Proteomics Study On Antiosteoporotic Mechanism Of Icariin And Interaction Of Epimedium Brevicornum And Curculigo Orchioides

Drug pair composed of Epimedium brevicornum Maxim.(Berberidaceae, whole herb)and Curculigo orchioides Gaertn.(Amaryllidaceae, rhizome), Warming kidney andReinforcing Yang, has been widely used for the clinical treatment of osteoporosis disordersand menopausal syndrome for several decades. Both Epimedium brevicornum and Curculigoorchioides, Traditional Chinese Medicine, were recorded in Pharmacopoeia of people'sRepublic of China. In the present study, we investigated the antiosteoporotic activity andmechanism of the main active constituents. Total Flavones of Epimedium and total phenolicglycosides of curculigo, icariin and curculigoside was the main constituent respectively, wererecongnized as effective drugs for osteoporosis in many published papers. The thesisincluding four parts:Ⅰ. Proteomic protocol involved of2-DE, image analysis and spectrometry detection wasset up. Proteomics studies of icariin on the target organs of HPA axies (includinghypothalamus, pituitary gland, uterus and bone) shows that: Proteins separation intwo-dimensional gel meet the requirement of PDQuest2-D Analysis Software.Bioinformatics and literature review showed that differentially expressed proteins functionsrelated to apoptosis, cell proliferation, cell differentiation, proteins aggregation, proteinsfolding, molecular chaperones, cytoskeleton formation, cell proliferation signals transduction,oxidative stress and mineral crystal fiber et al.Ⅱ. Icariin, icariside, baohuoganⅡ, Epimedin C, curculigoside, curculigoside A, Orcinolglucoside and Orcinol rhamnoside of10-8~10-10mol/L, were evaluated for their activity onosteoblast proliferation and differentiation in neonatal rat calvaria cultures, TRAP activitywere determined in the concentration of10-5~10-7mol/L on the multinucleated osteoclastsderived from rat marrow cells induced by1,25-(OH)2-D3and dexamethasone. ATP contentsand mitochondrial membrane potential and cytoskeleton of cells were also observed in theconcentration of10-6mol/L. Considering of all aspects, we chose icariin and curculigoside asthe compounds of effective components to study their interaction on osteoblast and osteoclast.Ⅲ. We selected the3months old ovariectomized rat model to investigate theanti-osteoporosis effect of total Flavones of Epimedium and total phenolic glycosides ofcurculigo, and analyzed the indexes include total femur bone mineral density,histomorphometry, the Ca, P, Cr content and ALP activity in serum and urine； Serumprogesterone levels; examine serum antioxidant enzymes Activity (glutathione peroxidase, superoxide dismutase, nitric oxide synthase) and MDA content. The results show that theindicators are effective when treated with total Flavones of Epimedium, total phenolicglycosides of curculigo and their combination, they show certain mutual enhancement. Wealso investigated the changes of metabolites in plasma and urine by metabolomics. The resultshows the osteoporosis-related metabolites mainly relevant with energy metabolism and lipidmetabolism.Ⅳ.Icariin and curculigoside could not only promote the proliferation and ALP activity ofOB, but also increase the quantity of bone nodules. They show mutual enhancement onosteoblastic activity. For osteoclasts, they can decrease the number of mature osteoclasts,suppress the marker enzyme activity (TRAP and Cathepsin K), reduce the bone resorption pitarea. The morphous of osteoclast and its nucleis (actin ring and nuclear staining), proteinexpression of key regulatory factor (OPG and RANKL), the relative proteins includingNF-κB,MAPK(ERK,P38,JNK),PI3K/Akt, belonging to the routes of RANK, were alsoevaluted. Results showed that icariin and curculigoside showed the strongest suppressingactivity. They show mutual assistance.The conclusion we made in these study is osteoporosis were relate with cytoskeleton andenergy metabolism. The interaction between total Flavones of Epimedium and total phenolicglycosides shows mutual enhancement on OVX rats. Icariin and curculigoside show mutualassistance in osteoclastic activity, mutual enhancement in osteoblastic activity.