, in the revised manuscript we have performed additional experiments to identify the relationship between ERK/JNK and NF-κB. The results showed that (1) treating cells with homocysteine induced IκB phosphorylation (new Figure 5D) and (2) pretreating cells with ERK and JNK specific inhibitor attenuated homocysteine-increased NF-κB p65 activity (new Figure 5E). We have also added these new data and statements regarding these results in the revised manuscript (Results: page 12, lines ; Figure legends: page 24, lines ; page 25, line 482). Major Comment (3): Out of the 32 patients enrolled, how many patients' monocytes were tested in Figures 1A, 2A, and 2C? Response: We thank reviewer for this comment and we apologize for the mistakes, the monocytes used in this study were isolated form buffy coat of normal volunteers (Taiwan Blood Center, TBSF, Taiwan), not from patients. We deleted the description of Subjects in the MATERIALS AND METHODS and add the statements about the source of buffy coat in the Cell culture (Materials and Methods: page7, lines ). Minor Comment (1): Line 117, "proved" should be "approved". Response: We thank reviewer for this comment. The Subjects, including Line 117, in the MATERIALS AND METHODS has been deleted in the revised manuscript. Minor Comment (2): Line 137, PGE2 ELISA assay kit is from which company? Response: We tha nk the reviewer for this comment. PGE2 ELISA assay kit was purchased from R&D Systems. This statement has been added in the revised manuscript (Materials and Methods: page 8, line 134). Minor Comment (3): Line 185, add "in a" before "dose dependent manner". 1

4 Response: It is much appreciated that the reviewer made this valuable comment. We have modified this sentence in the revised manuscript (Results: page 10, line 182). Minor Comment (4): Figure 4D, ERK panel is black, please provide a better blot. Response: It is much appreciated that the Reviewer made this valuable comment. We have provided another blot in the revised manuscript (new Figure 4C). 2

5 MS NO.: MS TITLE: Fulvic acid attenuates homocysteine-induced cyclooxygenase-2 expression AUTHORS: Shao-Ju Chien, Te-Chuan Chen, Hsing-Chun Kuo, Cheng-Nan Chen and Shun-Fu Chang Reply to the Reviewer--Cheng-Yang Huang: Comment (1): The molecular structure of fulvic acid may need to be included in this manuscript. This can improve the readership and further drug optimization. Response: It is much appreciated that the reviewer made this valuable comment. We have added the FA structure in the revised manuscript (new Figure 7). Comment (2): They have provided several lines of evidences that fulvic acid inhibits homocysteine-induced COX-2 expression in monocytes via a signaling pathway within ERK and JNK. The inhibition pathway(s) may be worth a figure for conclusion. Response: It is much appreciated that the reviewer made this valuable comment. We have added Schematic diagram in the revised manuscript (new Figure 7). 3