Research paper
The effects of Liuwei Dihuang on canonical Wnt/β-catenin signaling pathway in osteoporosis

https://doi.org/10.1016/j.jep.2014.01.040Get rights and content

Abstract

Ethnopharmacological relevance

The Liuwei Dihuang (LWDH), a wellknown classic traditional Chinese medicine formula, consists of six herbs including Rehmannia glutinosa Libosch. (family: Scrophulariaceae), Cornus officinalis Sieb. (family: Cornaceae), Dioscorea opposite Thunb. (family: Dioscoreaceae), Alisma orientale (G. Samuelsson) Juz (family: Alismataceae), Poria cocos (Schw.) Wolf (family: Polyporaceae) and Paeonia suffruticosa Andrews (family: Paeoniaceae). It has been used clinically in the treatment of many types of diseases with signs of deficiency of Yin in the kidneys for more than 1000 years in China. The purpose of this study was to observe the effects of LWDH on canonical Wnt/β-catenin signaling pathway in osteoporosis.

Materials and methods

Osteoporosis model was induced by ovariectomy (OVX) in 8-week-old female Sprague–Dawley (SD) rats. After 12 weeks of treatment with LWDH by intragastric administration, the rats were put to death in batch. The changes of alkaline phosphatase (ALP), osteocalcin (BGP) and estradiol (E2) in serum were determined, bone mineral density (BMD) and histomorphology of right femur were observed, biomechanics of lumbar vertebra were measured, and the expression of Lrp-5, β-catenin, Runx2, Osx involving the canonical Wnt/β-catenin signaling pathway were detected by RT-PCR. In addition, osteoblasts isolated from neonatal rat calvariae were used in this study to investigate the effects of LWDH on the canonical Wnt/β-catenin signaling pathway. Cell proliferation and differentiation were observed by the MTT test, ALP activity and calcified nodules. The expression of Lrp-5, β-catenin, Runx2, Osx mRNA of cells were also detected. All the data were analyzed by SPSS 13.0.

Results

Twelve weeks of treatment with LWDH could significantly decrease the level of ALP and BGP in serum, increase the BMD of femurs, and improve the biomechanical capabililty of vertebral body in maximum loading and elastic modulus. Concerning histomorphology, we found ordered arrangement of trabeculae, slightly thinning of trabeculae and none obvious slight fractures in femurs after twelve weeks of treatment with LWDH. In osteoblast, serum containing LWDH elicited significantly increase in cell viability (at day 6), alkaline phosphatase activity (at days 2, 4 and 6) and amount of calcified nodules. The expression of Lrp-5, β-catenin, Runx2 and Osx involved in the canonical Wnt/β-catenin signaling pathway were significantly up-regulated in the presence of LWDH both in vivo and in vitro experiment.

Conclusions

Our results suggest that Liuwei Dihuang could alleviate osteoporosis induced by ovariectomy, in part, through up-regulation of canonical Wnt/β-catenin signaling pathway of osteoblast.

Graphical abstract

Our results suggest that Liuwei Dihuang could alleviate osteoporosis induced by ovariectomy, in part, through up-regulation of canonical Wnt/β-catenin signaling pathway of osteoblast.

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Introduction

Osteoporosis is the condition in which a low bone mass and altered bone microarchitecture leads to increased risk of fragility fracture (Collins et al., 2011, Rachner et al., 2011). Clinically, osteoporosis has been divided into two types: primary and secondary osteoporosis. The former usually results from aging and decreased gonadal function, such as decreased level of estrogen, while the latter is often the complication caused by other health problems and drugs, such as the prolonged corticosteroid therapy (Rachner et al., 2011). Osteoporosis is considered by the World Health Organization (WHO) to be an important health issue secondary to coronary heart disease. About 40% of postmenopausal women are affected by osteoporosis, and with an ageing population, this number is expected to steadily increase in the near future (Melton et al., 1992, Burge et al., 2007). In addition, about 18% of women and 6% of men worldwide will experience hip fragility fractures resulting from osteoporosis (Piscitelli et al., 2007). Current therapeutics in clinical use included bisphosphonates, anti-RANKL, parathyroid hormone, and strontium ranelate, which may reduce the risk of first and recurrent fractures (Das and Crockett, 2013). The direct and indirect costs of osteoporosis were estimated at $21.9 billion in American in 1998 (Max et al., 2002). However, these drugs remain costly and are not afford for the majority of Chinese. Therefore, there is an urgent and increasing need for effective but lower-cost pharmacotherapy.

The Liuwei Dihuang (LWDH), a classic traditional Chinese medicine formula, has been used clinically in the treatment of many types of diseases with signs of deficiency of Yin in the kidneys for more than thousand years in China. LWDH is most early recorded in the book of “Xiaoer YaoZheng ZhiJue”, and consists of six Chinese crude herbs: Rehmannia glutinosa Libosch. (family: Scrophulariaceae), Cornus officinalis Sieb. (family: Cornaceae), Dioscorea opposite Thunb. (family: Dioscoreaceae), Alisma orientale (G. Samuelsson) Juz (family: Alismataceae), Poria cocos (Schw.) Wolf (family: Polyporaceae) and Paeonia suffruticosa Andrews (family: Paeoniaceae) as a proportion of 8:4:4:3:3:3 (Wu et al., 2007, Zhao et al., 2007, Wang et al., 2010, Liu et al., 2013).

LWDH is orally administered as a decoction or in the form of pills, which is applied for the declined functions related to aging process and geriatric diseases, especially for diabetes, cognition and memory in china and Japan (Yokozawa et al., 2004, Wang et al., 2010, Liu et al., 2013). Recently, a number of studies have revealed that LWDH has therapeutic effects on osteoporosis and some possible mechanisms of action have been put forward (Chen et al., 1994, Sun et al., 2006, Peng, 2007); however, the precise mechanism has not yet been fully revealed.

The activation of different bone cells in bone remodeling process is regulated by multiple pathways and these pathways are exploited in the development of new therapies for osteoporosis. The canonical Wnt/β-catenin signaling pathway, which controls multiple developmental processes in skeletal and joint patterning also be involved in the progression of osteoporosis (Fei and Hurley, 2012, Brunetti et al., 2013, Kim et al., 2013, Xu et al., 2013).

Since genetic association studies suggest that the canonical Wnt/β-catenin signaling pathway plays a critical role in the pathogenesis of osteoporosis, we hypothesized that the benefit of LWDH on osteoporosis based on the regulation of the canonical Wnt/β-catenin signaling pathway. In our study, the key molecules involved in the canonical Wnt/β-catenin signaling pathway, including Lrp-5, β-catenin, Runx2 and Osx, were detected after treatment with LWDH both in vivo and in vitro experiments.

Section snippets

Preparation of LWDH

The method of preparation of LWDH was obtained from Yang et al. (2006). In brief, Rehmannia glutinosa Libosch. (family: Scrophulariaceae), Cornus officinalis Sieb. (family: Cornaceae), Dioscorea opposite Thunb. (family: Dioscoreaceae), Alisma orientale (G. Samuelsson) Juz (family: Alismataceae), Poria cocos (Schw.) Wolf (family: Polyporaceae) and Paeonia suffruticosa Andrews (family: Paeoniaceae) were mixed as a proportion of 8:4:4:3:3:3, and the total dry weight was 2 kg. The mixture of herbs

General condition of osteoporosis model

The success of osteoporosis model was confirmed by serum ALP, BGP, E2 and bone mineral density examination after twelve weeks of ovariectomy. When compared with control group, the level of ALP and BGP in serum were significantly increased in OVX group (P<0.05). Comparison of E2 and BMD, those in OVX group were significantly reduced than that of control group (P<0.05) (Fig. 1a–d). During the period of experiment, mean body weight of rat consistently increased in each group, while there was no

The model of osteoporosis

Setting up an appropriate animal model is very important in researches or studies. An ideal animal model should be similar to the prototype, and through analysis of models we could obtain partial information about prototypes. In our study, we used a well described, estrogen deficiency related model, which mimics features of postmenopausal osteoporosis women (Kalu, 1991, Aoyagi et al., 1996, Goss et al., 2004, French et al., 2008, Fathilah et al., 2013). This model, set up firstly by Saville

Acknowledgments

This publication was made possible by the National Natural Science Foundation of China (Grant no. 81202710), State Administration of Tradition Chinese Medicine of Zhejiang province (Grant no. 2009YA001), Zhejiang Provincial Natural Science Foundation of China (Grant no. Y2111184), and the Science Technology Department of Zhejiang province (Grant no. 2012R10064).

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