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Anti-osteoporotic Effects of Deer Antler Base (Cervus Nippon Temminck) Extracts in Vitro

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Tutor: XuYongPing
School: Dalian University of Technology
Course: Biochemical Engineering
Keywords: Deer antler base extract,Osteoporosis,Osteoblasts,Osteoclasts,Press-shear assist
CLC: R259
Type: PhD thesis
Year:  2013
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Abstract:
Osteoporosis is a systematic skeleton disease characterized by low bone mineral density and structural deterioration of bone tissue leading to bone fragility and increased cases of fractures particularly of the hip, spine and wrist. It is a disease common in post-menopausal women and the elderly. The incidence of osteoporosis-related fractures is very high and has enormous impact on public health and on the quality of life of the patient. Nowadays, many medicines for osteoporosis have some disadvantages such as adverse side effects or a high cost, which including salmon calcitonin, bisphosphonates and human parathyroid hormone. Thus, alternative drugs of efficacy, safety and low cost, should be developed for the prevention and treatment of osteoporosis. Deer antler base (Cervus) has been recorded in the Chinese medical classics Shen Nong Ben Cao Jing2000years ago and is believed to tonify the kidney, invigorate the spleen, strengthen bones and muscles, and promote blood flow.Our previous study has showed that deer antler base has a good therapeutic effect on ovariectomy-induced osteoporosis in rats. But since now no studies have been carried out to evaluate the mechanisms by which deer antler base exerts its anti-osteoporotic effect. Therefore, the present study aimed to systematically investigate the effects of deer antler base extracts on the bone formation of osteoblasts and bone resorption of osteoclasts in vitro and its mechanisms of action. The main results are summarized as following.(1) The raw material of deer antler base was processed into PAI powders by the press-shear assisted interaction technology (PAI) in an energy-intensive vibrational mill. The particle size and the content of main functional ingredients related to osteoporosis were detected.To see what damage had been done to the cells, the PAI powders were examined using a laser diffraction particle analyzer and scanning electron microscopy. The results showed that the percentage of particle size ranged from1-30¦Ìm accounted for82.73%after PAI treatment, and the cells of deer antler base PAI powder were basically broken. The active ingredients were probably released upon mixing with cellular fragments. The tests for active ingredients showed that deer antler base contains328.79¡À34.21pmol/L estradiol,25.38¡À4.48nmol/L testosterone,17.56¡À3.45nmol/L progesterone,0.750¡À0.033¦Ìg/g IGF-1,16.17¡À0.52mg/L calcium citrate and133.61¡À3.55mg/L free calcium, but no calcium fumarate. Moreover, the contents of active ingredients were larger than those of coarse powder, indicating press-shear assisted interaction technology is not only conducive to the release of active ingredients, but also to raise the dissolution rate of active ingredients.(2) Influences of deer antler base extracts (aqueous extract and ethanolic extract) on the proliferation, osteogenic differentiation and mineralization of human osteoblast-like MG-63cells in vitro.The results of lactate dehydrogenase (LDH) activity and cell morphology indicated that deer antler base extracts did not show cytotoxicity. Cell growth and viability was assessed by crystal violet test, neutral red absorption test, trypan blue exclusion test and MTT test. And the cell differentiation was evaluated by alkaline phosphatase (ALP) activity. The results showed that deer antler base extracts not only stimulated the growth and proliferation of MG-63cells, but also promoted the differentiation of MG-63cells. The results of alizarin red-S staining and4-hydroxyproline test showed that deer antler base extracts promoted the bone matrix mineralization by stimulating the deposition of calcium and increasing the synthesis and secretion of type I collagen. In addition, the OPG and RANKL protein and mRNA levels were detected using an enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). The results indicated that deer antler base extracts can significantly increase the OPG protein and mRNA level, whereas decrease the RANKL protein and mRNA level, thereby increasing the ratio of OPG/RANKL and indirectly inhibit osteoclast differentiation and maturation. The findings suggest that deer antler base extracts promote bone formation of osteoblasts and play a role on prevention and treatment of osteoporosis.(3) The effects of deer antler base extracts on osteoclastogenesis and bone resorption activity were investigated using the macrophage cell line RAW264.7as a model of osteoclast precursors.The results of TRAP staining and cell counting showed that the RAW264.7cells can differentiate into osteoclast-like cells when stimulated by50ng/mL of recombinant mouse soluble RANKL. Moreover, deer antler base extracts could reduce the number of osteoclasts and the TRAP activity in a dose-dependent manner, indicating suppresses the RANKL-induced differentiation of RAW264.7cells. The results of LDH activity, PI staining and quantitative analysis of DNA fragmentation consistently indicated that deer antler base extracts could induce the apoptosis of mature osteoclasts. Furthermore, the RT-PCR results indicated that deer antler base extracts could down-regulate the osteoclastic phenotypic genes, TRAP and MMP-9mRNA expression levels. These results suggest that deer antler base extracts inhibit bone resorption of mature osteoclasts and play a role on prevention and treatment of osteoporosis. (4) The anti-osteoporotic mechanisms of deer antler base extracts were further investigated using MG-63cells treated with p38specific inhibitor SB203580.After treatment with p38specific inhibitor SB203580, MTT assay, ALP activity, alizarin red-S staining and4-hydroxyproline test were respectively used to evaluate the effects of SB203580on proliferation, differentiation and mineralization of MG-63cells. The results showed that deer antler base extracts can effectively promote the proliferation, differentiation and mineralization of osteoblasts, while the effects can be blocked by the p38specific inhibitor SB203580. Further, the up-regulation of OPG and down-regulation of RANKL at the protein and mRNA level were observed in deer antler base extracts treated MG-63cells using an enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). The results implied that deer antler base extracts can indirectly inhibit osteoclast differentiation and maturation by regulating the expression of OPG and RANKL in MG-63cells, while the inhibitory effect can be effectively blocked by the p38specific inhibitor SB203580. The findings suggest that deer antler base extracts could affect the dynamic balance of osteoblasts and osteoclasts, which may be mediated via the p38MAPK signal pathway.Taken together, our results confirmed that deer antler base extracts could promote bone formation of osteoblasts and inhibit bone resorption of mature osteoclasts through the p38MAPK signal pathway, which providing a theoretical basis for the prevention and treatment of osteoporosis.
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