Elsevier

Journal of Ethnopharmacology

Volume 191, 15 September 2016, Pages 107-114
Journal of Ethnopharmacology

Root bark of Sambucus Williamsii Hance promotes rat femoral fracture healing by the BMP-2/Runx2 signaling pathway

https://doi.org/10.1016/j.jep.2016.05.017Get rights and content

Abstract

Ethnopharmacological relevance

Sambucus Williamsii Hance (SWH) is a plant from a family of Caprifoliaceae, which has a long medical history of use as an effective folk treatment for fracture bruises.

Aim of the study

To evaluate the effects of 50% ethanol extracts of root-bark of Sambucus Williamsii Hance(EE-rbSWH) on fracture healing of rats and explore its mechanism of actions related to the BMP-2 signaling pathway.

Materials and methods

EE-rbSWH was orally administered at the doses of 340 and 680 mg/kg to adult Sprague-Dawley rats with operation of open femur fracture completely for 2, 4 and 8 weeks. And the rats of sham operation and Model groups were administered Vehicle (distilled water 0.8 mL/200 g/day). Firstly, the bone X-ray morphology and bone mineral density(BMD) of the fracture site were observed and measured after anesthesia the rats at weeks 2, 4, and 8 after surgery, then the serum levels of alkaline phosphatase(ALP) and osteocalcin (BGP) were measured; Secondly, the tissue morphology of the fracture site was observed after sacrificed the rats; Thirdly, the formation of mineralized nodules in bone marrow stromal cells(BMSC) were evaluated at week 2; Lastly, the genes levels of BMP-2 and Runx2 in the femur were detected at week 2 and 4, and the proteins expression of BMP-2 signaling pathway (BMP-2, BMPRIB, BMPRII and Runx2) in the femur also were detected at week 2.

Results:

EE-rbSWH remarkably accelerated fracture healing by promoting bone formation at all the time points of fracture healing. Mainly by increasing the BMD level at the fracture site, the levels of serum ALP and BGP, and also the numbers increasing of calcified nodules in BMSC. The mechanism studies, EE-rbSWH can promote fracture healing by enhancing the expressions of BMP-2 and Runx2 mRNA, and also the proteins of BMP-2, BMPRIB, BMPRII and Runx2 at the fracture site of rats.

Conclusions:

Our results suggested that 50% ethanol extracts of root-bark of Sambucus Williamsii Hance can accelerate fracture healing by recruitment of osteoblasts at the fracture site and through up-regulation of the BMP-2 signaling pathway.

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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