The Research On The Mechanism Of Cyanidin Chloride Effect Osteolytic Disease

Bone remodeling is a dynamic process including bone metabolism and anabolism throughout our lifetime. It is reported that more than 10% of the bone undergo remodeling and regeneration per year in adults. It attributes to two cells. One is osteoclast for bone absorption, another is osteoblast for bone synthesis. Osteoclast is derived from hematopoietic stem cells (HSCs) of the bone marrow monocytes. The mature osteoclast is multinucleate giant cell with the function of bone absorption. It is consensus that M-CSF and RANKL are two crucial cytokines for proliferation, differentiation and maturity of osteoclast. M-CSF mainly acts on monocytes macrophage and also towards proliferation and differentiation to precursor of osteoclast. Meanwhile, it contributes to the activation, adhesion and migration of mature osteoclast, whilst promoting the expression of RANK on precursor of osteoclast.RANKL, as a member of the tumor necrosis factor ligand super family, is designated as a type Ⅱ transmembrane protein, which plays the key role in further differentiation of osteoclast and bone absorption with assistant of M-CSF when it combined with specific receptor. Osteoblast derived from mesenchymal stem cells (MSCs), while mature osteoblast could secrete collagen and glycoprotein, as well as produce extracellular bone matrix and mineralized tissue. Besides, in the bone microenvironment, osteoblast also implicates in differentiation and function of osteoclast when osteoblast secrete numbers of osteoclast-related cytokines during the process of differentiation. As was mentioned above, RANKL and M-CSF which come from osteoblast mainly apply to the differentiation and proliferation of osteoclast. In addition, osteoblast also produces type I collagen, osteocalcin and other chemical tended factor, which are responsible to precursor of osteoclast in migration.Between 1997 and 1998, it is the first time that RANKL was independently verified as the ligand for RANK by molecular cloning technology, meanwhile, it was certified that RANKL was responsible to the differentiation, cell fusion and function through gene knock-out mice model. The RANKL genes encode two forms of type II transmembrane proteins (RANKL1/2) and one form of secreted protein (RANKL3). In fact, RANKL1 is the one that binds with specific RANK receptor on osteoclastic surface. RANKL is highly expressed in various tissues and organs of our body, such as lymph nodes, thymus, mammary gland and lung. As regards skeleton system, RANKL is produced by osteoblasts and bone stem stromal cells as we discussed above. Moreover, various hormones and cytokines affect the secretion of RANKL, for instance, positive regulators of RANKL are parathyroid hormone (PTH), prostaglandin E2, dexamethasone, inflammatory cytokines (interleukin-1,6,11), tumor necrosis factor a (TNF-a), 1,25-dihydroxyvitamin D3, and glucocorticoids. On contrast, estrogens, androgens, transforming growth factor β (TGF-β), IL-13, IL-18 and IFN-y, are considered as negative regulators. As extensive research reported that-induced excessive osteoclast genesis is the culprit of pathological bone lesion. Such as tumor-related bone destruction, inflammatory associated bone lesion, aseptic prostheses loosening of artificial joint replacement, postmenopausal osteoporosis, Paget’s disease, Psoriatic arthritis, ankylosing spondylitis and so on. Taken together, RANKL is considered as a potential therapeutical target in the treatment of osteolysic disease.As a water-soluble pigment and the aglycone of Anthocyanidin, Anthocyanin is responsible to the color of fruits and flowers in botany kingdom and exists in various types of subunits via different glycosylation and it belongs to flavonoid. The basic chemical structure of Anthocyanin is a kind of cation with 2-phenylbenzopyrylium. Anthocyanidin, is polyphenolic compound composing of 6 main subunits:delphinidin, cyanidin, petunidin, pelargonidin, peonidin, and malvidin. Moreover, with high bioavailability, anthocyanidin could be adsorbed by intestinal cells and enter into bloodstream immediately by oral administration. Therefore, anthocyanidin has a broad scope in future clinical application. Recently, number of study reported that anthocyanidin obtain great ranges of pharmacological function in anti-oncogenesis, anti-inflammation, anti-oxidative. Cyanidin, the main subunit of anthocyanidin, widely spread in vegetables, fruits, beans and wines. It is reported that daily intake of cyanidin is more than 180mg in western developed countries such as America and Europe. Other study suggested that consumption of cyanidin is benefit to reduce the morbidity of cancer, cardiovascular disease and diabetes.So far, both domestic and overseas researches have elucidated the chemical activity and underlying mechanism of cyanidin in the preclinical application toward related diseases. But most of the researches are focus on the variant of cyanidin-3-glycoside (C3G). However, as a natural extraction and the chloride form of cyanidin, cyanidin chloride was reported as having the function of protecting against gastrointestinal damage from the aspirin-induced membrana mucosa. Hitherto, the therapeutic effect and exact mechanism of cyanidin chloride in osteolysic disease are unknown. In our study, by means of cell biology and modern molecular biology technology, we investigated the influence of cyanidin chloride on osteoclast proliferation, differentiation and bone-resorptive function. Furthermore, we elaborated the underlying mechanism at the molecular level. Ultimately, we proved that cyanidin chloride efficiently relieved osteoclastic bone destruction through ovariectomy-induced osteoporosis animal model with mice.The experiment includes the following three parts.Part Ⅰ Establishment and identification of BMMs osteoclastogenic culture systemOBJECTIVE:To isolate and culture bone marrow macrophages, survey its morphological characteristics and detect the capacity of unidirectional differentiation to osteoclast.METHODS:The bone marrow macrophages of mice were isolated and extracted from Bone marrow cells through whole bone marrow adherence method. In the present of M-CSF and RANKL together, bone marrow macrophages were induced directionally to differentiate into osteoclasts. The mature osteoclast was stained with TRAP solution for morphological observation, and bone-resorptive capacity was assessed with bone slide.RESULTS:In the stimulation of M-CSF alone, bone marrow macrophages were proliferative and rapid grow with adherence in T-75 cm2 flask. Until confluence, bone marrow macrophages were seeded in 96-well cell culture plate, and treated with RANKL. After 7 days culture, bone marrow macrophages fused and formed TRAP-positive osteoclast-like multinucleated giant cells. On the other hand, bone marrow macrophages were plated on bone slices in 96-well plate with the same culture system. When osteoclast-like multinucleated giant cells were formation, the result of scanning electron microscope showed that osteoclast-like multinucleated giant cells leaded to bone resorption and produced resorption pits on the surface of bone slices.CONCLUSION:The whole bone marrow adherence method and BMMs osteoclastogenic culture system provide stable resource of seed cells for drug testing in vitro. bone marrow macrophages were seeded in 96-well cell culture plate, and treated with RANKL. After 7 days culture, bone marrow macrophages fused and formed TRAP-positive osteoclast-like multinucleated giant cells. On the other hand, bone marrow macrophages were plated on bone slices in 96-well plate with the same culture system. When osteoclast-like multinucleated giant cells were formation, the result of scanning electron microscope showed that osteoclast-like multinucleated giant cells leaded to bone resorption and produced resorption pits on the surface of bone slices.Part Ⅱ Culture and identification of osteoblastOBJECTIVE:To isolate culture primary osteoblast from calvarium of mice, to test the alkaline phosphatase activity and capacity of mineralization of osteoclast.METHODS:After removal the duramater, skin and cartilage, the calvaria was shredded and soaked in the digestive solution composed of 0.25% trypsin and 0.2% collagenase for 15 minutes in 37℃ incubator respectively. Then theDMEM/FBS was added to stop the enzyme activity, then the digestive solution was centrifuged, discarded the supernatant and resuspend with DMEM/FBS. Finally, the cell suspension was add toT-75cm flask until osteoblast confluence. In sequence, primary osteoblasts were plated in 12-well cell culture plate with differentiation medium for alkaline phosphatase activity assay and calcium nodule formation assay.RESULTS:Osteoblast is spindle cell-based, and vigorous grow adhering to the wall. After 7 days stimulation with differentiation medium in 12-well plate, osteoblasts were stained and lysed to qualitative assay and quantitative analysis for alkaline phosphatase activity. Whilst, treated with differentiation medium for 14 days, the conformation of sufficient bone nodule testified osteoblast mineralization capacity.CONCLUSION: The method of isolation and culture of calvarial osteoblast is the successful and stable resource of seed cells for tissue engineering experiment. In sequence, primary osteoblasts were plated in 12-well cell culture plate with differentiation medium for alkaline phosphatase activity assay and calcium nodule formation assay.Part Ⅲ The treatment effects of cyanidin chloride on ovariectomy-induced osteolysis and the underlying mechanismOBJECTIVE:To investigate the therapeutic effect of cyanidin chloride on ovariectomy-induced osteolysis and RANKL-induced excessive osteoclastogenesis, then elucidate the molecular mechanism.METHODS:OVX-induced osteoporosis animal model was established to test the effect of cyanidin chloride on RANKL-induced hyper-resorptive bone disease by comparative analyzing bone parameters with micro-CT. To test the biological effect of cyanidin chloride on osteoclast, at first, BMMs were extracted, isolated cultured and directionally differentiated to osteoclast-like multinucleated cells in the treatment of indicated concentration of cyanidin chloride, then stain with TRAP and count. MTS and apoptosis assay were exerted to evaluate the cytotoxicity of cyanidin chloride on progenitor of osteoclast. Resorption pit assay was performed to assess the influence of cyanidin chloride on osteoclast bone resorptive function. To clarify the potential mechanism, Luciferase reported gene assay, western-blot, RT-PCR and calcium oscillation assay were used to detect the involving transcription factor and signaling pathway of cyanidin chloride on the differentiation and function of osteoclast. At the same time, in the presence of cyanidin chloride, calvarial osteoblasts were isolated culture and examined the activity of alkaline phosphatase and capacity of mineralization.RESULTS:Cyanidin chloride relieved ovariectomy-induced osteoporosis in mice efficiently. Simultaneously, Cyanidin chloride suppressed RANKL-induced osteoclast formation and bone-resorptive function through slowing the degradation of IκB-α and attenuating the phosphorylation of extracellular signal-regulated kinases (ERK). Additionally, Cyanidin chloride hampered RANKL-induced influx of ionic calcium and abrogated the activation of nuclear factor of activated T cells calcineurin-dependent 1 (NFATc1) and c-Fos. Moreover, Cyanidin chloride benefit to up-regulate activity of alkaline phosphatase and formation of calcium nodule in vitro.CONCLUSION:Cyanidin chloride has the protective effect on OVX-induced osteoporosis. The underlying mechanism is that cyanidin chloride block the function of RANKL-induced excessive overgrowth of osteoclast and bone absorption, whilst promoting osteoblast differentiation and bone formation.