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The Study of Antiosteoporotic Effects and Mechanisms of Du-Zhong on Postmenopausal Osteoporosis

Author: ZhangRong
Tutor: MeiQiBing
School: Fourth Military Medical University
Course: Pharmacology
Keywords: Postmenopausal osteoporosis Osteoblasts Osteoclasts Eucommia Lignans DEXA μCT Three-point bending test OPG RANKL Bone turnover
CLC: R285
Type: PhD thesis
Year: 2008
Downloads: 924
Quote: 2
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Abstract

Middle-aged future research background, physiological degenerative changes caused by osteoporosis (osteoporosis, OP) morbidity increase with age and increased to decreased bone mass, the microscopic structure of the bone tissue degradation, increased bone fragility induced fractures characterized. OP is a global health problem, seriously affecting the quality of life of the elderly. Estrogen plays an extremely important role in the occurrence of the process of the OP, especially in post-menopausal osteoporosis (postmenopausal osteoporosis, PMO), hormone replacement therapy (hormonereplacement therapy, HRT) has been regarded as a prevention PMO effective means. The Women's Health Initiative study results show that long-term use of HRT has increased in patients with cardiovascular accidents and reproductive system cancer risk, severely limits the application of HRT in the prevention and treatment of PMO. Other anti-osteoporosis drugs, including the promotion of bone formation and inhibiting bone resorption drugs, although the exact PMO preventive or therapeutic effect, but long-term use is not only expensive, but may cause side effects. Therefore, there is an urgent need to find a safe, effective drug prevention and treatment of OP, to overcome the problem of existing drugs. Clinical practice shows that many natural medicines can safely control and OP similar clinical symptoms. Certain plants rich in many natural plant chemical substances that have important physiological activity. Soy isoflavones natural selective estrogen receptor modulators (selectiveestrogenreceptormodulators, SERMs), raloxifene can prevent bone loss in post-menopausal women or oophorectomy (ovariectomy, OVX) rats pharmacological effects similar to bone tissue . In recent years, plants rich in polyphenols also attracted the attention of researchers. Lignans from closed-linking, as isolated from linseed different pine resin alcohol diester to effective prevention and treatment of postmenopausal women lead to bone loss occurred; different yew lignans isolated from yew in animal OVX after effectively slow bone loss; separated from red Nan, lignans, can significantly promote the proliferation and differentiation of mouse osteoblasts (osteoblast, OB). In addition, studies have shown that flavonoids such as rutin can effectively inhibit the reduction of bone mass and bone strength decline of menopausal rats may be related to inhibition of bone resorption and promote bone formation. The research purposes eucommia is rich in lignans, phenolic acids and flavonoids, natural chemicals \Therefore, the subject copy OVX rat model of osteoporosis based on a systematic study of eucommia prevention PMO role; excavations isolated active ingredients - Eucommia lignans; vitro cultured primary rat osteoblasts at the cellular level, active pharmaceutical ingredients, analysis of the the Eucommia prevention PMO material foundation and its mechanism. Research methods select 3-month-old nulliparous females two Sprague - Dawley (SD) rats were randomly divided into sham-operated (SHAM) group, ovariectomized (OVX) group, Eucommia bark extract group (DZCE) 17β-estradiol (E2). Four weeks after the operation began intragastric administration for 12 consecutive weeks. During the week to observe the time changes of body weight in rats. Before the animals were killed to collect rats 24h urine collection of serum anesthetized animals. All the animals were killed and dissected, collected the following specimens were measured and analyzed: the quality of the rat uterus and other organs of the body and uterine pathological changes, analysis of drugs on the uterus and long-term medication organs of rats; using automatic biochemical analyzer serum and urine Ca, P and ALP bone metabolic markers, liver and kidney function parameters were analyzed; using ELISA and other immunological methods of bone turnover indicators in urine DPD and serum OCN measured.; using three-point bending experiments on The rat femur biomechanical performance analysis; using DEXA and μCT femur BMD, BMC and metaphyseal trabecular bone microstructure characteristic analysis. 2.50% ethanol heating reflux extraction of Eucommia total lignans, AB-8 macroporous resin of Eucommia total lignans were isolated and purified, followed by deionized water, 50% ethanol. To collect 50% ethanol elution fraction, and concentrated to pinoresinol diglucosides as a reference, using the colorimetric analysis. 3 using the multi-enzyme digestion method for primary rat osteoblasts cultured in vitro, the cell morphology was observed by phase contrast microscopy, and alizarin red staining and other methods of the obtained cells were identified with an alkaline phosphatase staining and calcium nodules. MTT, the mineralized nodules alizarin red staining method to observe Eucommia total lignans in rat primary osteoblast proliferation, differentiation and mineralization process. 5 using RT-PCR method to observe the Eucommia total lignans into rat primary the bone cells OPG and RANKLmRNA of expression. The experimental results of 1.300 and 500mg/kg/day the therapeutic dose DZCE 16 weeks of continuous treatment, can significantly improve the rat femur structural mechanics parameters, including bone stress and elastic modulus. Biomechanical properties change with DZCE increased BMD and improved trabecular bone micro-structural properties are closely related. Could dose-dependent inhibition of OVX caused BMD decreased DZCE after 16 weeks of continuous treatment, but also accompanied reflect Biochemical indicators of the decline in bone reconstruction, DZCE significantly reduce the level of ALP, OCN and DPD, reducing the OVX-induced rat urine Ca, P increases. The μCT analysis shows 500mg/kg/day the DZCE of the therapeutic dose of a significant increase in BV / TV, Tb.N, Tb.Th and Conn.D, to reduce Tb.Sp SMI index. The 16 weeks DZCE continuous therapy, not observed stimulation of the uterus, nor a significant impact on other organs and liver and kidney function. 2. Separation of purified of Eucommia total lignans, 50% ethanol eluate dried material the Eucommia total lignans purity up to 58.5% total solids. 3. Multiple enzyme digestion method to get rich in the number of cells, and the typical OB morphological characteristics, alkaline phosphatase staining and mineralized nodule staining were positive. 4 in the concentration range of 30 ~ 500μg/mL, of Eucommia total lignin dose dependent manner in primary rat osteoblast proliferation; 100 and 300μg/mL of Eucommia total lignans can significantly increase rat primary into osteoblasts ALP activity and mineralized nodule formation number. 5.100 and 300μg/mL of Eucommia total lignin significant adjustment in primary rat osteoblast OPG and RANKL gene expression, increased both mRNA expression than the value of the level of more than four times. Conclusion 1. Eucommia 16 weeks of treatment by increasing the rat femur BMD and improved trabecular bone micro-structural properties thereby preventing OVX caused the biomechanical performance degradation, compared with estrogen has the advantage of no uterine stimulation. 2 Eucommia total lignan dose-dependent manner in primary cultured rat osteoblast proliferation, differentiation and maturation. The lignan compounds may Eucommia play the active ingredient of the anti-rat PMO. 3 the Eucommia total lignan dose-dependent increase the rat primary bone cells of OPG / RANKL mRNA expression level ratio, eucommia total lignans may by regulation of OPG / RANKL expression on osteoclasts (osteoclast, OC) The differentiation and maturation of impact. Du Zhongfu containing polyphenolic compounds - lignans, can effectively prevent rat PMO bone loss, the destruction of the trabecular microstructure and biomechanical performance degradation, this pharmacological effect may Eucommia activity The ingredients promote OB bone formation or indirectly by regulating the OB the secretion of OPG and RANKL inhibition of OC differentiation and maturation. Eucommia lignans and its active ingredient compounds may become a new natural drug for prevention and treatment of the OP.

The Study of Antiosteoporotic Effects and Mechanisms of Du-Zhong on Postmenopausal Osteoporosis

Abstract

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