Effects of Pueraria mirifica Airy Shaw et Suvatabandhu and its mechanisms of actions on osteoporosis in aged cynomolgus monkeys : in vitro and in vivo approaches

Abstract

The effects and mechanism of action of Pueraria mirifica powder (PMP) on the development of osteoporosis were assessed in cynomolgus monkeys undergoing menopause. Initially, a cross-sectional study was conducted using pre, peri, as well as early- (0 to less than 5 years), mid- (5 to 10 years), and late- (more than 10 years) postmenopausal monkeys. Bone mineral density (BMD) and content (BMC) were measured by peripheral Quantitative Computed Tomography (pQCT) of the metaphysis and diaphysis of the distal radius and proximal tibia. This cross-sectional study also included assessment of serum and urinary osteotropic hormones and bone turnover markers. An abrupt decrease in metaphyseal trabecular BMD was observed as monkeys entered the perimenopausal period and afterwards. Cortical bone was progressively lost as a function of menopausal status. Bone loss was associated with elevated serum follicle-stimulating hormone (FSH) levels at early- and mid-postmenopause and with decreased serum 17beta-estradiol level at the late-postmenopausal period. Serum BAP and urinary NTX levels were negatively correlated with the metaphyseal trabecular BMC, while serum osteocalcin levels showed a negative correlation with the diaphyseal cortical BMD. Ten osteoporotic postmenopausal monkeys were selected and used further for longitudinal 16-month study of the effects of PMP. The mRNA expression of ALP, RANKL and OPG quantitated by qRT-PCR in cultures of osteoblasts isolated from iliac bone biopsies of these monkeys were also determined. Monkeys were fed either control standard diet (PMP0) or diet containing 1000 mg/kg BW/day of PMP (PMP1000) and parameters of bone structure and function were measured every 2 months. In addition to BMD, BMC and hormones and markers measured in the cross-sectional study, bones of the PMP-treated and control monkeys were also analyzed for cortical area, thickness, and periosteal and endosteal circumferences. The cortical BMD and BMC of the PMP0 monkeys continuously decreased throughout the 16-month period. PMP treatment ameliorated the bone loss, mainly at the cortical diaphysis, and this was associated with a decrease in bone turnover markers. Changes of cortical bone geometry (cortical thickness and area) was in the same line with the cortical BMD and BMC. The mRNA analysis of osteoblast cultures isolated from the treated and control monkeys revealed a significant decrease in the RANKL/OPG ratio in the PMP1000 group and a similar but smaller decrease in the PMP0 group. Lastly, the effect of PMP on healing of the iliac crest defect created during the biopsy procedure was assessed. X-ray radiography and 3D-CT scans indicated accelerated bone healing in the PMP1000 group which was confirmed by a histological examination indicating increased new bone formation and decreased number of chondrofibroblast cells. In conclusion, the cynomolgus monkey is an ideal animal model for osteoporosis in menopausal women. Furthermore, since PMP improves bone quantity and structural properties and accelerates bone healing (in part through the RANKL/RANK/OPG system), this work advocates investigation of PMP as a therapeutic agent for prevention of osteoporosis and for treatment of fractures in postmenopausal women.