Center for Herbal Medicine Improvement Research
Korea Institute of Oriental Medicine
483 Expo-ro, Yuseong-gu, Daejeon 305-811, Republic of Korea
To. Editor
In BMC Complementary and Alternative
Medicine
Jin-Yeul Ma, Ph.D.
Tel: +82-42-868-9466
Fax:+82-42-868-9573
E-mail: jyma@kiom.re.kr
November, 27, 2012
Dear, Editor
We have carefully read the reviewer’ comments and have included our responses below. We
thank the reviewers for improving the quality of this manuscript and hope that you consider the
revised manuscript for possible publication in BMC Complementary and Alternative
Medicine.
We look forward to hearing from you.
Thank you.
Best regards,
Jin Yeul Ma
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Reviewer 1:
1.1. Figure 3: Explain why authors did not quantify differences in mRNA expression and use
statistics to conclude the inhibitory effects of the different samples.
->We have quantified and analyzed the statistical difference of mRNA expression in
the “Results” section (page 10-11) of the revised manuscript like as below.
“HRT-BU significantly decreased mRNA levels of c-Fos about 2 folds less than
vehicle at day 1 and NFATc1 about 6 folds less than vehicle at day 2, respectively (Figure 3A).
Notably, fHRT-BU significantly decreased mRNA levels of c-Fos about 14 less than vehicle and
NFATc1 about 10 fold less than vehicle at same day. We also investigated mRNA levels of
NFATc1-regulated osteoclastogenesis related genes, including TRAP, ATP6v0d2, and cathepsin
K. HRT-BU significantly decreased mRNA levels of osteoclastogenesis related genes about 4
fold less than vehicle at 4 day. Furthermore, fHRT-BU significantly decreased mRNA levels of
osteoclastogenesis related genes about 13 to 40 fold less than vehicle at same day.”
->We analyzed the statistical significance of mRNA levels of genes by Student’s t-test
and described it in the “Method”. We have marked the statistical significance of mRNA
expression (Figure 3) in the “Figures” of the revised manuscript as the reviewer’s
recommendation.
1.2. Method paragraph 9; What is the rationale for choosing the dose of 0.3 g/kg of fHRT in the
in vivo experiment? Authors please add route of administration (Oral?) and single or multiple
doses used in this study.
->We have provided the rationale for using 0.3 g/kg as the oral administration dosage
and the route of administration in the “Methods” section (page 8-9) of the revised manuscript
like as below.
“Oral administration of samples once a day began 1 week and finished 3 months after
OVX surgery. Dosage for oral administration was based on maximum dosage for daily
administration of herbal medicine in traditional herbal medicine clinic. The same amount of
saline was orally administered to the sham and OVX groups.”
1.3. Discussion – paragraph 2, please explain why HRT-BU’s effect on osteoclastogenesis was
less potent in cocultured osteoclasts than in single culture BMM’s (Fig2) and not effective at all
in the in vivo model (fig6).
->In MNC counting, the inhibitory percentage of HRT-BU at 3 μg/ml on BMM
culture was 81 + 12. That of HRT-BU at 3 μg/ml on co-culture was 66 + 11. Although the
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inhibitory effect of HRT-BU in BMM culture was greater than that of HRT-BU in co-culture,
these values were within the standard deviation and there was no statistically significance. Thus,
we concluded that the inhibitory effect of HRT-BU at 3 μg/ml on MNC counting in BMM
culture was greater than co-culture system without statistical significance. We have described it
in the “Results” section (page 10) of the revised manuscript like as below.
“Although the inhibitory effect of HRT-BU at 3 μg/ml on MNC counting in BMM
culture was greater than that of HRT-BU in co-culture, there was no statistically significance.”
->We have suggested that the different efficacy between HRT and fHRT in OVXinduced bone loss model might be due to the effect of bacterial fermentation, which increases
the generation of active components, the absorption rate, or the amounts absorbed in body. We
have described it in the “Discussion” section (page 14) of the revised manuscript.
2. Minor essential revisions
->To improve the grammatical mistake, we proofreaded the manuscript as the
reviewer’s recommendation in the revised manuscript.
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Reviewer 2:
1. In cell-based and in vivo assay, fHRT more strongly inhibited the RANKL-induced formation
of TRAP-positive multinucleated osteoclasts compared to HRT. But, authors failed to show the
difference between both in mode of action study. Different with the inhibitory activity, c-Fos
was strongly inhibited by HRT. c-Fos is one of up-stream regulators of NFATc1. Also,
overexpression of NFATc1 did not rescue the inhibitory action of HRT or fHRT. With this
result, authors suggested that HRT-BU and fHRT-BU can act as an inhibitor of NFATc1
activity or downstream signaling. This suggestion is not acceptable to elucidate your study. In
conclusion, authors showed the phenotypic action of HRT and fHRT, but failed to elucidate the
mode of action.
->In this study, we found that fHRT-BU decreases NFATc1 expression more than
HRT-BU. Particularly, fHRT-BU inhibited RANKL-induced NFATc1 expression at 2 day, which
is a time point that NFATc1 profoundly amplifies NFATc1 itself. Furthermore, the inhibitory
effect of fHRT-BU on endogenous of NFATc1-induced and ectopic expression of Ca-NFATc1induced osteoclastogenesis was greater than that of HRT-BU. Therefore, we suggested fHRTBU might inhibit NFATc1 activity or its downstream signaling for osteoclastogenesis.
-> In this study, we also found that fHRT-BU partially decreases ERK and JNK
activation, which was not affected by HRT-BU. In addition, fHRT-BU decreased c-Fos
expression although it is less than HRT-BU. Based on these results, we suggested fHRT might
affected ERK and JNK dependent pathway to regulate AP-1 expression, which eventually
induces NFATc1 expression on osteoclastogenesis. Taken together these results, we described
the inhibitory mode of action by fHRT on c-Fos and NFATc1 expression in the “Discussions”
section (page 13) of the revised manuscript.
2. In figure 6, no correlation between values and image (G). In figure 6G, fHRT-treated sample
is osteopetrotic; cortical bone is thicker and the trabecular bone is denser than the control.
->We have replaced the micro-CT images of trabecular and cortical bone, which more
represents and correlates with the bone parameter in the “Figure 6” of the revised manuscript.
3. Statistical analysis is full required in all graphs.
->We analyzed the statistical significance of TRAP activity and mRNA levels of genes
by Student’s t-test. We also analyzed the statistical significance of bone parameter by one-way
ANOVA and Duncan multiple comparison test as described in the “Method”. We have marked
the statistical significance of mRNA expression (Figure 3) in the “Figures” of the revised
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manuscript as the reviewer’s recommendation.
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