The Effects And Mechanisms Of SIRT1Regulates Chondrocytes Autophagy In Osteoarthritis

Osteoarthritis is a kind of destruction of articular cartilage, and joint space narrowing formation is characterized by degenerative joint disease. Because of its pathogenesis is not fully known, it is difficult to early prevention and treatment. The current focus of study on the pathogenesis of osteoarthritis is how to maintain a dynamic balance of cell and tissue metabolism, and how these mechanisms to regulate the aging process. Recently, we found that autophagy is involved in the aging process of the body’s organs, engulfed by lysosomal degradation of their damaged structures to affect the synthesis of cellular material, degradation and re-use in order to maintain cellular homeostasis. SIRT1, a NAD+-dependent deacetylase, is a homologue of silent information regulator (Sir2), which was initially identified from studies in aging yeast. Numerous reports show that SIRT1activation may improve or retard age-related diseases such as diabetes and cardiovascular disease by regulating a variety of cellular processes including decreasing inflammation and inducing autophagy. However, it is involved in osteoarthritis cartilage aging and could adjust the level of chondrocyte autophagy. Therefore, this study first investigate whether autophagy involved in articular cartilage degeneration and chondrocyte autophagy in osteoarthritis of the possible roles, and then further clarify whether SIRT1and aging-related cartilage chondrocytes and whether autophagy regulation.The study consists of two parts. In part I, in order to study autophagy chondrocytes and cartilage aging and osteoarthritis correlation, we firstly collect osteoarthritis group, age group and youth group cartilage tissue for pathological score, immunohistochemical method to detect autophagy-related proteins LC3, Beclin-1expression in cartilage tissue. Then isolated and cultured chondrocytes identified chondrocytes in vitro rarely observed autophagy, and found no significant difference between the three groups of chondrocytes basis autophagy. In order to simulate the body joints hypoxic environment, we cultured chondrocytes in vitro hypoxic environmental conditions; we found that hypoxia-induced changes in chondrocyte autophagy, there are significant differences in the three groups of cells. The plasmid with GFP-LC3transferred cartilage cells, using GFP-LC3fusion protein to trace the formation of autophagy, the process of autophagy was observed in confocal, and youth groups in osteoarthritis chondrocytes were found in a large number of autophagy particle formation, while the older group chondrocyte autophagy particles no significant changes. These results suggest that autophagy cells decreased in aging cartilage. The results show that the chondrocytes hypoxic environment autophagy is activated in young group, and increasing the number of cell activity. Then we found that the electron microscope and confirmed in the youth group chondrocyte autophagy, the autophagy in cell organelles normal morphology was wrapped infer may be a protective autophagy, is encapsulated cells may be reused, which in the chondrocyte autophagy occurs when the body contains a large number of autophagic vacuoles and cellular debris, it may be induced autophagic death. This may be prompted autophagy in the youth group and osteoarthritis group opposite effects on cell viability basis. Thus, we conclude that aging cartilage of osteoarthritis and increase the level of autophagy may play a different role.In part II, to study whether SIRT1associated with osteoarthritis, immunohistochemical analysis of three samples of SIRT1expression, and the results show that the older and osteoarthritis group SIRT1protein expression was significantly too low, and then we used Western blot cultured chondrocytes in vitro validation of the results. To study the effects of SIRT1on autophagy chondrocytes, we used SIRT1agonist (Resveratrol) and inhibitors (Sirtinol) alter the activity of chondrocytes endogenous. SIRT1and deacetylation by SIRT1activity assay kit to detect the activity of SIRT1in SIRT1 activity under changing conditions detected osteoarthritis group and youth group autophagic capacity of chondrocytes change, the results showed that SIRT1activity was inhibited, and the older group chondrocyte autophagy capacity enhancement, while osteoarthritis chondrocytes autophagic capacity significantly weakened. Then, by using the agonists or inhibitors of autophagy autophagy base change detecting expression of SIRT1, chondrocytes results in the three groups, with the ability to change autophagy, SIRT1protein showed no significant change. Therefore, we used co-immunoprecipitation method to determine the autophagy-related protein SIRT1and whether interactions within the cell. The results showed that autophagy-related protein Atg7, LC3and Beclin-1, only Atg7interact with SIRT1. This further confirms our hypothesis that SIRT1relationship with a direct effect of autophagy-related proteins, and SIRT1involved in regulating chondrocyte autophagy.In summary, autophagy plays two different roles in osteoarthritis. With cartilage aging, autophagy ability is abated, cartilage repair capacity is also associated with decreased, and the mechanism of autophagy may initiate autophagic cell death. In the whole process, SIRT1is involved in the regulation of autophagy chondrocytes in the cartilage, early aging can enhance autophagy cells, chondrocytes through regulating autophagy defer cartilage aging; while in osteoarthritis later, by reducing the cartilage autophagic cell death hinder the further destruction of cartilage.