| Objective:The temporomandibular joint(TMJ)is a bilateral joint in the oral and maxillofacial regions,involved in various oral and maxillofacial activities such as chewing,swallowing,language,and expression.Temporomandibular joint osteoarthritis(TMJ-OA)is a chronic degenerative disease characterized by progressive impairment of temporomandibular joint function.TMJ-OA involves dysfunction caused by the remodeling of subchondral bone in the temporomandibular joint,leading to long-term joint inflammation,reduced joint synovial fluid volume,and progressive bone joint degeneration.The pathogenesis of TMJ-OA is very complex,involving joints and their surrounding structures.The exact pathogenesis and potential molecular mechanisms are still unclear.Sirt2 is a member of the Sirtuin family and is a dependent deacetylase mainly present in the cytoplasm.It can shuttle between the cytoplasm and nucleus during mitosis,and has the effect of inhibiting collagenase induced osteoarthritis response.Inflammation plays an important role in the occurrence and development of TMJ-OA.Inflammation can exacerbate the destruction and absorption of subchondral bone,causing bone defects and osteophyte formation in the subchondral bone,reducing the elastic deformation of the subchondral bone of the condyle,and weakening the absorption of impact force by the condyle.However,there are significant differences in the structure and tissue between the temporomandibular joint and the bone joint,so there is no definitive report on whether Sirt2 plays the same role in TMJ-OA.This study mainly explores the effect of Sirt2 on the response of temporomandibular joint osteoarthritis and its related mechanisms of action.Analyze whether Sirt2 can inhibit inflammatory factors and alleviate bone resorption caused by subchondral bone inflammation in the temporomandibular joint.To clarify the effect of Sirt2 on bone resorption factor cathepsin K,osteoclast inhibitor OPG,osteoclast differentiation factor RANKL and TGF-β/ The role of Smad3 signaling pathwayMethods:Part Ⅰ: Effects of inflammatory environment on bone resorption of Sirt2 and temporomandibular joint1.Explore the effects of inflammation on the expression levels of Sirt2,inflammatory factors,and bone resorption related factors,and establish an animal experimental model of TMJ-OA.Male KM mice aged 8-10 weeks were randomly divided into a control group and an arthritis model group.A TMJ-OA mouse model of temporomandibular joint osteoarthritis was constructed by injecting type II collagenase into the temporomandibular joint cavity.HE staining and Micro computed tomography(Micro CT)were used to detect the damage of the temporomandibular joint in mice;Tartrate resistant acid phosphatase(TRAP)staining was used to detect the number of osteoclast;Enzymelinked immunosorbent assay(ELISA)was used to detect the levels of Sirt2,IL-6,TNF-α,and cathepsin K;Immunohistochemical staining assay for detecting osteoprotegerin(OPG)and nuclear factor in subchondral bone κ Receptor Activator of Nuclear Factor Ligand-κ The expression of B Ligand,RANKL,sirtuin 2,cathepsin K,IL-1β,and IL-6.2.In vitro experiments were used to further verify the results of in vivo experiments.In the cell experiment,mouse osteoblast MC3T3-E1 was added with 10ng/ml IL-1β to simulate the inflammatory environment,The expression of OPG,RANKL,Sirt2,cathepsin K and IL-6 in the inflammatory environment at 0,1,2,3,4 and 5 hours was detected by using Western-blotting and Real-time q PCR experiments.Immunofluorescence staining was used to observe the secretion of cathepsin K at 0,1,2,3,4 and 5 hours.Part Ⅱ: Effects of Sirt2 on inflammatory factor secretion and expression of Bone resorption related factors1.To explore the effect of Sirt2 on inflammatory factors and subchondral bone absorption in inflammatory environment,and to construct an animal experimental model of TMJ-OA,which was specifically divided into control group,model group,model group+Sirt2 agonist group(NAD+),model group+Sirt2 inhibitor group(AK-7).The injury of temporomandibular joint of mice in each group was detected and observed by HE staining,hard tissue sectioning and Micro CT.TRAP was used to detect the number of osteoclast;ELISA assay was used to detect the levels of IL-6,TNF-α,IL-1β,and cathepsin K.The expression levels of Sirt2,cathepsin K,OPG,RANKL,and IL-1β were detected by immunohistochemistry.2.A cell experimental model of TMJ-OA was established by stimulating MC3T3-E1 cells with IL-1β for 5 hours to validate the research results of in vivo experiments.The experiment was divided into four groups: control group,IL-1β treatment group,IL-1β+Sirt2 overexpression group,and IL-1β+Sirt2 knockdown group.CCK8 and flow cytometry were used to detect the activity and apoptosis of MC3T3-E1 cells in an inflammatory environment.Western blotting and Real time q PCR were used to detect OPG,RANKL,cathepsin K,and IL-6.Immunofluorescence technology was used to detect changes in the expression of cathepsin K under overexpression or knockdown of Sirt2.Part Ⅲ: The regulatory effect of Sirt2 on the inhibition of osteoblast related factors in the TGF-β/Smad3 signaling pathway1.Exploring the relationship between inflammation and the TGF-β/Smad3 signaling pathway through in vitro experiments,using Western blotting experiments and Real time q PCR experiments to detect changes in TGF-β1,Smad3,and pSmad3 expression levels in MC3T3-E1 cells under inflammatory conditions at 0,1,2,3,4,and 5 hours.2.Explore whether reactivation of the TGF-β/Smad3 signaling pathway blocks the role of Sirt2 in alleviating inflammation and inhibiting osteogenic resorption.Firstly,an animal experimental model of TMJ-OA was constructed,which was divided into control group,model group,Sirt2 agonist+model group,Sirt2 inhibitor+model group.Immunohistochemical staining was used to detect the expression levels of TGF-β1,pSmad3,and Smad3 proteins.Then,MC3T3-E1 cells were stimulated with IL-1β for 5 hours to establish a cellular inflammation model.The experiment was divided into four groups: control group,IL-1β treatment group,IL-1β+Sirt2 overexpression group,and IL-1β+Sirt2knockdown group.The expression levels of TGF-β1,pSmad3,and Smad3 were detected using Western blotting experiments.Real time q PCR assay was used to detect the expression levels of TGF-β1 and Smad3.Immunofluorescence experiments were conducted to observe the effects of Sirt2 overexpression and knockdown on pSmad3.3.Study whether the protective effect of Sirt2 is influenced by the TGF-β/Smad3 pathway.A cellular inflammatory model was established by stimulating MC3T3-E1 cells with IL-1β for 5 hours.The experimental groups are as follows: control group,group IL-1β,IL-1β+Sirt2 overexpression group,IL-1β+Sirt2 overexpression+ TGF-β/Smad3 agonist group.Observing the changes in the expression levels of OPG,RANKL,and cathepsin K through Western blotting and Real time q PCR experiments,detecting the effects of Sirt2 and TGF-β/Smad3 pathway,and detecting the expression of inflammatory factors TNF-α,IL-6,and IL-1β through ELISA experiments.Results:Part Ⅰ: Effects of inflammatory environment on bone resorption of Sirt2 and temporomandibular jointThe HE staining results showed that in the model group,the surface of the mouse cartilage was smooth,the number of chondrocytes and small blood vessels was reduced,the cartilage lacunae were clear,the thickness of the whole layer of cartilage was reduced and the layers were blurred,the polarity was disordered,and the cartilage matrix was denatured.In the control group,the surface of mouse cartilage was smooth,the differentiation of chondrocytes was mature,the cartilage lacuna was clear,the cartilage layer was thick,and the structure was clear.The results of microscopic CT showed that compared with the control group,the BV/TV and Tb of subchondral bone in the model group decreased.SP increase(P<0.05).The TRAP results showed that the number of osteoclasts in the model group increased significantly(P<0.05).ELISA results showed that the expression level of Sirt2 in the control group was significantly lower than that in the model group,while the secretion level of TNF-α,IL-6 and cathepsin K was higher than that in the model group(P<0.05).The immunohistochemical results showed that the expression of cathepsin K,IL-6,RANKL and IL-1β in the model group was significantly higher than that in the control group,while the expression of Sirt2 and OPG was significantly lower than that in the control group(P<0.05).The results of cell experiment showed that the expression of RANKL,cathepsin K and IL-6 increased gradually after 1 to 5 hours of inflammatory stimulation,while the expression of OPG and Sirt2 decreased gradually(P<0.05).The immunofluorescence experiment showed that the expression of cathepsin K in MC3T3-E1 increased with the increase of IL-1β stimulation time(P<0.05).Part Ⅱ: The effect of Sirt2 on the secretion of inflammatory factors and the expression of bone resorption related factorsThe HE staining results of hard tissue sections and HE staining showed that compared with the model group,the subchondral bone structure of Sirt2 inhibitor group was disordered,the layers were unclear,and the chondrocyte layer was broken,while the subchondral bone morphology of Sirt2 agonist group was relatively complete,the absorption was less,and the bone trabeculae were arranged neatly.Micro-CT results showed that compared with the model group,BV/TV increased and Tb.SP decreased in the Sirt2 agonist group.On the contrary,BV/TV decreased and Tb.SP increased in the subchondral bone in the Sirt2 inhibitor group(P<0.05).The results of TRAP experiment showed that the number of osteoclasts in Sirt2 agonist group was less than that in model group,while the number of osteoclasts in Sirt2 inhibitor group was significantly higher than that in model group(P<0.05).The results of ELISA showed that the secretion levels of TNF-α,IL-6,IL-1β and cathepsin K in Sirt2 agonist group were significantly lower than those in model group.On the contrary,the secretion levels of TNF-α,IL-6,IL-1β and cathepsin K in Sirt2 inhibitor group were higher than those in model group(P<0.05).The results of immunohistochemistry showed that Sirt2 agonist group could reduce the expression of cathepsin K,RANKL,IL-1β protein,and increase the expression of Sirt2 and OPG;The experimental results of Sirt2 inhibitor group and Sirt2 agonist group were contrary(P<0.05).Through cell experiments,we confirmed that Sirt2 overexpression group can reduce the apoptosis of osteoblasts and increase the activity of osteoblasts.In Sirt2 knockdown group,the number of osteoblasts apoptosis increased and the cell activity decreased(P<0.05).The results of Western-blotting and Real-time q PCR showed that Sirt2 overexpression group reduced the expression levels of RANKL,cathepsin K,IL-6,promoted the expression of OPG,and increased the OPG/RANKL ratio.The results of Sirt2 knockdown experiment were contrary to those of Sirt2 overexpression experiment(P<0.05).Immunofluorescence test showed that the expression of cathepsin K decreased when Sirt2 was over-expressed,and increased when Sirt2 was knockdown.Part Ⅲ: The regulatory effect of Sirt2 on the inhibition of osteoblast related factors in the TGF-β/Smad3 signaling pathwayThe results of immunohistochemistry showed that the expression level of TGF-β1,pSmad3 and Smad3 in Sirt2 inhibitor group was higher than that in model group,while the expression level of TGF-β1,pSmad3 and Smad3 in Sirt2 agonist group was lower than that in model group(P<0.05).The trend of cell experiment was basically consistent with that of animal experiment,but the expression level of Smad3 did not change significantly(P>0.05).The immunofluorescence results showed that the fluorescence intensity of pSmad3 decreased when Sirt2 was overexpressed,indicating that the expression of pSmad3 decreased,while the fluorescence intensity increased when Sirt2 was knockdown,suggesting that the expression of pSmad3 increased.When Sirt2 overexpression group was induced by TGF-β/Smad3 agonist for 3 hours,the results of Western-blot and Real-time q PCR showed that the expression of OPG decreased in the group with TGF-β/Smad3 agonist,while the expression of RANKL and cathepsin K increased.The expression of TNF-α,IL-6 and IL-1β increased by ELISA.Conclusion:1.Inflammatory reaction can reduce the secretion of Sirt2,promote the absorption and destruction of osteogenesis,and play an important role in temporomandibular joint osteoarthritis.2.Increasing the expression of Sirt2 can inhibit the secretion of inflammatory factors,improve the imbalance between the expression of OPG and RANKL,and reduce the destruction and absorption of osteogenesis.3.Sirt2 alleviates inflammation,inhibits osteogenesis and reduces TGF-β/Smad3 signal pathway. |
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