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

Ethnopharrnacological 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: Comaceae), 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/beta-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, beta-catenin, Runx2, Osx involving the canonical Wnt/beta-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/beta-catenin signaling pathway. Cell proliferation and differentiation were observed by the MTT test, ALP activity and calcified nodules. The expression of Lrp-5, beta-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, beta-catenin, Runx2 and Osx involved in the canonical Wnt/beta-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/beta-catenin signaling pathway of osteoblast. (C) 2014 Elsevier Ireland Ltd. All rights reserved.