Effects Of Hyperbaric Oxygen On The JNK/c-Jun Pathway Of The Inflammatory Mandibular Condylar Chondrocytes Induce By Interleukin-1 Beta

Objective:Explore the effect of hyperbaric oxygen on Sox-9 and COL2 in the inflammatory condylar chondrocytes induce by IL-1 beta via JNK/c-Jun signaling pathway.Methods:Mandibular condylar chondrocytes were separated form 3-4 weeks SD rats'condylar cartilage and cultured in vitro.In an bioclean environment,the anti phosphate buffer solution(PBS)washing and stripping cartilage,repeated shear condylar cartilages to 1mm3,first of all,with the 0.25%trypsin,digestion for 30 minutes in 37 ? enviroment.Then 0.2%collagenase type ?,oscillation digestive 1-2 hours to digest at 37 ? environment.Separate and culture primary chondrocytes,the growth and morphological changes of primary chondrocytes,observed by microscope.Use the cell morphology and type ? collagen immunohistochemistry staining to identify the primary condylar chondrocytes.Use the second generation in of condylar cartilage cells were divided into four groups:with 0 ng/ml,0.1 ng/ml,1 ng/ml,10ng/ml IL-1 beta inducing 24 hours,JNK,p-JNK,c-Jun,p-c-Jun,Sox-9 and type ? collagen protein were detected and JNK,c-Jun,Sox-9 and type ? collagen mRNA expression were detected.We screene the IL-1 beta optimal concentration of condylar cartilage cell inflammatory state;Take the second generation of cells,we divided cells into three groups:1,control group,cells do not any treatment;2,IL-1 ?group,10ng/ml IL-1 treating cells 24 hours;3,IL-1 ? + inhibitor group:lOng/ml IL-1 beta treatment for 24 hours,40 min early to use 40 um SP600125(JNK inhibitor).Use western blotting to detect JNK,p-JNK,c-Jun,p-c-Jun,Sox-9 and type ? collagen protein expression;RT-qPCR detects mRNA expression of JNK,c-Jun,Sox-9 and type II collagen expression in each group.Take the second generation of condylar cartilage cells were divided into four groups:1,control group,cells do not any treatment;2,IL-1 ? group,10ng/ml IL-1 treating cells 24 hours;3,IL-1 ? +HBO group,10ng/ml interleukin-1 ?(IL-1 ?)at 24 hours after use HBO every 48 hour handle 90 minutes.4,IL-1 ? + inhibitor group:lOng/ml IL-1 treatment for 24 hours,40 minter early to use 40um SP600125(JNK inhibitor).Western blotting,immunofluorescence detected by JNK,p-JNK,c-Jun,p-c-Jun,Sox-9 and type ?collagen protein expression;RT-qPCR detects mRNA expression of JNK,c-Jun,Sox-9 and type ? collagen expression in each group.Results:(1)The primary mandibular condylar chondrocytes were extracted and cultured,and the lens was regulated round,after 1 day the majority of adherent cells were polygonal shape.Cultured for 5-7 days,the number of cells increased obviously,cells covered the bottom,showing a cobblestone appearance.After passage,the cell adherence rate of 24h cells was significantly higher than that of the primary cells,and the distribution was relatively uniform.After 5 to 7 days,the cells were fused into pieces.Type ? collagen immunocytochemistry staining showed that there were brownish type ? collagen yellow granules in cytoplasm.The proliferation of chondrocytes in the first three generations was grown fast.After the 3rd generation,the cell appearred long spindle shape and the condylar chondrocytes proliferation rate was slowed down significantly.(2)Compared with the control group,the levels of p-JNK and p-c-Jun expression were gradual higher significantly in the three IL-1?groups with the increasing concentration of IL-1 ?.The level of JNK,was no obviously changed,the levels of c-Jun was no obviously changed between the IL-1 beta induced groups,but compared with control group,the level of c-Jun of IL-lbeta induced groups were increased obviously.Compared with the control group,levels of type II collagen and Sox-9 expression were significantly gradual lower in the three IL-1? groups with the concentration of IL-1 ? increasingly.(3)The level expression of Sox-9 and type ? collagen were significantly higher and the expression of p-JNK and p-c-Jun were significantly lower in the IL-1?+inhibitor group compared with the IL-1? group,the expression of JNK and c-Jun were no obviously changed.The decreased expression of Sox-9 and type II collagen in inflammatory condylar chondrocytes via active JNK/c-Jun signaling pathway.(4)The level expression of Sox-9 and type ? collagen were higher significantly and the expression of p-JNK and p-c-Jun were significantly lower in the IL-1?+HBO group compared with the IL-1? group,the protein expression of JNK and c-Jun were no obviously changed,it has the same tread with the IL-1?+inhibitor group.Conclusion:Hyperbaric oxygen can increase the expression of Sox-9 and type ?collagen in inflammatory condylar chondrocytes induce by IL-1 beta via inhibit JNK/c-Jun signaling pathway,and provide a theoretical basis for TMJ-OA treatment.