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Rosavin suppresses osteoclastogenesis in vivo and in vitro by blocking the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways

Background Bone homeostasis is mediated by osteoblast-related bone formation and osteoclast-related resorption. The imbalance of bone homeostasis due to excessive osteoclastogenesis or reduced osteogenesis can result in various disorders, such as postmenopausal osteoporosis (PMOP). The receptor activator of nuclear factor-κB ligand (RANKL)-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways are essential in osteoclastogenesis. In this study, we aimed to investigate the effects of rosavin, an alkylbenzene diglycoside compound from the traditional Chinese medicine Rhodiola Rosea L, on RANKL-induced osteoclastogenesis in vitro and in vivo. Methods The effects of rosavin on osteoclastogenesis were assessed by TRAP staining of bone marrow monocyte cells (BMMCs) and RAW 264.7 cells. The effects of rosavin on osteogenesis were determined using alkaline phosphatase (ALP) and alizarin red staining, as well as real-time quantitative reverse transcription polymerase chain reaction. Actin ring formation and bone formation experiments were performed to evaluate osteoclast function. Western blotting was carried out to determine the expression of osteoclastogenesis-related genes, and the activation of the NF-κB and MAPK pathways was evaluated by performing western blotting and immunofluorescence staining. Ovariectomized mice were used to explore the effect of rosavin on bone loss. Results Rosavin could inhibit osteoclastogenesis, suppress the function of osteoclasts, and decrease the expression of osteoclast differentiation-related genes, including tartrate-resistant acid phosphatase (TRAP), cathepsin K, matrix metalloproteinase-9 (MMP-9), calcitonin receptor (CTR), TNF receptor-associated factor 6 (TRAF-6), receptor activator of nuclear factor-κB (RANK), and colony-stimulating factor-1 receptor (c-fms). Rosavin inhibited RANKL-induced phosphorylation of p65 and inhibitory subunit of NF-κB alpha (IκBα), and suppressed p65 nuclear translocation. Rosavin was also found to inhibit the phosphorylation of extracellular-signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Furthermore, rosavin promoted osteogenesis in bone marrow mesenchymal stem cells (BMSCs). In vivo experiments showed that treatment with rosavin could alleviate ovariectomy-induced osteoporosis in mice. Conclusions Our results indicated that rosavin suppressed RANKL-induced osteoclastogenesis in vivo and in vitro by blocking the NF-κB and MAPK pathways. Rosavin treatment is a potential therapy for the clinical treatment of osteoclastogenesis-related disorders.

Comment 6: In Figure 2D (page 14, line 6), rosavin does not appear to suppress the expression of NFATc1. The author should explain. Reply 6: Thank you for your suggestions. In the immunofluorescent staining of NFATc1, we intended to explore the cellular location of NFATc1 before and after treatment of rosavin. In Figure 2D, we demonstrate that before the treatment, NFATc1 is mainly localized in the cytoplasm. RANKL induction translocated NFATc1 from the cytoplasm to the nucleus for the gene transcription while rosavin significantly ablated the translocation. Since it could easily lead to misunderstandings, and we have examined the expression with PCR, we removed this result.
Comment 7: In Figure 3 (page 14, line 14-line 17), the experimental procedure is unclear. For "early stage" and "late stage", describe in detail how rosavin was applied. Reply: Thank you for your suggestions. We have added more details. Changes in the text: To investigate the process of osteoclastogenesis influenced by the rosavin, after induction with M-CSF and RANKL, we treated BMMCs and RAW264.7 cells with rosavin on day 1, 3, or 5 after the induction. TRAP staining results showed that rosavin mainly suppressed osteoclast formation on the first day (**p<0.01). However, when given on day 3 for RAW 264.7 cells and day 5 for BMMCs, rosavin could not inhibit osteoclast formation ( Figure  3A and 3B). Figure 4 (page 15, line 2-line 10), rosavin does not appear to suppress the nuclear translocation of p65. The graph is also small and it is difficult to determine whether it is significantly suppressed. Reply 8: Thank you for your advice. We have adjusted the graph and make it clear to see. In Control group, we could see that the p65 is mainly localized in the cytoplasm with almost no fluorescence in the nucleus. RANKL incubation almost drives all p65 into the nucleus while rosavin significantly blocks the translocation. Changes in the text: Figure 4A. Comment 9: In Figure 5B (page 16, line 9-line 10), it is difficult to determine the inhibitory effect of rosavin on micro CT, so it should be described in more detail. Reply: Thank you for your advice. We have added more detail in the micro CT results. We added the 3D reconstruction representative images and the BV/TV, BMD, Tb.N and Tb. Area analysis. Changes in the text: Figure 5B. Page 16, line 9.

Comment 8: In
Comment 10: Overall, the graph is too small, and the effect of rosavin in immunostaining is difficult to understand. Therefore, it is necessary to improve the quality of the figure. There are many mistakes in the paper, and all of them cannot be list. All mistakes should be corrected. Reply: Thank you for your comments. We have improved all the images and corrected the mistakes in the text.

Reviewer B
Zhang et al seeks to identify mechanism of rosavin regulated bone formation. The study would benefit from increased clarity on methods used and time points when experiments were performed. Reply: Thank you for your comments. We have made the corresponding changes based on your suggestions.
Major comments: Comment 1: Statistics should be added to MTT assay for figure 1A. Reply: Thank you for your advice. We have supplemented the statistics in MTT assay.
Comment 2: Graphs for figure 1, 2 are cloudy, small, and need to be higher resolution. Reply: Thank you for your advice. We have selected clear graphs for figure 1 and 2. Comment 4: To improve clarity, text for figure 2 should identify time point for treated with rosavin and induction. In addition, 2C says RANKL + 1d, 3d, 5d. There is no reference to treatment with rosavin or concentration used however results mention use of rosavin. Reply: Many thanks for your comments. We have moved precious Figure 2C to Figure 3C because it shows the effects when rosavin was given on 1, 3 and 5. Changes in the text: Figure 3C.
Comment 5: Methods should be included on how BMMCs and BMSC were acquired in addition to identify how the cells were validated prior to use. In addition are Bone marrow monocytes (BMMCs) and BMSCs: are cells from a single mouse donor or are triplicates representative of multiple mice? What is western blot representative of-tissues, cells? Reply: We have added the isolation methods for BMMCs and BMSC. Cells are triplicates representative of multiple mice. Western blotting results represent cells. We have clarified in the text. Many thanks. Changes in the text: Page 7, line 15-18. BMMCs were washed from the femurs of the C57 mice. Cells were cultured in α-MEM (1% penicillin, 1% streptomycin and 10% FBS). Cells were