Total saponin from Anemone flaccida Fr. Schmidt abrogates osteoclast differentiation and bone resorption via the inhibition of RANKL-induced NF-κB, JNK and p38 MAPKs activation
Authors: Xiangying Kong
Hongyan Zhao
Hongye Wan
Wenbin Wu
Xiaomin Li
Michun Zhong
Na Lin
Shiwei Jia
Xiaohui Su
Yue Yang
Dahong Ju
Journal: Journal of translational medicine (2015)
Discipline: CLINICAL MEDICINE
Keywords: Osteoclasts; bone destructive diseases; natural plant-derived products; total saponin; Anemone flaccida Fr. Schmidt
Affiliations: Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, No. 16, Nanxiaojie, Dongzhimennei, Beijing, 100700, China. ., Institute of Basic Theory, China Academy of Chinese Medical Sciences, Beijing, 100700, China. ., Guangzhou Kanghe Pharmaceutical Limited Company, Guangzhou, 511440, China. .

Abstract:  Osteoclasts, bone-specialized multinucleated cells, are responsible for bone destructive diseases such as rheumatoid arthritis and osteoporosis. Natural plant-derived products have received substantial attention given their potential therapeutic and preventive activities against bone destructive diseases. In the present study, we investigated the effects of total saponin (TS) from Anemone flaccida Fr. Schmidt, on receptor activator of nuclear factor-κB ligand (RANKL)-induced in vitro osteoclast differentiation. We observed that TS concentration-dependently inhibited RANKL-induced osteoclast formation from RAW 264.7 cell and bone marrow-derived macrophages (BMMs), as well as decreased extent of actin ring formation and lacunar resorption. The RANKL-stimulated expression of osteoclast-related transcription factors were also diminished by TS. Moreover, TS blocked the RANKL-triggered TRAF6 expression, phosphorylation of mitogen-activated protein kinases (MAPKs) and IκB-α, and inhibited NF-κB p65 DNA binding activity. Furthermore, TS almost abrogated the nuclear factor of activated T cells (NFATc1) and c-Fos expression. Taken together, our results demonstrated that TS suppresses RANKL-induced osteoclast differentiation and inflammatory bone loss via the down-regulation of TRAF6 level, suppression of JNK and p38 MAPKs and NF-κB activation, and subsequent decreased expression of c-Fos and NFATc1. Therefore, TS may be a potential agent and needs to be more evaluated in vivo or in clinical trials to become a therapeutic for lytic bone diseases. 

Retracted: No

PubPeer comments:
 Figure 5A and 6A. * Blue boxes: Four lanes in the GAPDH panels of Figure 5A and 6A appear to show an overlap. The figures are showing the same experiment (TS concentration series) but the lanes are shifted so the individual lanes no longer represent the same conditions. ![file] , 
 The same GAPDH blot appears to be visible in another paper by the same research group that was submitted about a year later, i.e. Kong X et al. Int. J. Biol. Sci. 2015, Vol. 11, DOI: 10.7150 ijbs.12296. See: https: pubpeer.com publications 5FFC5075F0E59D3307E8EC9B2EA255 . In this older paper, the blot represents a TS concentration series, while in the other newer paper, the blot represents a W3 concentration series. Here are Figures 5A and 6A from this older Kong et al. paper (left) compared to Figures 7A and 8A from the other newer paper (on the right) ![file] , 
 Figure 1A from this paper appears to have been reused in Figure 2 of the newer paper, Kong X et al. Int. J. Biol. Sci. 2015, Vol. 11, DOI: 10.7150 ijbs.12296. The experiments are the same, but the TS 2.5 image is rotated 180 degrees. ![file] , 
 Fig 4B:  TS abrogates expression of TRAF6 in RANKL-induced RAW 264.7 cells. RAW 264.7 cells were pre-treated with RANKL (50 ng mL), and cell lysates were collected at... various concentrations of TS (0.1, 0.5 and 2.5 u03bcg mL) for 24 h. Below: Figure 7A.  TS down-regulates RANKL-induced expression of NFTAc1 and c-Fos in RAW 264.7 cells. RAW 264.7 cells were preincubated with indicated concentrations of TS for 2 h, and then treated with 50 ng mL RANKL for 24 h. Total protein was extracted and subjected to Western blot analysis using antibodies against NFATc1 and c-Fos (A). ![file] The conditions appear to be different (pre-treatment vs post-treatment with RANKL), but the loading control bands are very similar - albeit with spaces between lanes in 7A. , 
 #3 Fig 4B of this paper *[below]* also seems to share a GAPDH loading control with *[above]* 6B from [Kong et al (2015)] . The two figures represent increasing doses of TS and of W3 respectively. ![file] , 
 One more for the sake of completeness. Below, Fig 6A:  TS suppresses RANKL-induced NF-u03baB activation in RAW 264.7 cells. Confluent RAW 264.7 cells were pre-treated with various concentrations of **TS** (0.1-2.5 u03bcg mL) for 2 h followed by RANKL (50 ng mL) for 30 min. Above, Fig 8 from [Kong et al (2015)] :  W3 suppresses NF-u03baB activation in RANKL-induced RAW 264.7 cells. (A) Confluent RAW 264.7 cells were pre-treated with various concentrations of **W3** (0.1-2.5 u00b5g mL) or vehicle for 2 h followed by RANKL (50 ng mL) for 0.5 h . Different sets of conditions but same GAPDH loading control. ![file] , 
 Dear Dr.Bik, I just got the message from the administration department of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, and informed me that you raised some inquiries on my research papers via the Pubpeer. Many thanks for your kind concern and sorry for later response due to not checking the Pubpeer website recently. Regarding to the inquiries and concerns in the papers mentioned, I will take the highest priority and immediately start investigations, including carefully checking the manuscripts, raw data and experimental records. And also I will contact with the editorial offices of the relevant journals as soon as possible, if there isany risk to the highest degree of accuracy of our scientific findings. After a quick check on some inquiries and concerns (the careful check is still ongoing), I am confident in the validity of our scientific findings published in those articles. Nevertheless, there is no excuse for any mistakes in the papers as thecorresponding author, a lab leader and supervisor. I will be more than happy to make the responses accordingly with careful double-checking via the Pubpeer. Again, many thanks for your concerns which definitely alert me and help me in my future research. Also I must sincerely apologize for any mistakes, including oversight and inconvenience caused, to my students, colleagues and collaborators.I will take this as an invaluable learning opportunity, and keep the accuracy and integrity of science as my first priority in my research. Best regards! Yours, sincerely, Na LIN , 
 Correction, May 2021: https: translational-medicine.biomedcentral.com articles 10.1186 s12967-021-02868-5  *After publication of this article [1] it was brought to our attention that there were errors in Figs. 4a, b, 5a, 6a and 7a. We found that the GAPDH control bands were mistakenly placed. There were also some errors in the description of cell treatment in the legend of Fig. 4. The corrected figures and figure legend are shown below. These corrections do not change the results or conclusions of the article. We apologise to readers for these errors.* ,