The Non-Nuclear Effects Of Icariin In The Proliferation Of The Osteoblasts

Background: As aging diseases, spinal degeneration and osteoporosis often occur at the same time, and influence each other. Osteoporosis(OP) is a systemic disease characterized by low bone mass and micro structure destruction of bone tissue, which leads to increased bone fragility and increase the risk of fracture.These destructions of bone structure in spine often take place more early and more broadly than in peripheral bone. As a result, these can lead to some changes of vertebral morphology, physiological curvature of the spine, and the mechanical properties of the spine.In addition,osteoporosis can aggravate the degeneration and deformation of endplate, leads to the degeneration of intervertebral disc increased, and These pathological changes eventually aggravate the degeneration of intervertebral disc. The relationship between the two diseases has become more and more concerned by clinicians. Therefore, how to find effective drugs and its mechanism from the perspective of the whole two diseases to treat osteoporosis is still the basic clinical problems must be considered by clinical.Part I Construction, identification and purification of the conjugate of Icariin and Bovine serum albuminObjective:To construct the conjugate of Icariin and Bovine serum albumin, then to study the effect of the non nuclear effect of ICA on the proliferation of osteoblast by using this conjugate.Methods:Make a combination of Icariin and Bovine serum albumin using the hydrogen bonding and van der Edward force between ICA and BSA. Finally the ICA-BSA conjugates were separated by using Sephadex G-25 gel chromatography technology from ICA, BSA and ICA-BSA mixed solution.Results:By ultraviolet spectrophotometry testing we found that BSA has a strong fluorescence emission peak at 282 nm,ICA at 291 nm and ICA-BSA at 305 nm. Compared with the ICA and BSA, the ICA- BSA waveform and the peak absorbance has changed. In addition, in chromatographic separation experiment part, we have found UV- visible absorbance values of tube numbered 4 was the highest compared with other samples, especially at 300 nm and 310 nm.Conclusion : The experiment successfully constructed ICA- BSA conjugate,conducted the purification and identification of ICA-BSA conjugate,and obtained the ICA- BSA concentration( 60.5 ug/m L)by BSA assay method.Part Ⅱ The non-nuclear effects of Icariin in the proliferation of the OsteoblastsObjective:To detect the role of nonnuclear effects of ICA in the proliferation of the osteoblast cells, and explore the relationship between nonnuclear effects of ICA and estrogen receptor by studying the effects of estrogen receptor inhibitor on the proliferation of osteoblast cells induced by ICA.Methods:Using enzyme digestion method to obtain primary osteoblasts of rats,and staining and identification.Then coupling the icariin and bovine serum albumin, after the successful coupling, with the Sephadex G-25 gel chromatography technology to desalination and purification, and Osteoblasts were randomly divided into three groups:A(ICA) group、B(ICA-BSA) group、C(ICI182780 +Icariin–BSA) group. The MTT method and Flow cytometry were used to detect the proliferation of osteoblasts at 1 hour after administration of the medication.Results:Bone cells, identified as osteoblasts by histochemical staining of alkaline phosphatase and calcified nodules were obtained by sequential digestion. Icariin and bovine serum albumin were coupled, desalinated and purificated successfully. MTT and Flow cytometry results show that B group can quickly promote the proliferation of osteoblasts, and had significant difference compared with A and C groups(P <0.05).Conclusion:This study shows that the impermeable membrane ICA—E2-ICA has the same effect of promoting the proliferation of osteoblasts with ICA, indicating the existence of the non nuclear effect of ICA. And this action could be interfered by ER antagnon—ICI182780, suggestion that the non-nuclear effects of ICA on proliferation is ER dependent.Part Ⅲ The study of signal pathway of the nonⅢ-nuclear action of Icariin in osteoblastsObjective:The activation of extracellular signal-regulated kinase(ERK1/2) and concentration of intracellular free Ca2+ were measured to investigate the signaling pathway activated by non-nuclear action of Icariin.Methods : Two osteoblasts were incubated with ICA and ICA-BSA, then the activation of ERK1/2 was measured by means of western blotting expreiments; the intracellular free Ca2+ concentration([Ca2+]i) of Fura-3 loaded osteoblasts was examined by immunofluorescent labeling and confocal microscopy. The inhibitory effects of ICI 182780 and PD 98059 on the activation of ERK1/2 and [Ca2+]i were observed, which were incubated previously in ICA-BSA treated osteoblasts.Results:The experimental results show that ICA and ICA-BSA induced a significant increase of ERK1/2 phosphorylation and the concentration of intracellular free Ca2+, but and these changes in osteoblasts could be inhibited by ICI 182780 and PD 98059.Conclusion:Just like ICA, membrane-impermeable E2-BSA could activation the ERK1/2 and increase the intracellular free Ca2+ concentration of osteoblasts. These non-nuclear effects of ICA could be inhibited by ICI 182780 and PD 98059, Which indicated that in the osteoblasts, the non-nuclear effects of ICA were mediated by ER, and The MAPK signaling pathway plays an important role in the process.