Root bark of Sambucus Williamsii Hance promotes rat femoral fracture healing by the BMP-2/Runx2 signaling pathway
Graphical abstract
Introduction
Fractures were one of the common orthopedic diseases. Nowadays, the drug therapy, which contained chemical drugs and traditional Chinese medicine (TCM), was the first choice in clinical use for treatment fracture, due to its properties of confidently use and good effects. Though chemical drugs had made some achievements in cure fracture, such as selective estrogen receptor modulators (SERMs), parathyroid hormone (PTH 1-34), bisphosphonates, and calcitonin (Gerstenfeld et al., 2003; Jung et al., 2007; Delaney et al., 2006; Gass et al., 2006). However, they were unsuitable using in long-term for their toxic side effects, the US Food and Drug Administration had recently recommended it carry a black-box warning because of its association with an increased risk of osteosarcoma in rats by using PTH 1-34 (John et al., 2002). So, people were gradually turning to develop the TCM to treatment fracture, which had small toxic side effects and suitable for long-term use in fractures patients (Ngueguim et al., 2013). Therefore, to find the new drugs of TCM with more efficacy on curing fracture was become an problem to solve urgently.
Sambucus Williamsii Hance (SWH) was a plant from a family of Caprifoliaceae (Tropicos, 2011), which was a deciduous shrub or small tree widely distributed in China, and called in Chinese “Jie-gu-mu”, “Da-jie-gu-dan”, “Gong-lao-dao”, etc (Han et al., 2008). The root bark of Sambucus Williamsii Hance (rbSWH) was a folk medicine with a long history of use in China for treatment fractures and osteoporosis (Committee of Chinese Herbals, 1999; Han et al., 2008), the earliest application of SWH in the treatment fractures was recorded in “Tang Materia Medica”. In our present work, we had determined the healing fracture effective fraction was 50% ethanol extracts of rbSWH (EE-rbSWH) (340 and 680 mg/kg) on fracture rabbits, and also found that Lignans and Iridoids compounds were the main active components in it (Han et al., 2013a, Han et al., 2013b). However, no systematic study was available to explain the mechanism of actions in healing fracture by EE-rbSWH in rats.
As we known, osteoblast function played an important role on regulating bone metabolism (Rodan and Martin, 2000, Seeman and Delmas, 2006), deficiency in osteoblast function may lead to negatives effects on bone formation and delaying the progress of fracture healing (Dutmanee et al., 2007). Osteogenic therapy had become one of the most popular methods for treatments bone disease (Liu et al., 2011). Recently, the herbal drug extracts of Sanguis Draconis resin (Ngueguim et al., 2013) and Ructus Ligustri Lucidi (Lyu et al., 2014) were found to cure bone disease by promoting osteogenic differentiation. BMP-2 protein was reported with an important effect on promoting osteogenic differentiation and maturation in skeletal and joint patterning(Noel et al., 2004). In our previous studies, we found that EE-rbSWH can promote the proliferation and differentiation on MC3T3-E1 cell mainly by BMP-2 signaling pathway in vitro (Yang, et al., 2015). In this study, we hypothesized that EE-rbSWH promoted fracture healing of rats mainly regulation by the BMP-2 signaling pathway in vivo, and we designed the experiments in fracture rats and BMSC.
Section snippets
Preparation of EE-rbSWH
Fresh plants of SWH were collected from Founder County of Harbin. The plant was authenticated by Ruifeng Fan of Heilongjiang University of Chinese Medicine. A voucher specimen (2011079) was deposited in Heilongjiang University of Chinese Medicine Harbin, China. Dry rbSWH (5 kg) was cut into 3–4 cm slices, and extracted with 95% ethanol (75 L) two times, for 2 h each time. The mixture was filtered and concentrated under reduced pressure to obtain a total extract 264 g power. The extracts (264 g) were
Compounds from EE-rbSWH
Twenty-five compounds (1–25) were isolated from EE-rbSWH, it contained 16 known compounds (1–8, 14–19, 24, 25) and 9 new compounds (9–13, 20–23). Thirteen Iridoids (1–13) were identified as α-morroniside (1), β-morroniside (2), Caryoptoside (3), Ligstroside (4) (Song et al., 2014), 7α-O-ethylmorroniside (5), 7β-O-ethylmorroniside (6), 7-dehydrologanin or 7-ketologanin (7), 7-formyloysecologanin (8) (Han, 2006), Williamsoside A (9), Williamsoside B (10) (Wang et al., 2011), Williamsoside C (11),
Discussions
Sambucus Williamsii Hance was a traditional herb medicine for treatment fracture (Committee of Chinese Herbals). In the present study, firstly, we observed the changes of bone X-ray and tissue morphology at the fracture site to determine whether EE-rbSWH could promote fracture healing. Then the BMD at the fracture site was measured to assess the quality of the new formation callus after treatment EE-rbSWH. Secondly, we measured the serum indicators of bone formation to determine whether
Conclusions
Taken together, our findings suggested that the 50% ethanol extracts of root-bark of Sambucus Williamsii Hance at the dose of 340 mg/kg can accelerate fracture healing of fracture rats, the mechanism of actions may be enhancing the recruitment of osteoblasts to the injury site and regulating by the BMP-2/Runx2 signaling pathway. Indicating that EE-rbSWH was a promising traditional Chinese medicine treatment for fractures. Our study provided a theoretical basis for the clinical use of root-bark
Conflict of interest statement
The authors declare there is no conflict of interest regarding the publication of this paper.
Acknowledgments
This publication was made possible by the National Natural Science Foundation of China (Grant no. 81173501), Harbin Application Technology Research and Development Projects (Grant no. 2014AB3AS045).
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