Circadian Rhythm Protein Bmal1 Modulates Cartilage Gene Expression In Temporomandibular Joint Osteoarthritis Via MAPK/ERK Pathway

Objectives:Temporomandibular joint osteoarthritis(TMJ-OA)is the most common joint degenerative disease.Under the influence of mental and psychological inducing factors,TMJ-OA is more likely to occur when occlusal trauma occurs.The purpose of this study was to explore the relationship between circadian rhythm disturbance(CRD)and TMJ-OA,the expression changes of circadian clock gene Bmall and MAPK/ERK signal pathway and their downstream transcription factors under circadian rhythm disturbance,and the mechanism of extracellular matrix degradation and arthritis development,and to clarify the mechanism of circadian clock gene Bmal1 regulating temporomandibular osteoarthritis mediated by MAPK/ERK signal pathway.Methods:After detecting the circadian expression rhythm of the rat rhythm gene Bmall,the rats' normal sleep rhythm was disrupted by the experimental Modified multiple platform method(MMPM)for the appropriate time period.Serum cortisol(CORT)and adrenocorticotropic hormone(ACTH)levels in Wistar rats were measured to assess the effect of a model of chronic sleep rhythm disorder established in rats.HE staining was used to observe the extent of cartilage destruction in the TMJ joint in rats.Immunohistochemistry,Western blot,and RT-qPCR were used to observe changes in the expression of circadian clock gene Bmall,MAPK/ERK signaling pathway,and joint inflammatory factor IL-6 in the inflammatory state of condyles of rats with circadian rhythm disturbance.After isolating cultured rat condylar chondrocytes(MCCs)in vitro and using the inflammatory factor interleukin 6(Interleukin 6,IL-6)to induce the inflammatory state of condylar chondrocytes,the ERK-specific inhibition of U0126,Bmall-siRNA,and B mall-plasmid subgroups were added to the in vitro TMJ-OA model of chondrocyte culture medium,respectively.The morphological characteristics of chondrocytes were observed by Alicin blue staining,the growth-proliferative state of chondrocytes was detected by CCK-8,and the expression changes of the clock-rhythm gene Bmall,MAPK/ERK signaling pathway and downstream matrix metalloproteinases and extracellular matrix were observed by Western blot.Specifying the mechanism by which the circadian clock gene Bmall regulates the MAPK/ERK signaling pathway-mediated temporomandibular osteoarthritis.The effect of the circadian clock gene Bmall on the TMJ in the sleep rhythm disordered state was further validated in vivo by again administering an adenovirus containing the Bmall overexpressing plasmid into the joint lumen of rats in the sleep rhythm disordered state,using Western blot and RT-qPCR to observe changes in the expression of the clock rhythm gene Bmall,MAPK/ERK signaling pathway and downstream matrix metalloproteinases and extracellular matrix.Results:We used MMPM to disrupt normal sleep rhythms in rats after successfully causing a stress state in rats with sleep rhythm disorders.The serological results showed that with the prolongation of sleep disturbance,the serum content of CORT and ACTH was significantly increased(p<0.05)in the rats in the circadian rhythm disturbance(CRD)and recovery(REC)groups compared with the subgroups in the control group(control,CON).And the damage worsens as the duration of the sleep disturbance increases.Immunohistochemistry,Western blot and RT-qPCR results showed that both IL-6 and p-ERK expression were upregulated in the CRD and REC groups compared to the CON group.And it increases with the duration of sleep rhythm disorders.IL-6 and p-ERK expression was reduced when the cessation of sleep rhythm disruption was adjusted to normal circadian rhythms.The clock gene Bmall,on the other hand,was reduced in both the CRD and REC groups compared to the CON group after sleep rhythm disruption.Bmall expression was up-regulated when cessation of sleep rhythm disruption was adjusted to normal circadian rhythms.The inflammatory state of condylar chondrocytes was successfully isolated and cultured in vitro in rats and induced with IL-6.After inhibiting the phosphorylation of ERK using U0126,Western results showed downstream expression of MMP3,MMP13,and ADAMTS5,upregulation of Col2al expression,and no statistical significance(p>0.05)for the difference in expression of the clock-rhythm gene Bmall.Alcian blue staining showed increased chondroitin sulfate secretion with U0126 in the inflammatory state.CCK-8 results showed decreased cell proliferation rate in the inflammatory state and increased proliferation rate with U0126.Western results using Bmall-siRNA showed up-regulation of p-ERK,MMP3,MMP13,ADAMTS5 expression and down-regulation of Col2al expression,whereas the opposite results were obtained using Bmall-plasmid.The CCK-8 results showed a further decrease in cell proliferation rate after the addition of Bmall-siRNA and an increase in cell proliferation rate after the addition of Bmall-plasmid in the inflammatory state.Adenoviruses containing Bmal 1 overexpressing plasmids were administered intra-articularly to rats in a sleep rhythm disordered state,and Western blot and RT-qPCR results showed down-regulation of p-ERK,MMP3,MMP13,and ADAMTS5 expression and up-regulation of COL2?1 expression in the injected group compared to the control group.Conclusions:When the sleep rhythm is disrupted,the clock rhythm gene Bmall is dysregulated,resulting in activation of the MAPK/ERK signaling pathway,progressively higher phosphorylation levels of ERK,and elevated expression levels of the inflammatory factor IL-6,which is closely associated with the development of joint inflammation,and the enzymes MMP3,MMP13,and ADAMTS5,which are associated with the degradation of cartilage matrix.And down-regulation of COL2?1 associated with the integrity of extracellular mesenchymal structures,followed by inflammatory pathological changes in condylar protrusions.This study explains the important role of the clock rhythm gene Bmall in the pathogenesis of osteoarthritis in the sleep rhythm disordered state by exploring the mechanism of action of the sleep rhythm disordered state in regulating OA cartilage degeneration.To lay the foundation for future research on the pathophysiology of OA related to the neuraxial axis of the brain joint.