The Study Of Antiosteoporotic Effects And Mechanisms Of Radix Dipsaci On Postmenopausal Osteoporosis

BackgroundOsteoporosisisakindofsystematicskeletondiseaseinwhichbonemass isreduced, bone microstructure is degenerated, bones become fragile and morelikely to break. Postmenopausal osteoporosis is the main type of primarycultured osteoporosis. Osteoporosis has become a major public health threat allthe country of the world. Up to now, in our country, 90 million chineses or 7%of the people have osteoporosis. Almost 50% women over 50 years old wereaffected by the disease. The up-to-date data of the National OsteoporosisFoundationshows that in theUSAtoday,10 millionindividuals are estimatedtoalready have the disease and almost 34 million more are estimated to have lowbone mass, placing them at increased risk for osteoporosis. Osteoporosis oftencauses serious complications and lowers a patient's quality of life. The rate ofosteoporosis-related fracture is very high, fractures due to osteoporosis causevery serious results even cause the death of the patient. Osteoporosis-relatedfractures cost alargeamount ofeconomicand healthresources. For a longtime,hormone replacement therapy (HRT) by estrogen and progestogen has been themain route for the prevention and treatment of postmenopausal osteoporosis. But several large scale clinical trials showed that long-term application of HRTincreased the risks of cardiovascular events and cancers of reprodutive system.Some trials were ceased for the serious side effects. This limited the applicationof HRT for the prevention and treatment of postmenopausal osteoporosis.Researchers and patients put their eyes on the selective estrogen receptormodulators (SERMs). This kind of drugs can prevent bone loss withoutstimulating effects on the reprodutive system. Also the risks of cardiovascularevents and cancers of reprodutive system are not increased. These drugs evencanbeusedtotreatsomecancers.Buttheystillhavegastrointestinalsideeffectsand can cause thrombus in deep vein. A series of new drugs such asbisphosphonates, salmon calcitonin (CT) and recombinated human parathyroidhormone (rhPTH) show good effects on the prevention or treatment of thepostmenopausal osteoporosis in clinical trials. The shortages such as only targetone site, can not be applicated in combination due to conflict mechanism, andhave very high price limit the application of these drugs. Thus alternative drugsof proven efficacy and more safety and economic should be developed for theprevention and treatment of postmenopausal osteoporosis. Traditional ChineseMedicine (TCM) treat postmenopausal osteoporosis from multiple aspects andhas less side effects. The principle of TCM is consistent with the desire ofpeople to return to the nature. What's more TCM has more abundant resourcesand lower price compared with chemical drugs, and fit the situation of ourcountry.ObjectiveThe objective of this subject is that observe the effects of Radix Dipsaci,which is often used in the clinical practice in orthopaedics, on OVX-induced postmenopausal osteoporosis comprehensively, systemically and scientifically;isolate and purify the Radix Dipsaci total saponins and observe the effects ofRadix Dipsaci total saponins on osteoblast; discuss the mechanism of theantiosteoporotic effects of Radix Dipsaci on postmenopausal osteoporosis;provide the theoretic witness for the advanced research of Radix Dipsaci andexperimentalevidenceforthenatureproductbasednewdrugdevelopment.Methods1. OVX or sham operations were performed on sixty 3-month-old virginSprague-Dawley rats. Observe the effects of Radix Dipsaci extract (RDE) onpostmenopausal osteoporosis by using 17β-estrodiol as positive control. Ratsthat were divided into six groups: sham control group (sham, n=10); OVXcontrol group (OVX, n=10); 17β-estradiol treatment group (E2, n=10); threeRadix Dipsaci extract treatment groups RDE100 (n=10), RDE300 (n=10) andRDE500 (n=10). The treatment began 4 weeks after the surgery and lasted for16 weeks. The rats received RDE 100, 300, 500mg/kg/d or 17β-estrodiol25μg/kg/d respectively. Collect 24h urine, serum, uterus, heart, liver, spleen,lung, kidney, thymus, brain and femur samples. Organs were weighed andcomputed the organ index. Biochemical parameters and bone turnover markerswere analyzed with automatic biochemical analyzer or ELISAassay. Bone masswas analyzed by DEXA, bone biomechanical properties was measured by threepoint bending test and the trabecular bone microarchitecture was evaluated byMicroCT.2. Radix Dipsaci total saponins was isolated and purified by 60% ethanolrefluxing and D101 macroporous resin. Total saponins was analyzed bycolorimetricmethodbyusingasperosaponinⅥasstand. 3. Rat primarycultured osteoblasts were obtained byenzyme digestion andidentifiedbymorphological,histochemicalandfunctionalproperties.4. Observed the effects of Radix Dipsaci total saponins on proliferation,differentiation and calcification of rat primary cultured osteoblasts by usingMTT assay and Alizarin Red S staining. Observed the effects of Radix Dipsacitotal saponins on the expression of OPG and RANKL mRNA in rat primaryculturedosteoblasts.Results1. 16 weeks treatment of RDE slowed down the body weight gain inducedby the OVX; reversed the increasing of urinary Ca, P excretion and boneturnover markers such as serum ALP, OCN and urinary DPD induced by theOVX; The treatment could also enhance the bone strength and prevent thedeterioration of trabecular microarchitecture; increased the bone volume,trabecular number, thickness and connected density, decreased the trabecularspace and SMI. The effects mentioned above were not accompanied withstimulating effects on uterus. Long-term using of RDE had not influcence onotherorgansandtheliverandkidneyfunctions.2. Isolated and purified Radix Dipsaci total saponins successfully, and thepurityoftheRadixDipsacitotalsaponinsis76.5%.3. Obtained the rat primary cultured osteoblasts successfully, and the cellshadthemorphological,histochemicalandfunctionalpropertiesofosteoblast.4. Radix Dipsaci total saponins increased the proliferation of rat primaryculturedosteoblasts upto180%at thelargedosageinadose-dependent manner.Radix Dipsaci total saponins also increased ALPactivityof rat primaryculturedosteoblasts up to 2.5 folds, and also increased the amount of the calcification bonenodulesintheratprimaryculturedosteoblasts.5. Radix Dipsaci total saponins modulate the expression of OPG andRANKL mRNA in the rat primary cultured osteoblasts, the ratio ofOPG/RANKLmRNAwasincreasedupto8folds.Conclusions1. Radix Dipsaci had potential antiosteoporotic effects on OVX inducedpostmenopausal osteoporosis, could slow down the body weight gain due toOVX, prevent bone loss, maintain BMD, enhance the bone strength. Comparedwithestrogenithadnostimulatingeffectsonuterus.2. Radix Dipsaci total saponins increased the proliferation, differentiationand activation of rat primary cultured osteoblasts in a dose-dependent manner.This may be the cytological mechanism of the antiosteoporotic effects on OVXinducedpostmenopausalosteoporosis.3.Radix Dipsaci total saponins increased theratioofOPG/RANKLmRNAin a dose-dependent manner. It may affect the osteoclast formation andactivationviathispathway.4.Ourstudyrevealedtheantiosteoporoticeffects andmechanismsofRadixDipsacionpostmenopausalosteoporosis.Discoveredtheantiosteoporoticeffectsof Radix Dipsaci total saponins and proved that the effects were due to thepromoting effects on proliferation, differentiation and calcification ofosteoblasts. Radix Dipsaci total saponins could act on the bone resorption byregulating the OPG/RANKL system. Our study not only revealed the elementsandmechanism ofthe effects ofRadix Dipsaci onpostmenopausal osteoporosis,also found the new effects and potential target site of the Radix Dipsaci totalsaponins. Our study not only provided the experimental evidences for the application of the Radix Dipsaci and its total saponins, also gave a new clue onthedevelopmentoftheantiosteoporoticdrugs.