| Objective:Temporomandibular Disorders(TMD)is a common oral and maxillofacial disease.In this disease state,there is a relatively severe type,called TMJ osteoarthritis(TMJ-OA).The etiology of TMJ-OA is complex,so far,its pathogenesis is still unknown,and it is in a state of contention and blooming of a hundred flowers.Some scholars believe that the abnormal mechanical changes in the joint area may be one of the pathogenic factors of TMJ-OA.Abnormal occlusal or poor oral behavior habits cause long-term unbalanced stress in the TMJ region,which leads to changes in the expression of forced-sensitive factors.Piezo1 is a piezoelectric ion channel protein that dramatically increases the mechanical sensitivity of subchondral bone cells when stimulated by mechanical forces,resulting in increased bone wear.However,whether it plays a role in the whole inflammatory pathway of TMJ-OA still needs to be further studied.Il-1β is an important member of the IL-1 family and has received much attention due to its important role in inflammation-related diseases.IL-1β has a variety of functions,such as its strong pro-inflammatory activity,which can induce a variety of pro-inflammatory mediators,such as cytokines and chemokines.Studies have shown that it may promote the process of TMJ-OA to a certain extent,making related inflammation further aggravated.The purpose of this study is to analyze the activation of Piezo1 by IL-1β inflammatory pathway,so as to explore the changes of Piezo1 in the course of TMJ-OA.Firstly,the specific action sites of Piezo1 were analyzed by proteomics,and the related pathways mediated by PIEZO1 activated by IL-1β were investigated in vitro.Meanwhile,the TMJOA model of Sprague Dawley(SD)rats mediated by Complete Freund’s Adjuvant(CFA)was successfully constructed.The patients were treated with Piezo1 inhibitor Gs MTx4 and Platelet Rich Plasm(PRP).Finally,the pro-inflammatory effect of Piezo1 on TMJ-OA was clarified,which provides a new way for the subsequent treatment of TMJ-OA.Methods:1.Proteomic study of osteoblast inflammation model in vitro.MC3T3-E1 cells were coincubated with IL-1β.CCK8 was used to study the activity of MC3T3-E1 cells and the expression of IL-6 was detected by ELISA.After the successful establishment of the cell model in vitro,the related proteomics analysis was performed.2.To investigate the relationship between IL-1β inflammatory environment and Piezo1 pathway in vitro.In this part,firstly,Piezo1 expression in MC3T3-E1 cells induced by IL-1β was detected by WB and RT-PCR.Then the expression of IL-6 in the supernatants of NC,DMSO,IL-1β,IL-1β+Yoda1,IL-1β+si-NC,IL-1β+ si-Piezo1 were detected by ELISA,m C3T3-E1 cells were used to investigate the mechanism of Piezo1 activation in IL-1β pathway by WB method.3.In vivo experiments to explore Piezo1 and the correlation of the pathway.In this chapter,the TMJ-OA model of SD rats induced by CFA was established,and then Piezo1 was inhibited by Gs MTx4 injection,then micro-CT,HE staining,fuchsin green staining,immunohistochemistry(Piezo1 and pSmad3),TRAP staining and WB staining were used to detect the synergistic changes between pSmad3 and Piezo1 in CFA-induced TMJ-OA model.The expression of Piezo1,Smad3 and pSmad3 was detected by immunohistochemistry and WB.4.Evaluation of therapeutic effect of PRP on CFAinduced TMJ-OA in SD rats and effect of Piezo1.The animal model of TMJ-OA was evaluated and its Piezo1 effect was investigated by PRP-mediated CFA-induced TMJ-OA treatment.Further,by Piezo1 immunohistochemistry and WB experiments.The quantitative data were Normality test.The normal distribution of quantitative data was described by mean±SD,quantitative data that did not conform to the normal distribution were described by median(interquartile range)m(P25,p75)and categorical data were described by case number and percentage n(%).According to the normality and homogeneity of variance of the data,for the quantitative data conforming to the normal distribution,the comparison between the two treatment groups was performed by independent sample t test.Comparisons among three and more treatment groups were tested with One-way ANOVA.When differences were statistically significant,LSD-t test was further used for post hoc pairwise comparisons.Nonparametric test is used for quantitative data that do not conform to normal distribution.The categorical data were compared between groups by chi-square test.Statistical analysis of the data was performed by Graph Pad Prism 5 software,and the difference was statistically significant at P<0.05.Results:1.Proteomic study of osteoblast inflammation model in vitro: after IL-1β stimulation,the activity of MC3T3-E1 cells was not affected,but the content of IL-6 in cell supernatant was significantly increased.Piezo1 plays a positive role in IL-1β-mediated inflammation of subchondral osteocytes,and the expression of Piezo1 is obvious along with the process of inflammation.2.In vitro study on the relationship between the Piezo1 pathway and the inflammatory environment of IL-1β: the expression of Piezo1 in IL-1β group was significantly increased by WB and ELISA.Afterward,Yoda1,an agonist of Piezo1,was added to IL-1β-stimulated MC3T3-E1 cells,and the expression of Piezo1 was significantly increased in IL-1β+Yoda1-treated cells compared with the IL-1β group.In contrast,the expression of Piezo1 stimulated by IL-1β can be inhibited by the si RNA added Piezo1.The expression of IL-6 in the supernatant of IL-1β+Yoda1 group was the highest,and IL-1β+siPiezo1 was down-regulated compared with IL-1β+si-NC group.These results suggest that the expression level of IL-6 increases and decreases with the increase of Piezo1 expression,which proves that Piezo1 plays a key role in IL-1β-mediated subchondral osteocyte inflammation.At the same time,the results of cell migration showed that IL-1β + Yoda1 group had the highest cell migration rate,which indicated that IL-1β+Yoda1 group could effectively promote the development of cell inflammation,and had a significantly enhanced effect than IL-1β group.In order to further explore its mechanism of action,the phosphorylation of pSmad3 in IL-1β+Yoda1 group did not increase significantly,but the phosphorylation of pSmad3 increased significantly.These results suggest that IL-1β can activate Piezo1 expression in MC3T3-E1 osteoblasts and further regulate the inflammatory environment by affecting the phosphorylation of Smad3.This provides a new idea for the treatment of osteoarthritis in the future.3.In vivo study of Piezo1 and related pathways:Gs MTx4 can reduce the inflammatory damage induced by CFA.After injection of Gs MTx4,bone volume fraction(BV/TV)was significantly higher and trabecular separation(Tb.Sp)was significantly lower than that in CFA Group.The number of subchondral osteoclasts increased in the CFA Group,and the number of osteoclasts in the CFA+ Gs MTx4 group was lower than that in the CFA Group.In addition,the expression of Piezo1 and pSmad3 increased after CFA treatment compared with the control group.However,after Gs MTx4 treatment,expression of Piezo1 and pSmad3 decreased compared with CFA injection group.WB results also demonstrated that Piezo1 can modulate the osteoarthritis environment by altering pSmad3 levels.4.Therapeutic evaluation and Piezo1 effect of PRP on CFA-induced TMJ-OA in SD rats: with the application of PRP,TMJ-OA was significantly relieved,both micro-CT and HE staining showed good bone damage and low inflammatory reaction.The therapeutic effect of PRP on TMJ-OA was fully explained.Furthermore,the results of Piezo1 immunohistochemistry and WB experiments show that PRP effectively inhibits Piezo1 pathway,which is similar to that of Gs MTx4.Thus,PRP may act through its anti-inflammatory component,the effective suppression of Piezo1 pathway was realized,and the therapy of TMJ-OA was realized.Conclusion:1.Through proteomics and cell experiment,it is confirmed that IL-1β can activate Piezo1 expression effectively in the inflammatory model in vitro.After further up-or downregulation of Piezo1,the degree of inflammation also changed.After further study of the signaling pathway,Piezo1 was found to modulate the inflammatory environment by affecting the expression level of pSmad3.2.The high expression of Piezo1 in the condylar cartilage and subchondral bone of TMJOA rat model induced by CFA was demonstrated,TMJ-OA may affect the disease progression by affecting the expression of pSmad3.The application of Piezo1 inhibitor Gs Mtx4 has produced an effective treatment for TMJ-OA,which reveals that the suppression of Piezo1 pathway may be a new method for the treatment of TMJ-OA.3.It was found that PRP had a good therapeutic effect on TMJ-OA model rats,and it inhibited Piezo1 pathway.It is revealed that PRP may inhibit Piezo1 pathway effectively through its anti-inflammatory component,thus realizing the treatment of TMJ-OA. |
PDF Full Text Request