The Research Of Anti-osteoporosis Activity With QOA-8a

As a disease plagues patients all over the world, especially in elderly patients, osteoporosis is actually a disease with a low bone mass, and the most common cause is the increased bone absorption, that exceeds the bone formation which can not be a corresponding increase in order to fully compensate for bone absorption. Common manifestations are reduced bone mass, damage of micro-structural in bone, decrease in bone strength, increase in bone fragility, and the easier to fracture. Osteoporosis usually attacks elderly patients, especially in women. This closely relates to menopause and reduced estrogen level. The treatment of osteoporosis generally focus on either promoting bone formation, which is increasing the quantity and activity of osteoblasts, or inhibiting bone absorption, which is reducing the quantity and activity of osteoclasts. At present, there are no available drugs on the market that have the dual effects, and the hormone replacement therapy commonly has severe side effects. Therefore, it’s particularly important to look for anti-osteoporosis drugs, which can be safe, reliable, with dual effects.Objective:1. To assay the effects of QOA-8a on the differentiation and activity of human osteoblasts cells hFOB1.19, and osteoclasts formation induced from human peripheral blood mononuclear cells in vitro.2. To understand the impact on skull growth in mice, and bone microstructure and biochemical indices in ovariectomized rats with QOA-8a in vivo.3. To explore the possible mechanism about QOA-8a through inhibition of the synthesis of5-HT, and the influence of BMP-2pathway. Methods:1. Human osteoblasts cells hFOB1.19treated with QOA-8a at the concentrations of2nM,20nM,200nM,2μM and20μM for48h, respectively. The cell proliferation was analyzed with MTT assay, and the alkaline phosphatase (ALP) levels, as a marker of osteogenesis cell function and differentiation were also assayed. Osteoclasts were generated from human peripheral blood mononuclear cells in the presence of TNF-a, RANKL and M-CSF. The inducing process was intervened with20nM,50nM,200nM,2μM and20μM of QOA-8a. After14days, the multinucleated osteoclasts (TRAP+) were identified with TRAP staining.2. Subcutaneous injection with0.1,1,5,10and20mg/kg/d of QOA-8a was conducted on the right side of the skulls of three-week ICR mice. The skulls were took out and stained at10days. The ovariectomy (OVX) or sham surgery female SD rats were employed and divided into the sham, vehicle, the positive control,0.1,1and10mg/kg/d of QOA-8a experimental groups. QOA-8a was administrated orally for60days after two months of OVX operation. And the uterus slice, bone microstructure, bone biomechanical properties were analyzed, and serum N-terminal middle of osteocalcin (N-MID-OT), type I procollagen amino terminal propeptide (PINP) and other indicators were also detected.3. The RBL-2H3cells in vitro that had an endogenous expression of tryptophan hydroxylase-1(TPH-1) were cultured and followed by a treatment with0.2μM,2μM and20μM of QOA-8a for48h. After culture, the5-HT contents in cell lysates were measured with HPLC with fluorescence detector. hFOB1.19with a continuous treatment of0.2μM,2μM and20μM of QOA-8a for48h, the levels of osteopontin, osteocalcin and bone morphogenetic protein (BMP-2) in the supernatant were assayed.Results:1. In vitro, QOA-8a showed no effect on proliferation of hFOB1.19, while increased the ALP content significantly at a concentration of20nM and above in a dose-dependent manner, by100-300%, respectively. Meantime, QOA-8a significantly inhibited the formation of osteoclasts induced from human PBMC at a concentration of2μM and above. It was a50%inhibition rate nearly at2μM compared with that of the control group.2. In vivo,26-55%increases of the right skulls’thickness after continuous10days’ injection of QOA-8a with the dose of lmg/kg/d and above were observed, but without dose-dependent manner. This phenomenon was not found in the left side of the skull. In ovariectomized rats, with60d’s oral administration of QOA-8a with lmg/kg/d and above significant improvements on the BV/TV, trabecular thickness and trabecular gap of the femur bone compared with vehicle group were observed, but did not reach to the level of sham group, while the dose of1Omg/kg/d, vertebrae had a decreasing trend of trabecular gap. Among serum biochemical indices, PINP levels showed a20%increase with the dose of0.1mg/kg/d and1mg/kg/d. N-MID-OT displayed no similar trend. In addition, the uterine slices showed no hyperplasia or atrophy changes in the endometrial glands, and no infiltration of inflammatory cell in smooth muscle cells among treated groups.3. The5-HT contents of the RBL-2H3cell lysates were115.49%,71.51%,77.64%of the control group’s at concentrations of with0.2μM,2μM and20μM of QOA-8a, respectively. While only the content of BMP-2was increased significantly among three indicators related to the BMP-2protein pathway. The results suggested that QOA-8a might not affect the extracellular matrix mineralization, but to participate in the osteoblasts differentiation, which might relate to an early proess of the BMP signaling pathway. Meanwhile, the mechanism of QOA-8a on anti-osteoporosis might be via the inhibition of the TPH-1, thereby reducing the5-HT level, which inhibited the bone formation in vivo.