| Objective Eucommia ulmoides Oliv., also called Du-Zhong, is a herbal medicine withstrong pharmacological effects such as kidney-tonifying and skeleton-strengthening activities.It has been extensively used in traditional formulas and modern medications to treatbone-related diseases. Based on our previous studies, the present study was designed to screenthe osteogenic constituents in Eucommia ulmoides Oliv., to investigate its preventive effect ondisuse osteoporosis using a hind limb suspension rats model, and further to explore thepossible mechanisms behind the anti-osteoporosis effect.Methods Extraction efficiency of total lignans in Eucommia ulmoides Oliv. wasemployed as main index. The major impact factors including solvent, concentration, time,temperature, extraction times, extent of pulverizing and material to solvent ratio werescreened using Plackeet-Burman experimental design. Response surface methodology wasfurther utilized to optimize the method to obtain higher extraction efficiency.Disuse-induced osteoporosis was simulated using a Hind limb suspension (HLS) ratmodel. Eucommia ulmoides Oliv. cortex extract or40fraction was administrate by oralgavage for6weeks (2weeks prior to and during the4weeks of HLS). Alendronate (Aln) waschosen as a positive reference drug. At the end of the treatment, bone mineral density (BMD)of the whole right femur was measured by dual energy x-ray absorptiometry (DEXA). Thetoxic and side-effect was investigated by organ morphology and index. Bone metabolicmarkers were analysed by an automatic biochemical analyzer and ELISA kits. Three-pointbending test was employed to determine the effect of various treatments on bone strength. Thebone microarchitecture was assessed by microCT and histomorphometry analysis.The osteogenic fractions of Eucommia ulmoides Oliv. cortex extract were screened inMC3T3-E1and primary osteoblastic cells. Cell proliferation was analysed by MTT method.The osteoblast differentiation was evaluated by the activity of alkaline phosphatase (ALP)using ALP assay kit. The osteoblast mineralization was evaluated by the area of alizarin redstained nodule. High performance liquid chromatography (HPLC) was employed to quantifythe content of pinoresinol di-O-β-D-glucopyranoside (PDG), aucubin (AU) and geniposide (GP) in40fraction.Primary osteoblastic cells were treated by40fraction, PDG, AU or GP and the mRNAand proteins levels of several key molecules in bone metabolism signal pathways weremeasured by the real time PCR and western blot analysis.Results Plackett-Burman experiments showed that ethanol concentration, extraction timeand temperature were the main factors that affect the extraction efficiency of total lignansfrom Eucommia ulmoides Oliv.. The main factors were further optimized by response surfacemethodology, then the optimum technology was gained: Eucommia ulmoides Oliv. cortexpulverized to40mesh, added10times volume of62%ethanol, then heated to72°C refluxextracting111min for twice. The yield of Eucommia ulmoides cortex extract was12.35±0.26%, and the content of total lignans in Eucommia ulmoides cortex extract was1.86±0.012%.Although the body weight of the HLS rats were significantly lower than control group,the organ morphology and index was no significant difference between various group. After4weeks HLS, the level of bone resorption markers (tartrated resistant acid phosphatse,deoxypyridinoline, C-terminal crosslinked telopeptides of collagen type I, N-terminalcrosslinked telopeptides of collagen type I) were significantly increased in the HLS groupcompared with the control group. On the other hand, bone formation markers (ALP andosteocalcin) levels were significantly decreased in response to HLS. Eucommia ulmoides Oliv.cortex extract and40fraction could prevent both the increasing of bone resorption markersand the decreasing of bone formation markers, while alendronate trentment could onlyprevent the increasing the bone resorption marker. The Eucommia ulmoides Oliv. cortexextract and40fraction treatment enhanced the biomechanical strength of femur, prevented thedeterioration of trabecular bone microarchitecture, exhibited the effect of promote boneformation.Eucommia ulmoides Oliv. cortex extract further was separated by petroleum ether,dichloromethane, ethyl acetate and aqueous. Among the four fractions, aqueous fractionexhibited the most potent effect to promote cell proliferation, differentiation and mineraliztionin MC3T3-E1and primary osteoblastic cells. The aqueous fraction was adsorbed inmacroporous resin, then eluented by20%,40%,60%and80%ethanol (ratio with water). The 40%ethanol fraction (40fraction) could significantly stimulate cell proliferation,differentiation and mineraliztion formation in MC3T3-E1and primary osteoblastic cells.HPLC results show that the contents of PDG, AU and GP in the40fraction were28.63±1.46%,17.24±0.89%and14.87±0.94%, respectively. PDG, AU or GP treatment couldalso increase cell differentiation and maturation.The osteogenic effect of PDG, AU, GP or40fraction was associated with increasing thelevel of bone morphogenetic protein-2(BMP-2), p38, P-p38, ERK, P-ERK and RUNX-2,which indicated that Eucommia ulmoides Oliv. might promote bone formation through theBMP-2signal pathway. At the same time, PDG and40fraction can increase osteoprotegrin(OPG), but decrease NF-kB ligand (RANKL) gene expression in primary osteoblastic cells.Conclusion The Eucommia ulmoides Oliv. cortex extract or its effective fraction (40fraction) could effectively prevent disuse-induced osteoporosis in hind limb suspension ratswith the activities to increase BMD, to enhance the biomechanical strength of femur, and toprotect the microarchitecture of trabecular bone. Fraction40was the effective constituents inEucommia ulmoides Oliv. and the major components included PDG, AU and GP. BMP-2andOPG/RANKL/RANK signal pathways were responsible for the bioactive components inEucommia ulmoides Oliv. to exhibit the effect of promote bone formation and prevent boneresorption. |
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