Mechanisms Of M6A Methylation In Chondrocyte Apoptosis Induced By Functional Orthopedic Force Overload

Objective:Adolescent class Ⅱ malocclusion is a common disease in orthodontic clinic.For mild and moderate adolescent bony protrusion patients,functional orthodontic appliance is used to treat them,so as to reposition the position of the mandible and rebuild the normal occlusal relationship.Clinical data found that patients wearing functional orthodontic devices to lead the mandibular forward,which is conducive to the adaptive reconstruction of condyle,and if the functional orthodontic force is too large,the mandibular overextension,TMJ will appear uncomfortable symptoms,and even TMJ osteoarthritis in severe cases.Studies by some scholars have shown that ER stress is one of the primary responses of cells to external stimuli.Excessive or prolonged stimulation can activate downstream apoptotic signaling molecules,in which the PERK/CHOP signaling pathway plays a key role.M6 A methylation is one of the most abundant modifications in eukaryotic RNA epigenetics and is regulated by M6 A methyltransferase and demethylase.Compared with the regulation of the upstream and downstream gene information of the classic "central rule",whether there is M6 a epigenetic modification involved in the regulation of mandibular condylar orthopaedic force overload has not been reported.This topic from the TMJ condylar cartilage,aims to study PERK/CHOP signaling pathways in the expression of functional orthopedic force overload of chondrocytes,and through after the transcription level to explore the function of m6 A methylation in orthopedic force overload mechanism research of the process,for clinical rational use of orthopedic force,effective prevention and treatment of TMJ OA provide new theoretical basis,open up new research ideas.Methods:1.In this study,we built an animal model of mandibular hyperextension in rats to simulate the external environment of functional orthopedic force overload.At three time points 2 weeks,4 weeks and 8 weeks after the model was constructed,the condylar samples were collected,and hematoxylin/eosin(HE)staining,TRAP staining and saffine O/ solid green(SO)staining were used to evaluate the reconstruction of temporomandibular joint and the morphological and histological changes of condyle cartilage and subchondral bone in the experimental group and the control group.2.The mechanical overload microenvironment was constructed to simulate the cellular environment of functional orthopedic stress overload,and the effects of stress loading on M6 a methylation level,endoplasmic reticulum stress and apoptosis of chondrocytes were studied.First of all,overloading mechanical force was applied to cartilage precursor cells.CCK8 was used to detect the proliferation activity of cartilage precursor cells after 0,3,12 and 24 hours of stress,and WB and PCR were used to detect the changes of GRP78,PERK,CHOP and other related factors.Apoptosis was detected by FITC/PI double staining and Hoechst33342 flow cytometry.Similarly,WB and PCR were used to detect the changes of M6 A methylation related factors Mett L3,FTO,ALKBH5 and Mett L14 of chondrocytes in the 24 h stress group and the blank group under stress conditions.The M6 A methylation level of total RNA in each group was detected by the M6 A RNA quantitative detection kit(fluorescence method).Results:1.Condylar herniation showed degenerative changes and bone resorption at 2,4,and 8weeks after construction.He staining indicated disordered arrangement of bone trabeculae,decreased density of chondrocytes and unclear boundaries.The saffron O/solid green staining indicated that the chondrocytes were damaged and the release of proteoglycan was lost in the after stress group,which gradually increased with time.The number of osteoclasts in subchondral bone and bone marrow cavity was significantly increased by TRAP staining,suggesting enhanced osteoclast activity.2.When mouse chondrocytes were subjected to excessive mechanical tension,the proliferative activity of the chondrocytes decreased gradually with time from 0 to 24 years.3.In the in vitro experiment,fluorescence quantitative PCR and WB showed that compared with the control group,the expression levels of GRP78,PERK and CHOP in chondrocytes in the stress group were significantly increased,and the expression levels were gradually increased over time,suggesting endoplasmic reticulum stress and apoptosis of chondrocytes.4.FITC/PI double staining and Hoechst staining results showed that compared with the control group,chondrocytes in the experimental group appeared apoptosis after loading too much mechanical tension.With the increase of stress loading time,cell apoptosis gradually increased.5.The results of quantitative determination of m6 A RNA showed that the methylation level of m6 A decreased in total RNA of chondrocytes after augmentation.6.In the in vitro experiment,fluorescence quantitative PCR and WB showed that compared with the control group,the expressions of methyltransferase met TL3 and met TL14 in the chondrocytes of the augmentation group were significantly decreased,while the expressions of dimethy transferase FTO and alk BH5 did not change significantly.Conclusions:In the process of TMJ OA caused by functional orthopedic force overload,condylar prolapsed degeneration,bone destruction and chondrocyte damage occurred in rats.The activation of the PERK/CHOP signaling pathway mediated by ER stress plays an important role in the apoptosis of chondrocytes.METTL3/14 mediated M6 A methylation significantly changes in this process.Given that the pathogenesis and pathogenesis of TMJ OA are not yet clear,as the most common post-transcriptional epigenetic modification,it is of great significance to explore the mechanism of action of functional orthopedic force overload in the condyle from the perspective of m6 A methylation.It also provides a new theoretical method for the effective prevention and treatment of TMJ OA.