| Osteoarthritis(Osteoarthritis, OA) is a chronic joint diseases which characters are joint cartilage degenerative diseases and secondary bone hyperplasia,in it’s pathological process can cause synovial hyperplasia and the occurrence of synovial inflammation which can disorder the steady of joint cavity mico-enviroment and will deteriorate the progress of OA disease.The pathological features mainly include: the increase of protease activity lead to the degradation of extracellular matrix(ECM) in the organization, cells secrete a large number of inflammatory factors promote the development of OA, cell apoptosis worsen joint cavity micro-environment. Therefore, the degradation of ECM, the secretion of cytokines, cell apoptosis in the OA disease are the important targets to study the pathogenesis of OA.About the treatments of OA, drug treatment is largely adopted for its advantages such as been convenient to use, better curative effects, relatively low cost and other characteristics. Therefore, searching for more safe and effective drugs for the treatment of OA received great attention. Icariin as a substance derived from traditional Chinese medicinal herbs, it has been proven have effect to promote cartilage cell proliferation, osteoblast differentiation, reduce inflammatory factory and matrix metalloproteinase.In the development process of OA, synovial tissue may occur inflammation and secondary hyperplasia,Whether Icariin can regulate OA synovial cells so as to have curative effect on OA is unclear and which is worthy of further studying.Therefore, in order to provide references about new drug treatment approaches for OA, this study we use OA synovial cells, with cell viability, cell cycle, cell proliferation, different cytokines expression and cell migration as index to explore the regulation and mechanism of Icariin on OA synovial cells.Our main work and conclusions are as follows:(1) Using explant culture method to isolate human osteoarthritis synovial fibroblast-like cells from osteoarthritis patients, and expanding the primary isolated cells to establish the synovial cell banks for the subsequent experiments.(2) After being treated by different concentration of Icariin,using MTS method to detect the influence of Icariin to OA FLSs cytoactive and to explore the toxicity effect of Icariin, selecting the good drug concentrations: 0nM, 100 nM, 500 nM, 1μM.Then use flow cytometry method to detect the cell cycle, the results showed that Icariin can accelerate the amount of OA FLSs into S phase and G2 / M phase which indicating that Icariin can promote cell proliferation.Using EdU method to detect if OA FLSs proliferate or not, the results showed that Icariin can promote OA FLSs proliferation in a range of concentration.(3)Collecting the total RNA and protein of FLSs after being treated by different concentration of Icariin, by Real-time PCR to detect the change of gene expression about MMP14 and GRP78; By Western blot to detect the change of protein expression about MMP14 and GRP78; by immunofluorescence assay to detect IL-1β and MMP14 expression. The results showed that Icariin can inhibit the expression of gene and protein of MMP14 and GRP78, and can also reduce IL-1β expression level.(4)By transwell assay to detect cell migration after being treated by different concentration of Icariin, the result showed that Icariin can inhibit OA FLSs migration, and with the increasing concentration of Icariin the inhibitory effect was enhanced.In summary,by using different concentration of Icariin to treat OA FLSs, we demonstrated that Icariin has no cytotoxicity and it can reduce the expression level of MMP14, GRP78 and IL-1β, thereby actively regulate OA synovial cells and then change the articular cavity microenvironment which have therapeutic effect on osteoarthritis. |
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