| [Objective]To investigate the mechanism of leptin expression inside the condylar cartilage acting on the mandibular cartilage during temporomandibular joint osteoarthritis(TMJOA)-like pathogenetic process,and to provide a new direction of study on TMJOA pathogenetic mechanism.[Method]1.Twelve 8-week-old male C57BL/6 mice were used in this study for the mouse model of temporomandibular joint osteoarthrits.Using the animal model of temporomandibular joint compressive loading device(patent N.O.:201120210396.4),which was designed by our research group,an upward and backward compressive stress(20 g)was exerted on mouse condylar cartilage of the force-applying groups for 7 days.Compare the condylar cartilage morphology and thickness of the force-applying group and control group by Safraning O Staining.Immunochemistry(IHC)was performed to detect the protein expression of IL-1β,MMP-3,leptin and GGPPS in the mandibular condylar cartilage;2.Twelve 8-week-old male C57BL/6 mice were used in this study.They were randomly divided into experiment group and control group,which were fed with high fat diet and normal diet respectively.We weighed them weekly and recorded the body weight(g).After 12 weeks of different diets for the two groups,the morphology and thickness of the two groups’mandibular condylar cartilages were compared by Safraning O Staining,and the expression of IL-1β,MMP-3,leptin and GGPPS in the mandibular condylar cartilages of the two groups were compared by Immunochemistry(IHC);3.The mandibular condylar chondrocytes isolated from 3-week-old male SD Rats were cultured in vitro and treated with IL-1β for 24h to acquire inflammatory condylar chondrocytes model.CCK-8 was performed to detect the optimal concentration and time of simvastatin treatment on the condylar chondrocytes.The chondrocytes were divided into four groups(control group,IL-1β group,statin group,IL-1(3+statin group),and were treated with or without IL-1β and simvastatin in the optimal concentration for the optimal period of time.The mRNA expression of Collagen Ⅱ,MMP3,MMP13,leptin,Ob-Rb and GGPPS were detected with Quantitative real-time Polymerase Chain Reaction(qRT-PCR);4.Twelve GGPPS conditional knockout mice were used in our study.The experiment group include six GGPPS knockout mice(GGPPS-KO),and the control group include six backgroup-matched ordinary mice.Two weeks after tamoxifen inducing GGPPS gene knock out,the expression of GGPPS,IL-1β and leptin in the condylar cartilage were examined by IHC,and the mandibular condylar cartilage morphology and thickness of GGPPS-KO mice and control mice were compared.[Results]1.Compared with control,the condylar cartilage of force-applying mice was thinner,and the cartilage matrix was less.The IHC results showed up-regulation of IL-1β,MMP-3,and leptin in the condylar cartilage of force-applying mice(P<0.05);2.Mice fed with high fat diet gained more weight,and got obvious mandibular condylar cartilage destruction and higher OARSI scores by 4.5 times than those with normal diet.The expression of IL-1β,MMP-3,leptin in the condylar cartilage of high fat diet mice were all significantly increased when comparing with the normal diet mice(P<0.05);3.The results of qRT-PCR showed that the condylar chondrocytes treated with IL-1β had a decreased mRNA expression of collagen Ⅱ by 71%compared with control(P<0.05),significantly increased mRNA expression of MMP-3 and MMP-13(P<0.05),up-regulated mRNA expression of leptin by 1.5 times(P<0.05),and down-regulated mRNA expression of Ob-Rb by 77%(P<0.05).After treated with simvastatin,compared with IL-1β group,the IL-1β+statin group showed that the mRNA expression of leptin decreased by 60%(P<0.05),the mRNA expression of Ob-Rb increased by 85%(P<0.05),which were almost normal level(control);Meanwhile,the mRNA expression of collagen Ⅱ was up-regulated by 1.5 times(P<0.05),and the mRNA expression of MMP-3 and MMP-13 were significantly down-regulated(P<0.05);4.The IHC results showed that the expression of GGPPS protein in the TMJOA-like condylar cartilage was down-regulated both in force-applying mice(MF)and high fat diet induced obese mice(HFD)when compared with their respective control mice(P<0.05).The results of qRT-PCR showed that the condylar chondrocytes treated with IL-1β had a decreased mRNA expression of GGPPS by 26%compared with control(P<0.05),while after treated with simvastatin,the IL-1β+statin group showed that the mRNA expression of GGPPS increased by 33%compared with IL-1βgroup(P<0.05);The expression of GGPPS in the condylar cartilage of GGPPS-KO mice showed significant decrease(P<0.05).The the expression level of IL-1β and leptin in the GGPPS-KO mice’s condylar cartilage was higher(P<0.05),the matrix was degraded,and the cartilage was thinner(P<0.05)than those in control.[Conclusion]1.In the excessive compressive mechanical stress-induced TMJOA model,the force-applying mice with TMJOA-like pathological changes showed condylar cartilage destruction and the matrix degradation metabolism was active.The expression of leptin inside the condylar cartilage of force-applying mice was also increased,which suggested that the abnormal expression of leptin inside the condylar cartilage participated in the pathogenetic process of excessive compressive mechanical stress-induced TMJOA-like changes;2.High fat induced obesity gave rise to temporomandibular joint osteoarthritis that resulted in condylar cartilage matrix degradation,the mechanism of which might be relevant to the abnormal increased expression of leptin inside condylar cartilage.3.Inflammatory condylar condrocytes also showed active cartilage matrix degradation metabolism and increased expression of leptin.With statin down-regulating the leptin expression,the cartilage matrix degradation metabolism was alleviated and the matrix synthesis was recovered.These phenomenon also indicated that the abnormal increased expression of leptin played an important role in the inflammatory destruction in the condylar cartilage;4.Whether in vivo or in vitro study,the excessive leptin expression related TMJOA-like pathological changes were positively relevant to the down-regulation of GGPPS,which suggested that the pathogenetic process of excessive leptin expression induced TMJOA-like changes might be regulated by GGPPS mediated signal transduction pathway... |
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