| Objectives:Autonomic circadian clocks have been found in part of the cartilage of mice,however the role of the cartilage clock mechanism in temporomandibular joint(TMJ)remains unclear.This study aimed to investigate the role of clock gene brain and muscle ARNT-like-1(BMAL1)in interleukin-1?(IL-1?)-induced rat's mandibular condylar chondrocytes and the potential molecular mechanisms.Methods:The condylar chondrocytes of 4-week old Wistar rats were cultured in vitro.Under strict aseptic conditions,bilateral condylar tissue was removed from the rat and rinsed twice with phosphate buffered saline(PBS)containing diabody and the dissected condylar cartilage was cut into 1 mm3-sized tissue blocks and shredded.The tissue was placed in a 0.25%trypsin digestion at 37 ? for 30 minutes and then 0.2%collagenase type II was added.After 1-2 hours of digestion,the primary chondrocytes were isolated and cultured,and the progress of growth and morphology of the chondrocytes were observed by light microscopy.The results of culture were identified by cell morphology,toluidine blue staining and type ? collagen immunofluorescence staining.The second generation of condylar chondrocytes were randomly divided into five groups and treated with IL-1? at 0,1,5,10 and 100 ng/ml for 24 hours,respectively.The protein content and mRNA expression levels of P-catenin,BMALl,collagen ?(COL2)and Aggrecan(AGG)were measured,and the optimal concentration of IL-1? that could cause inflammation of the condylar chondrocytes was selected.The second generation of condylar chondrocytes were randomly divided into 4 groups:control group,untreated cells;IL-1? group,IL-1? concentration of 10ng/ml for 24 hours;blank plasmid group,adding empty plasmid;siRNA group,interference plasmids containing BMAL1 siRNA added.Transfection of BMAL1 siRNA was observed by fluorescence microscopy.Western Blotting was used to detect the protein expression of BMAL1,?-catenin,COL2 and AGG in each group.The expression of BMAL1,?-catenin,COL2 and AGG mRNA in each group were detected by real-time quantitative polymerase chain reaction(RT-qPCR).Results:(1)After the condylar chondrocytes were extracted and cultured,the cells were observed as small circles with regular morphology.After 1 day,the cells were mostly adherent,and the cells were found to be polygonal or star when observed microscopically.After cultured for 4-5 days,the cells were observed as "paving stones".After a passage,the rate of adherent cells significantly accelerated.By toluidine blue staining,the condylar chondrocytes became purple-red and the background was light blue.It was found by immunofluorescence staining of type II collagen that brownish yellow particles were observed in the cytoplasm.(2)The results showed that with the change of IL-1? concentration,the expression of COL2 and AGG in mandibular condylar chondrocytes changed to different extents.When the effect of IL-1? at 10ng/ml on the expression of COL2 and AGG in condylar chondrocytes was the best,and the expression of BMAL1 in condylar chondrocytes induced by IL-1p(lOng/mL)was significantly decreased.(3)After the expression of BMAL1 was down-regulated,the expression of?-catenin,a key protein of Wnt signaling pathway,was increased,leading to a significant decrease in the expression of extracellular matrix products COL2 and AGG.Conclusions:Circadian rhythm plays an important role in the condylar chondrocytes of temporomandibular joint.Inhibition of BMAL1 expression in an IL-1?-induced osteoarthritis model in vitro,whereas imbalance of rhythmic BMAL1 expression leads to disturbances in the dynamic homeostasis of condylar chondrocytes,resulting in OA-like changes,and this change may play a role by Wnt/p-catenin signaling pathway.This experiment found that rhythm plays a key role in the normal daily function and integrity of temporomandibular joint condylar cartilage,providing more research directions for the development mechanism of TMJ-OA,and more clinical studies are needed to confirm this effect. |
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