Effect of Zuoguiwan on osteoporosis in ovariectomized rats through RANKL/OPG pathway mediated by β2AR

The deficiency of kidney Yin is the main pathogenesis of postmenopausal osteoporosis (PMOP) according to traditional Chinese medicine (TCM). Zuoguiwan (ZGW) is among the classical prescriptions in TCM and has been applied to various diseases that are due to deficiency of kidney Yin, including osteoporosis, fractures, menopausal syndromes. However, the underlying mechanism of ZGW in treating PMOP remains poorly understood. ZGW, a traditional Chinese prescription, has been used to nourish Yin and reinforce the kidney since ancient times. The investigation aimed to explore the mechanism of ZGW via the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) signaling pathway as mediated by the β2-adrenergic receptor (β2AR) in an osteoporosis rat model. An osteoporosis model induced by ovariectomy was established in rats. A total of 40 female Sprague–Dawley rats were randomly assigned into bilateral ovariectomy group (OVX), sham operated group (Sham), 17β-estradiol-treated positive group (E2, 25 μg/kg/d), ZGW low-dose group (ZGW-L, 2.3 g/kg/d lyophilized powder) and ZGW high-dose group (ZGW-H, 4.6 g/kg/d lyophilized powder). The serum markers of bone turnover were measured using enzyme-linked immunosorbent assay (ELISA). The morphological structure changes in bones were detected through H&E staining. Local bone mineral density (BMD) and trabecular bone microarchitecture of the right distal femur were measured and evaluated by using micro-CT. Furthermore, the mRNA and protein expressions levels of β2AR, OPG and RANKL were measured by qPCR and Western blot analysis. Compared with the OVX group, ZGW groups showed significantly reduced levels of serum tartrate-resistant acid phosphatase 5b (TRACP-5b) and β-cross-linked c-telopeptide of type I collagen (β-CTX) (P ＜ 0.01), increased levels of serum bone-specific alkaline phosphatase (BALP) (P ＜ 0.01) and OPG (P ＜ 0.05), prevention of OVX-induced bone loss, and improved microarchitecture of the trabecular bone of distal femur. Moreover, ZGW mediated the osteoporosis syndrome by reducing the empty bone lacunae, promoting the ordered arrangement of trabeculae structure, and increasing the trabeculae structure thickness. Furthermore, in ZGW groups, the protein expression of OPG in the tibia was notably up-regulated (P ＜ 0.01), whereas the mRNA and protein expression of β2AR in the hippocampus (P ＜ 0.01), and the protein expressions levels of β2AR (P ＜ 0.01) and RANKL (P ＜ 0.05) in the tibia were down-regulated compared with OVX group. ZGW through its protective effects, stimulates bone formation and suppresses bone resorption. The underlying mechanism of ZGW in improving perimenopausal syndrome and increasing bone mass might be attributed to the regulation of RANKL/OPG, as mediated by β2AR. Therefore, ZGW may be used as an alternative treatment for PMOP.