The Study Of Antiosteoporotic Effects And Mechanisms Of Du-Zhong On Postmenopausal Osteoporosis

BackgroundThe incidence of osteoporosis increases dramatically with life expectancy. Thisdisease is characterized by a reduction in bone mass and microarchitecturaldeterioration of bone tissue, resulting in skeletal fragility and susceptibility tofractures. Osteoporosis has become increasingly recognised as a major healthcareproblem which will affect the lives of a considerable number of individuals.Because hypoestrogenemia after menopause is an important cause of osteoporosis,hormone replacement therapy (HRT) was used to be a popular regime forprevention and treatment of postmenopausal osteoporosis. Ironically, data fromthe Women's Health Initiative (WHI) Trial indicated that long-term acceptanceand/or compliance of HRT is low due to potentially malignant effects onreproductive tissue. Although traditional therapic agents that stimulated boneformation (e.g., sodium fluoride, growth hormone, and anabolic steroides) andantiresorptive agents (e.g., calcitonin and bisphosphonates) may prevent furtherbone loss in established osteoporosis, their costs are too high to benefit a largepopulation in the developing or even the developed countries for prevention andtreatment of osteoporosis. Consequently, it is necessary to develop"natural"products or synthetic substance with less undeirable side effects that cansubstitute or reduce the need for drugs used currently.Through thousands of years of human experimentation, belief in the safetyof"natural"products has contributed to the fairly widespread use ofcomplementary therapies among women to relieve postmenopausal symptoms. Indeed, many of commonly consumed foods, herbs and spices contain a complexarray of naturally occurring bioactive molecules called phytochemicals, whichmay confer health benefits. Soy food and isoflavones have received considerableattention for their potential role in preventing osteopenia induced by ovariectomyin rats or by menopause in women. They have been characterized as naturallyoccurring selective estrogen receptor modulators (SERMs) with similar beneficialeffects to raloxifene on bone. Very recently, attention also has focused on thepossible role of other polyphenols. Lignans, secoisolariciresinol diglycoside fromflaxseed and isotaxiresinol from Taxux yunnanensis prevented bone loss inpostmenopausal women or ovariectomized model, respectively. Arylnaphthalenelignans isolated from Machilus thunbergii increased mouse osteoblastdifferentiation by increasing ALP activity, collagen content and mineralization.Flavonoids, rutin have been shown to inhibits estrogen deficiency-induced boneloss in OVX rats, both by slowing resorption and by increasing osteoblasticactivity, resulting in increased femoral strength.ObjectiveDu-Zhong, be rich in polyphenolic compounds such as lignans, phenolicacid, and flavonoids, is a kidney-tonifying herbal medicine with a long history ofsafe use for treatment of bone fractures and joint diseases in China. Consequently,the aim of the present study was to systematically evaluate the ability ofDu-Zhong cortex extract (DZCE) consumption to prevent osteoporosis inducedby ovariectomy (OVX) in rats, and isolate and purify Du-Zhong total lignans andobserve the effects of total lignans on osteoblast; discuss the mechanism of theantiosteoporotic effects of Du-Zhong on postmenopausal osteoporosis.Methods1. Eighty 3-month-old female Sprague-Dawley rats were used and randomly assigned into sham-operated group (SHAM) and five ovariectomy (OVX)subgroups, i.e. OVX with vehicle (OVX); OVX with 17α-ethinylestradiol (E2,25ug/kg/day); OVX with DZCE of graded doses (100, 300, or 500mg/kg/day).Daily oral administration of DZCE or E2 started on week 4 after OVX for 16weeks. The body weight of the animals was recorded weekly during theexperimental period. efore euthanizing the animals, urine and serum sampleswere collected for biochemical analysis. The femur were dissected for evaluationof bone mineral content (BMC) and bone mineral density (BMD), bonemechanical competence and trabecular microarchitectural properties.2. Dried and powder Du-zhong cortex were refluxed with 50% alcohol. Then theextract was concentrated and subjected to AB-8 macroporous adsorptive resinseluted using distilled water, 50% alcohol. The total lignans (TL) wereconcentrated in the 50% fraction quantitativly determined using colorimetricmethod by using pinoresinol diglucoside as stand.3. Primary cultures of rats osteoblasts were obtained by enzyme digestion andidentified by morphological features, the alkaline phosphatase staining andAlizarin Red S staining of calcified nodules.4. The effects of TL on the proliferation and differentiation of rat osteoblast wereevaluated by the MTT method, measuring the activity of alkaline phosphatase(ALP activity) and Alizarin red staining.5. The effects of TL on the expression of OPG and RANKL mRNA in rat primarycultured osteoblasts by RT-PCR.Results1. Treatment with DZCE at higher doses (300 or 500 mg/kg/day) was found to beable to significantly prevent OVX-induced decrease in biomechanical quality offemur such as maximum stress and Young's modulus. The mechanical changes were associated with the prevention of a further BMD decrease or even withsome improvements in microarchitecture. DZCE dose-dependently inhibited totalBMD decrease in the femur caused by OVX, which was accompanied by asignificant decrease in skeletal remodeling, as was evidenced by the decreasedlevels of the bone turnover markers osteocalcin (OC), alkaline phosphatese(ALP), deoxypyridinoline (DPD), and urinary Ca and P excretions.μCTanalysis of the femoral metaphysis showed that DZCE at the highest doses(500mg/ kg /day) significantly prevent decrease in bone volume/tissue volum(BV/TV), connect density (Conn.D), trabecula number (Tb.N) and trabeculathickness (Tb.Th), and increase in trabecular separation (Tb.Sp) and structuremodel index (SMI) in OVX rats. DZCE at all dose levels did not prevent theincrease in body weight induced by ovariectomy in rats. Long-term using ofDZCE had not influcence on uters, other organs and the liver and kidneyfunctions.2. Extracted and purified Du-zhong total lignans successfully, and the purity oftotal lignans was up to 58.5 % in the dried part of 50 % ethanolic elution.3. The cultured cells possessed exhibited the typical morphological featurehistochemical and functional properties of osteoblast.4. The osteoblast proliferation was significantly increased dose-dependentlywhen the cells were treated with TL (30μg/mL to 500μg/mL). The activity ofALP of the osteoblasts in the presence of TL at 100μg/mL or 300μg/ml for 3days increased significantly. TL at 100μg/mL or 300μg/mL could significantlystimulate the formation of calcified nodules.5. TL modulate the expression of OPG and RANKL mRNA in the rat primarycultured osteoblasts, the ratio of OPG/RANKL mRNA were significantlyincreased by 100μg/mL or 300μg/ml TL. Conclusions1. 16 weeks of DZCE treatment improve bone biomechanical quality throughmodifications of BMD, and trabecular microarchitecture without hyperplasticeffect on uterus.2. Total lignans from Du-zhong could promote the proliferation, differentiationand of rat primary cultured osteoblasts in a dose-dependent manner, and totallignans might be one of the active constituents facilitating antiosteoporotic effectson OVX induced postmenopausal osteoporosis.3. Total lignans from Du-zhong increased the ratio of OPG/RANKL mRNA in adose-dependent manner. It may affect the osteoclast formation and activation viathis pathway.4. Du-Zhong, be rich in polyphenolic compounds such as lignans, can preventsthe estrogen deficiency-induced bone loss and deterioration of trabecularmicroarchitecture, thereby maintaining biomechanical competence of bone, bothby slowing down resorption and increasing osteoblastic activity. Du-Zhong andlignans might be a potential alternative medicine for treatment of postmenopausalosteoporosis.