Effects Of Progressive Reduction In Crown Height Of Unilateral Posterior Teeth On The Structure Of Rat Occlusal Muscles And Related Mechanisms

Objective: To investigate the effects of progressive occlusal disorder on the occlusal muscle by establishing a rat model of progressive unilateral posterior tooth crown height reduction,observing the microstructure of rat occlusal muscle,and applying RNA Sequencing(RNA-Seq)to analyze the transcriptional profile of rat occlusal muscle,and screening the key genes and pathways associated with occlusal muscle damage.Materials and methods: 1.Modeling: A total of forty-eight 8-week-old male SD rats,randomly grouped into 4 groups(12 rats per group): 4-week control and experimental groups(4w-CON and 4w-EXP),8-week control and experimental groups(8w-CON and 8w-EXP).All experimental groups were adjusted and grinding the clinical crowns of the left upper posterior teeth in 3 times in the 1st,2nd and 3rd weeks of the experiment,and the distance of each adjustment was about 0.3～0.4mm until the gingival level,and the crowns were lowered about 0.9～1.2mm in total.All controls were kept in the same environment without tooth adjustment.4w-CON and 4w-EXP were taken at week 4,8w-CON and 8w-EXP at week 8.The rats were collected at week 4 and8w-CON and 8w-EXP at week 8.After sampling,the rats were subjected to HE staining and transmission electron microscopy.2.The expression changes of transcript levels in rat bite muscle tissue were analyzed by RNA-Seq technology to screen the signaling factors and differentially expressed genes related to rat bite muscle injury,and the main enriched pathways and biological functions of key genes were analyzed by bioinformatics,and finally,real-time fluorescence quantitative reverse transcription polymerase chain reaction(qRT-PCR)was performed to verify the key genes.PCR)to verify the key genes.In this study,a two-way ANOVA was performed on the data of each group by Graph Pad Prism 9.0 for the study and was considered statistically significant when P < 0.05.Results: 1.HE staining showed that progressive unilateral reduction in crown height of posterior teeth caused changes in the microstructure of the rat occlusal muscle.4w-EXP staining showed disorganized muscle fiber arrangement,uneven thickness,and disorganized transverse muscle striations with focal inflammatory cell infiltration.4w-CON,8w-CON,and 8w-EXP all showed neatly arranged occlusal muscle fibers,uniform thickness,and uniform connective tissue between muscle fibers,without inflammatory cell infiltration.No inflammatory cell infiltration2.The transmission electron microscopy revealed that the ultra-microstructure of the rat occlusal muscle was altered.4w-EXP showed swollen skeletal muscle fibers,more disordered arrangement of myogenic fibers,more disordered arrangement of Z-lines,significant swelling of intracytoplasmic organelles,larger mitochondria,disappearance of cristae,and irregular changes in shape.8w-EXP showed slight swelling of skeletal muscle fibers,neater arrangement of myogenic fibers than 4w-EXP,neater arrangement of Z-lines,and less disordered arrangement of intracytoplasmic organelles.In the4w-CON and 8w-CON groups,the skeletal muscle fibers were neatly arranged without mitochondrial swelling.3.RNA-Seq results showed that there were 77 significantly different genes between4w-EXP and 4w-CON,including 42 up-regulated genes and 35 down-regulated genes;54different genes between 8w-EXP and 8w-CON,including 7 up-regulated genes and 47down-regulated genes;225 different genes between 4w-EXP and 8w-EXP,including 111up-regulated genes and 114 down-regulated genes.GO analysis revealed a significant enrichment of gene functions mainly in response to external stimuli,regulation of biological processes,immune response,metabolic processes,and so on.k EGG analysis revealed an enrichment of signaling pathways mainly including inflammation-related pathways,such as JAK-STAT,AMPK,Fox O,MAPK,PI3K-Akt signaling pathways,and others.4.The qRT-PCR results showed that 17 genes,Cdr2,Alas2,Ucp2,Dbp,Socs3,Cish,Slc4a1,Srebf1,Slc25a37,Fgfr2,Fhl1,Hba-a2,Fbp2,Igf1,Bhlhe40,Bhlhe41,Trib1,were significantly differentially expressed in 4w-EXP and(P < 0.05);14 genes,Cdr2,Alas2,Ucp2,Slc4a1,Srebf1,Hba-a2,Il6 r,Igf1,Slc25a37,Slc4a1,Socs3,Srebf1,Trib1 and Ucp2,showed significant differences in 8w-EXP and 8w-CON There were significant differences in expression(P < 0.05).Besides,the expression of 12 genes,Cdr2,Alas2,Ucp2,Dbp,Socs3,Cish,Slc4a1,Srebf1,Slc25a37,Fgfr2,Fhl1,and Hba-a2,were all significantly lower in 8w-EXP than in 4w-EXP(P < 0.05),while Bhlhe40,Trib1,Il6 r,and Foxo1,the four genes with significantly higher expression in 8w-EXP than in4w-EXP(P < 0.05).Conclusion: Progressive unilateral reduction of crown height in posterior teeth caused occlusal muscle injury in rats,with inflammatory injury in the early stage of the lesion(4w-EXP)and some adaptive modification in the late stage of the lesion(8w-EXP).The RNA-Seq sequencing and qRT-PCR showed that several genes and signaling pathways were associated with occlusal muscle injury and adaptive remodeling.