Function And Preliminary Molecular Mechanism Of Endoplasmic Reticulum Stress In Temporomandibular Joint Osteoarthritis

Purpose: This study aims to establish the temporomandibular joint osteoarthritis(TMJOA)rat model induced by unilateral anterior crossbite(UAC).On this basis,the differential gene expression profile of TMJOA was screened by high-throughput whole transcriptome sequencing,and then the role and preliminary mechanism of endoplasmic reticulum stress(ERS)in TMJOA were further clarified.Methods: 1.Rat TMJOA animal model was induced by UAC,and the pathological changes of condylar cartilage in UAC group and control group at 2 weeks,4 weeks and 8 weeks were detected by type microscope observation and histopathological staining to determine the successful establishment of TMJOA model.2.Whole transcriptome sequencing was performed on the condylar cartilage of rats in the 8-week UAC group and the control group to describe and analyze the gene expression profiles of these groups.Further GO and KEGG enrichment were used to analyze the function and related pathways of differentially expressed genes,and the competitive endogenous RNA(ce RNA)network was constructed by Cytoscape software.Immunohistochemical,q RT-PCR and Western blot were used to verify the expression of ERS marker molecules(GRP78,ATF4),markers of ERS induced apoptosis(CHOP,CASP12)and key molecules in the classic PI3K/AKT signaling pathway in the UAC group and the control group.4.Condylar chondrocytes were cultured in vitro and divided into control group,LPS group,LPS+LY294002(inhibitor)group and LPS+740Y-P(activator)group.In LPS+740Y-P group,the expressions of P-PI3 K,P-AKT,GRP78 and CHOP were increased,while the expressions of ATF4 and CASP12 were decreased.Results: 1.TMJOA model was successfully established.Postural microscope observation showed that that compared with the control group,in UAC group the surface of cartilage was slightly redness at 2 weeks,shallow depression appeared in the middle of cartilage at 4 weeks,and the condylar surface was uneven and irregular at 8 weeks.Histopathological observations showed that the chondrocyte arrangement was disordered at 2 weeks,the boundaries of chondrocytes in each layer were not clear at 4 weeks,and the number of cartilage cells in the mast layer decreased;at 8 weeks,the condylar cartilage fiber layer showed uneven changes,and the hyperplasia layer,pre-hypertropia layer and hypertrophic layer chondrocytes were significantly reduced,and local acellular areas appeared.2.Whole transcriptome sequencing of TMJOA.A total of 137 m RNAs,65 mi RNAs,132 lnc RNAs,and 29 circ RNAs were differentially expressed between the control group and the UAC group.GO and KEGG enrichment analyses suggested that the negative regulation of endoplasmic reticulum stressinduced intrinsic apoptotic signaling pathway,immune response,apoptosis,PI3K/AKT signaling pathways and PPAR signaling pathways were the main pathways involved in TMJOA.3.Verification of endoplasmic reticulum stress inducing cartilage apoptosis.The results of immunohistochemistry,q RT-PCR,and Western blot showed that the expression of GRP78,ATF4,CHOP,and CASP12 in the UAC group was higher than that of the control group at 2 weeks,4 weeks,and 8 weeks,and the expression of GRP78,ATF4,CHOP,and CASP12 in the UAC group gradually increased with the increase of modeling time.The expression of p-PI3 K and p-AKT in the UAC group was higher than that in the control group in the same period.4.A preliminary exploration of the mechanism of endoplasmic reticulum inducing apoptosis of chondroitocytes.Expression of GRP78,ATF4,CHOP and CASP12 in the LPS group was higher than that in the control group.Compared with the LPS group,the protein expression levels of p-PI3 K,p-AKT,GRP78,ATF4,CHOP and CASP12 in the LPS +LY294002 group were significantly reduced.The expression levels of p-PI3 K,p-AKT,GRP78,and CHOP and ATF4,CASP12 in the activator group did not change significantly.Conclusion: 1.This study successfully established the TMJOA model induced by UAC,and identified the differential gene expression profile of TMJOA by whole transcriptome sequencing for the first time,which laid a foundation for screening diagnostic markers and potential therapeutic targets of TMJOA.2.Through bioinformatics analysis and molecular biology experiment,it is confirmed that ERS induced apoptosis plays an important role in UAC induced TMJOA model,and it is preliminarily found that PI3 K / AKT signal pathway may play an important regulatory role in ERS induced apoptosis.