The Expression And Significance Of Interleukin 18 And Prostaglandin E2 In Synoviocyte With Osteoarthritis

BackgroundsOsteoarthritis (OA) has been become the most common articular disease over the world.From the investigation we know:The incidence rate of gonarthritis was 49% above 60ys in our country, now the rate has become higher and younger. OA maked the older disable and it is a heavy burden for society. The pathogenesis isn't clear up to now. The current research show that the pathogenesis of OA involves kinds of cytokines and growth factors, eg:BMPs, TGF, TNF and IL-1. The main pathogenesy of OA is disequilibrium between anti-inflammatory factor and proinflammatory factor, it may be involved in the degradation of cartilage matrix and synovium of the inflammatory lesion process. From the morphology study we know: OA destruction the cartilage and synovium of joint. But in cartilage and synovium, the reactivate and recovery potential are very limited. It is nearly an irreversible process. So, it is very important to intervention and avoidance the occurrence of OA in an early stage. Interleukin-18 (Interleukin-18, IL-18) is a new proinflammatory factor, it is cloned from the toxic shock mouse'liver by Okamura in 1995, it is the protein induced by the stress. Because the peptide can induce helper T lymphocytes produce interferon-y (IFN-y), it can enhance the ability of splenic T cell proliferation and splenic NK cell activity, and interleukin-12 (IL-12) the biological activity very similar, it was originally named IFN-y inducible factor (IFN-y IGIF). The primary function of IL-18 is to mediate the inflammatory reaction on tissue, it also relates to some autoimmune diseases. Current study showed that, IL-18 is not only found in synovial fluid, meniscus, cartilage cells of patients with arthritis, also be found in the serum, but the content is lower than in synovial fluid. Gracie found the IL-18 content of synovial fluid and synovial membrane of rheumatoid arthritis higher than osteoarthritis, and the osteoarthritis patients'content was higher than normal, but the expression level of IL-18 was closely related to and activity period of osteoarthritis,this prompted us IL-18 played an important role in the occurrence and development of osteoarthritis. Prostaglandin E2 (PGE2) is a kind of product of fatty acid metabolism, secreted by megalophage. It is also play an important role in the pathogenesis of OA. It could induce bone absorption in subchondral, influence the metabolism of bone and cartilage, prevent the composition of proteoglycans. Finally it makes the destruction of collagen typeⅡand articular cartilage. Recently research showed:IFN-γ, induced by IL-18 in the synovial fluid can significantly stimulate IL-6, NO, IL-1β, PGE2, prevent the composition of proteoglycans and decrease the expression of IL-8 and IL-10 level. Above all, in OA, IL-18 is a most probable cytokine which could induce the expression of PGE2.The etiology of OA mainly displays in two factors:one is destruction of articular cartilage and change of physico-chemical property in cartilage cell, the other is synovium of the inflammatory lesion process and release of inflammatory factor. In OA, after the articular cartilage was breakdown by etiological factor, it could release cartilage chips to synovial fluid. This can stimulate inflammatory reaction of synovium. Synovium of the inflammatory lesion process could release many inflammatory factors, which degradation cartilage. As time goes, it becomes an infernal circle. So we think the inflammatory reaction of synovium is the point of cartilage losed and OA chronic to last. Now the focal point of study is to approach the change of inflammatory factors which excreted by synoviocyte in synovium of the inflammatory lesion process. For precluding the influence of immune system and endocrine system, we use vitro cultured synoviocyte (OA patients and health adult) and make use of immunocytochemical method and flow cytometric analysis identify synoviocyte. We detected the expression of IL-18 and PGE2 in supernatant liquid of cultured cell, and analysed the variability and dependability. Then identify if IL-18 and PGE2 join in the synovium of the inflammatory lesion process. The research approaches how IL-18 and PGE2 affect the mechanism of OA from molecular level. It offers a theory for the clinical treatment and later period research of OA.Objectives1. Study the changes of expression of IL-18 and prostaglandin E2 in the pathogenesis of OA in articular synoviocyte of the patients with OA, find out IL-18 in the OA disease process and to analyze the differences of their expression in normal articular organize. And to observe the changes of IL-18 and prostaglandin E2 expression in the time and space, in order to explore the role of IL-18 and PGE2 in the process of the molecular mechanism of OA, fine out the new way for OA diagnosis and treatment.2. To get wareness of the morphological changes a of the synoviocyte in the passage.Make understand of the difference between normal and abnormal in morphology. Select the eq. synoviocyte for our later research and bserve the dedifferentiation of synoviocyte culture.Methods1. Selected the normal and OA synovium. Then culturing normal synoviocyte and OA synoviocyte (mainly from the patients with TKA)with block culture method. Subcultured and observation the differences in Morphological. Make use of immunocytochemical method and flow cytometric analysis identify synoviocyte.2. Detecting the expression of cytokines (IL-18 and PGE2) in the supernatants by ELISA. Comparing the differences between IL-18 and PGE2, find out the dependability between them.Then identify if IL-18 and PGE2 join in the synovium of the inflammatory lesion process. Approach the mechanism of OA from molecular level.3. Data presented by X±S, statistics done with SPSS 13.0. Inter-group comparison group using t-test, use correlation analysis between IL-18 and PGE2. P<0.05 presents statistics different.Results1. The pathological section of synovium showed:compared to normal synovium, the OA synovium had evident hyperplasy in vessel and infiltrating by inflammatory factors. This is the character of chronic inflammation. The survival rate of OA synoviocyte is lower than that's of normal synoviocyte. The time of OA synoviocyte in the form of adherence and primary culture is longer than the normal synoviocyte. Primary synoviocytes are mainly fusiform shape, some are macrophage-like cells. The synoviocytes occur dedifferentiation after passage 3. Make use of immunocyto-chemical method identify passage 3 synoviocytes with the antibody of Collagen type 1. we find most cytoplasm's coloretue of the postive synoviocytes (with one-antibody) is deeper, with brunneus. But the amount of cell is decrease and the shape is anomalism. Identified synoviocyte with flow cytometric analysis,93.1% of cells is excitated by fluorescence, which affiliated with antibody of VCAM-1 (marked by FITC). In control group, only 1.55% of cells is excitated by fluorescence (marked by FITC, without antibody of VCAM-1), this proves less than 90% of cells are synoviocytes. The result about identify of IL-18 in synoviocytes is similar to immunocytochemical method identify synoviocytes with the antibody of Collagen type I, the postive cells (with one-antibody) is deeper, with brunneus. The negative (without one-antibody) is tasteless.2. The level of IL-18 and PGE2 in OA synoviocytes is higher than that in normal synoviocytes. In OA synoviocytes the average expression of IL-18 is 63.858±21.401pg/ml while 17.735±1.467pg/ml in normal cells(t=10.044, P<0.001). In OA synoviocytes the average expression of PGE2 is 437.224±325.284pg/ml while 24.020±20.591pg/ml in normal cells(t=5.926, P<0.001).They have a statistical significance.3. The expression of IL-18 and PGE2 in Synoviocytes has a relationship. Correlation analysis showed that IL-18 and PGE2 (r= 0.863, P<0.001) have a positive correlation.Conclusions1. The level of IL-18 and PGE2 in OA synoviocytes is higher than in normal synoviocytes. This may reflect IL-18 and PGE2 direct join in the synovium of the inflammatory lesion process.2. We find the expression of IL-18 and PGE2 in supernatant liquid of cultured cells has a positive correlation. This may prove that IL-18 and PGE2 interaction in process of OA.3. The morphology different between normal synoviocytes and OA synoviocytes aren't conspicuous, but the synoviocytes occur dedifferentiation after passage 3. So if we want to study the etiology of OA, we should use primary or 1-2 passage synoviocytes.