Boiogito, a Japanese Traditional Herbal Medicine, Inhibits the Osteoclast Differentiation and Proliferation in the Subchondral Bone of an In Vivo Knee Osteoarthritis Rat Model
, Hideshi Ikemoto 1, Naoki Adachi 1, Haruka Takemura 1,2, Midori Mochizuki 1, Kanako Izukashi 1,2, Koji Kanzaki 2 and Masataka Sunagawa 1
Round 1
Reviewer 1 Report
The authors reported that Boiogito, a traditional Japanese herbal medicine, may be inhibiting knee osteoarthritis progression by inhibiting osteoclast differentiation and proliferation in subchondral bone, as evidenced by an increase in TRAP-positive multinucleated cells. This research would be beneficial to both medical practitioners and researchers working on bone health. However, to supplement the authors' findings, I propose testing biomarkers of osteoclast differentiation.
Minor revisions
Line 131: Change "reared" to "treated".
Line 132: When you say periodically, is it weekly, or monthly? be specific.
Author Response
Dear Reviewer 1,
Line 131: Change "reared" to "treated".
Line 132: When you say periodically, is it weekly, or monthly? be specific.
Thank you for pointing those up. We have checked and appropriately changed.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The introduction is very well written and provides all the important information for the reader. Aim of the research is clearly stated – authors want to examine if BO administration in food might decrease damage in DMM model of KOA. In principle results of experiments performed in present study are indicating that the use of BO in even a low dose is beneficial for rats with the DMM model of KOA. The overall merit of the study is positive – with the important assumption that authors underline that it is basic research. Still some issues require clarification by the authors of this study:
1. Authors refer to the beneficial effect of curcumin, oleanolic acid, and Osteoking on cartilage regeneration. Nevertheless, they refer to literature in the very laconic way in this field. Are authors sure that curcumin treatment is such beneficial for this purpose? It would be responsible to provide wider view on those topics also considering limitations and possible challenges associated with those compounds.
2. Authors assume long term benefit of their therapeutic approach on depletion of osteoclasts proliferation. While such approach is clearly beneficial for treatment of KOA question remains, how depletion of osteoclasts will affects whole organisms functioning? Second question is how use of BO affects homeostasis of organism?
3. How exactly authors counts multinucleated osteoclasts (TRAP-positive). Was it manual counting from microscopic images or authors applied macro? Please provide more details. Also statistic for this type of quantification is missing.
4. Figures quality must be improved. Please increase resolution for 300 dpi, also consider boldening lines e.t.c.
5. Authors show improvement after BO treatment – what is straightforward visible from their data. Nevertheless, a wider explanation of the mechanisms of BO action is missing in this study. For instant inhibition of particular pathways engaged in pathological processes which might be affected by BO. Please refer to this issue.
6. Authors state that time-course changes will be considered in future studies. What time scale is planned in future research?
Author Response
Reviewer 2
Thank you for your detail check and suggestions. We have modified the manuscript and answered your comments below. Please check them.
- Authors refer to the beneficial effect of curcumin, oleanolic acid, and Osteoking on cartilage regeneration. Nevertheless, they refer to literature in the very laconic way in this field. Are authors sure that curcumin treatment is such beneficial for this purpose? It would be responsible to provide wider view on those topics also considering limitations and possible challenges associated with those compounds.
Thank you for pointing this out. As you mentioned, it is controversial whether these compounds are really beneficial to the patient. In particular, the data that are only reported in in vitro are of limited usefulness, and the fact that the doses and safety at which they are effective for human remain unclear. From that point of view, it cannot be determined at this time whether curcumin is truly a beneficial therapeutic agent. What distinguishes BO from these compounds is that this kampo medicine is actually used clinically.
- Authors assume long term benefit of their therapeutic approach on depletion of osteoclasts proliferation. While such approach is clearly beneficial for treatment of KOA question remains, how depletion of osteoclasts will affect whole organisms functioning? Second question is how use of BO affects homeostasis of organism?
Thank you for your suggestions. First, subchondral bone responds primarily to increased or decreased mechanical loading of the articular surface. The hypermetabolism of that subchondral bone causes catabolic changes due to the proliferation of osteoclasts, which leads to the formation of bone cysts near the joint line. Because lesions within the bone cysts resulting from osteoclast activation are vulnerable to loading stress and cause joint destruction, some modification of the hypermetabolism of the subchondral bone is necessary to control this process. In addition, as considered in many herbal medicines, the effect of BO in maintaining organ homeostasis may be based on its anti-inflammatory action. Differentiation and proliferation of osteoclasts requires inflammatory mediators on macrophages or osteoclast progenitor cells, and BO likely inhibits part of this process. As you may have suspected, depletion of osteoclasts causes a variety of problems. However, as can be seen from the results in figure 4, the administration of BO did not significantly reduce the number of osteoclasts compared to the control group, so we do not think that there will be any major problems in BO administration. In fact, it has not been reported that long-term BO administration causes pathological changes in bone.
- How exactly authors count multinucleated osteoclasts (TRAP-positive). Was it manual counting from microscopic images or authors applied macro? Please provide more details. Also, statistic for this type of quantification is missing.
As you suggested, counting of the osteoclasts was done manually with the microscope images. To ensure intra-rater reliability, counts were performed three times for each sample, and the mean of the three was calculated. To ensure inter-rater reliability, the same measurements were performed again by a third party, and the intraclass correlation coefficient was confirmed as greater than 0.80.
- Figures quality must be improved. Please increase resolution for 300 dpi, also consider boldening lines e.t.c.
Thank you for pointing it up. We have improved the quality of Figures.
- Authors show improvement after BO treatment – what is straightforward visible from their data. Nevertheless, a wider explanation of the mechanisms of BO action is missing in this study. For instant inhibition of particular pathways engaged in pathological processes which might be affected by BO. Please refer to this issue.
A constantly debated issue in research on herbal medicines such as Kampo medicine is the fact that the research compounds contain so many chemicals that it is easy to speculate on the involvement of multiple pathways when investigating the mechanism of the action. What the above means is that a detailed study for the mechanism of kampo medicine would require numerous pathways to be intercepted and a huge amount of experimental cost. We see this as a very challenging and important issue, though. This means that in order to elucidate the anti-inflammatory effects of kampo medicine, we have to engage in a comprehensive view, including changes in the intestinal microbiota and its effect on the autonomic nervous system. One of the possible answers to your point is the inhibition of NLR family pyrin domain containing 3 (NLRP3) inflammasome formation in bone marrow macrophages or osteoclast precursor cells, or the activation of Sirtuin 1 (SIRT 1) in chondrocytes, but to be honest, we are not sure how beneficial such a detail-issued study would be for BO or herbal medicine.
- Authors state that time-course changes will be considered in future studies. What time scale is planned in future research?
Thank you for your question. It has been reported in previous reports that the articular cartilage is largely worn and degenerated approximately 8 to 10 weeks after the DMM rat model was created, and the pathophysiology of KOA becomes severe. In this study, a 4-week rearing time was set to capture the initial pathology, but an 8-week rearing time should be considered in the future. At the same time, we believe that it is worth investigating whether the effect of oral administration of BO after 4 weeks the DMM rat model has been established will also have an inhibitory effect on the progression of KOA.
Author Response File: Author Response.pdf
