The Role Of Sirt6in Osteoarthritis And Its Underlying Mechanisms

Osteoarthritis (osteoarthritis, OA), also known as degenerative arthritis, is a posttraumatic or age-related degenerative disease of the joint which is mainly characterized by the progressive degradation of cartilage with tissular inflammatory phase, along with joint pain and stiffness. So far, the pathogenesis and molecular mechanisms of OA are still unclear. It is founded that aging and inflammation are closely related to OA. Suppression of chondrocyte senescence or inflammation induced by IL-lbeta can significantly delay the occurrence of osteoarthritis. Therefore, studies on the mechanisms of aging or inflammation in joint may help shed some light on understanding the pathogenesis or getting new therapeutic target of OA.Sirt6is a NAD+dependent histone deacetylases that has been implicated in inflammatory, aging and gene stabilization pathways. Currently the function of sirt6in cartilage has not been reported. In this research we will study in cell and OA animal models using gene transfection and gene silencing technology to clarify the role of sirt6in osteoarthritis. The research contains:1) the expression of sirt6in osteoarthritis cartilage;2) effects of sirt6on chondrocyte senescence;3) effects of sirt6on chondrocyte degeneration under Inflammatory stimuli;4) the molecular mechanism of sirt6on regulation of OA;5) validated the role of sirt6in osteoarthritis in animal models. this study explore the role of sirt6in the pathogenesis of OA and may supply a new target for OA therapy. Part one The expression of Sirt6in osteoarthritis cartilageObjective:Clarify the expression of Sirt6in normal and OA cartilage.Method:Human normal and osteoarthritis cartilage were analyzed for the expression of Sirt6proteins by immunofluorescence and Western blot, the sirt6mRNA was analyzed RT-PCR. Take2and16-month-old C57mice, compare the expression of sirt6in knee joint by immunofluorescence analysis.Result:The sirt6fluorescence intensities are reduced in the knee articular cartilage of OA relative to normal. To confirm this result, primary human knee articular chondrocytes cultivated from normal and OA patient were studied by western blotting and RT-PCR. The protein and mRNA level of sirt6was significantly decreased in the articular chondrocytes of OA patients. immunofluorescence stains of the cartilage indicated an aging-related reduction in fluorescence intensities of sirt6Conclusion:The expression of sirt6was significantly reduced in osteoarthritis and nature degeneration cartilage. Part two The role of sirt6on chondrocyte senescenceObjective:Clarify the role of sirt6on chondrocyte senescence.Method:Senescence chondrocytes were analyzed for the expression of sirt6proteins by Western blot. First we overexpression or knock down sirt6in chondrocyte by lentivirus, then compare the the maximum number of passages and percentage of senescence cell between different groups. Senescence cells were marked by beta-gal staining.Result:Cellular senescence was found at the20th passage. Sirt6expression was dramatically decreased from passage2to20by Western blot. The percentage of SA-β-gal staining positive cells was dramatically increased (92.3%at10th passage) in sirt6knock down cells compare to the normal(31%). Overexpression of sirt6significantly reduced the percentage of aging cells at the15th passage. The maximum number of passages in sirt6overexpression chondrocytes was30.Conclusion:Decreased expression of sirt6is one of the main reasons for chondrocyte senescence. Overexpression sirt6can significantly inhibit chondrocyte aging. Part three The role of sirt6on chondrocyte degeneration induced by IL-1βObjective:Clarify the role of sirt6on chondrocyte degeneration induced by IL-1β.Method:Primary chondrocytes were treated by IL-1β(10ng/ml) for0,1,3and5days. Then we analyzed the expression of sirt6by Western blot and immunofluorescence. Chondrocytes were induced to express wild-type sirt6using lentivirus (Lentivirus-sirt6), after that, we examined the effect of sirt6in the regulation of MMP-13and type II collagen under IL-1β(10ng/ml) treatment for24hResult:The western blotting analysis revealed that IL-1β treatment reduced the expression of sirt6in a time-dependent manner. IL-1beta significantly increased MMP-13level in chondrocytes. However, this up-regulation was inhibited by the overexpression of sirt6. Moreover, IL-1βdecreased the expression of colleagn II and this effect was also significantly attenuated by overexpression sirt6.Conclusion:IL-lbeta reduces the expression of sirt6in chondrocytes. Overexpression sirt6significantly inhibits chondrocyte degeneration induced by IL-lbeta. Part four The molecular mechanism of sirt6on regulation of cartilage degenerationObjective:Clarify the molecular mechanism of sirt6on regulation of cartilage degeneration.Method:(1)Chondrocytes were transfected with sirt6wild type plasmid, thereafter cells was treated with IL-1β (10ng/ml) for24h. The intracellular distribution of endogenous Ik-Ba and NF-kB-p65was studied by immunofluorescence and Western blot. In order to clarify the role of sirt6on NF-kB pathway chondrocytes were transfected with sirt6wild type or sirt6mutant plasmid, then we examined the expression of NF-kB target genes (IL-1, MMP9*NFKBIA) and MMP-13and type II collagen proteins.(2) Chondrocytes was transfected with sirt6wild type plasmid. The intracellular autophagosme was analyzed by immunofluorescence. Protein LC3II/LC3I was analyzed by Western blot.Result:(1) NF-kB-p65traslocated to the nuclear both in the chondrocytes with sirt6-WT transfection and non-transfection. Meanwhile, the degradation of Ik-Ba was noted in both kinds of chondrocytes after IL-1β treatment. These results was confirmed by Western bolt. Overexpression wild type sirt6could suppres NF-kB target genes (IL-1、MMP9、 NFKBIA) and MMp13expression, meanwhile, it could inhibit type II collagen reduction. There were no effects in chondrocyte which overexpressed the mutant sirt6.(2) The number of autophagosome was significantly increased in chondrocyte transfected with Sirt6plasmid. The LC3II/LC3I ratio also increased significantly.Conclusion:Sirt6inhibits IL-1β-mediated osteoarthritic changes in chondrocytes by suppressing NF-kB pathway and improving autophagy. Part five Overexpression sirt6ameliorates osteoarthritis development in vivoObjective:Confirm sirt6ameliorates osteoarthritis development by using mice AO models.Method:Fifteen C57mice were divided into sham group, experimental group and control group, each with five mice. After modeling, the experimental groups was injected with sirt6lentivirus in the knee joint, the sham and control group were injected with blank virus. One month after modeling, the same lentivirus was injected for another time. SO staining and OARIS system was used to evaluate cartilage degeneration.Result:Cartilage destruction was demonstrated by Safranin Orange staining and OARIS system. The control group mice joints displayed the most severe OA phenotypes, such as cartilage damage or even loss, reduced Safranin O staining and Joint space. However, experiment group showed great protective effect on cartilage.Conclusion:Overexpression sirt6ameliorates osteoarthritis development in vivo.