Role Of Hippo Pathway In Degradation Of Mouse Cartilage Extracellular Matrix Induced By Fluoride

Objective Endemic fluorosis can cause articular cartilage degenerative changes,and the degradation of extracellular matrix（ECM）plays an important role in articular cartilage degeneration.According to previous reports,Hippo pathway could sense and regulate ECM homeostasis,but its role in the pathogenesis of articular cartilage degeneration induced by fluorosis is still unclear.Therefore,this study screened out the signal pathway significantly associated with fluoride induced bone injury through high-throughput data and established the mouse chondroblast（ATDC5）exposure model to explore the role of Hippo pathway in chondrocyte ECM degradation induced by fluoride,and provide a theoretical basis for the pathogenesis and prevention of skeletal fluorosis.Methods 1.Environmental response gene screening and pathway enrichment analysis of fluoride-induced bone injury The environmental response genes of interaction between fluoride and bone injury were screened by CTD database,and then these genes were annotated by DAVID and STRING for Gene Ontology（GO）analysis,KEGG pathway enrichment analysis and protein-protein interaction network analysis.The hub gene（top 20）was screened by Cytoscape software.2.Construction of in vitro model and selection of fluoride exposure concentration ATDC5 cells（mouse chondroblast cell line）were used to construct an in vitro model,and the chondrocytes were identified by morphological observation and the expression of type II collagen（COL2A1）.The cells were cultured with different concentrations of NaF（0,5,10,15,20,25,30,35,40 mg/L）for 48 hours,and CCK-8 kit was used to detect the proliferation activity of ATDC5 cells.The half maximum inhibitory concentration(IC₅₀)and exposure concentration of NaF were determined according to the cell activity.3.The effect of fluoride exposure on ATDC5 cells Control group（0 mg/L NaF）and exposure group（10,15,20 mg/L NaF）were set up.After 48 hours of exposure,the cell cycle detection kit was used to detect cell cycle,then Flow Jo software was used to analyze the cell cycle.Malondialdehyde（MDA）content,total antioxidant capacity（T-AOC）,total superoxide dismutase（T-SOD）activity and glutathione peroxidase（GSH-PX）activity in ATDC5 cells were detected by oxidative stress detection kit.4.The effect of fluoride exposure on ATDC5 cell matrix and Hippo pathway related gene expression qRT-PCR was used to detect mRNA levels of cartilage matrix（COL2A1,ACAN and MMP13）and Hippo pathways（MST1,LATS1 and YAP1）related genes.Western blot experiment was used to detect the protein expression of COL2A1,ACAN,MMP13,p-MST1/2,p-LATS1/2,p-YAP and YAP1.The localization and expression of COL2A1 and YAP1 protein in cells were detected by cell immunofluorescence experiment.5.Statistical analysis Using EXCEL software to organize the data and SPSS 22.0 software for analysis.The differences in the mean of multiple groups of continuous data were compared by one-way analysis of variance（one-way ANOVA）,and then the differences between groups were tested by Dunnet-t method,with the test level α=0.05.Using Graph Pad Prism 6.0 software to visualize the data.Results 1.Environmental response gene screening and pathway enrichment analysis of fluoride-induced bone injury The CTD database was used to screen 145 environmental response genes,including 83 up-regulated genes and 58 down regulated genes（4 genes with unclear expression）.KEGG enrichment analysis yielded 160 pathways,and 24 pathways significantly related to bone injury were screened,among which Hippo pathway was significant.Six proteins were found involved in the enrichment of Hippo pathway in the protein interaction network.2.The effect of fluoride exposure on the proliferation activity and oxidative stress level of ATDC5 cells CCK-8 assay showed that 5 mg/L and 10 mg/L fluoride exposure stimulated cell proliferation activity,and the viability of ATDC5 cells was significantly decreased when exposed to ≥15mg/L fluoride concentration（P<0.05）.The IC₅₀ of NaF was about 20 mg/L.After exposure to different levels of fluoride for 48 h,the cell morphology changed.Compared with the control group,the proportion of cells in G0/G1 phase was significantly decreased and the proportion of cells in G2/M phase was significantly increased in 15mg/L and 20mg/L NaF exposure groups（P<0.05）.At the same time,MDA content in 20mg/L NaF exposure group was significantly higher than that in the control group,T-SOD activity in 15mg/L and 20mg/L NaF exposure groups was significantly lower than that in the control group,and T-AOC and GSH-PX activities in each exposure group were also significantly lower than that in the control group（P<0.05）.3.Hippo pathway involved in the chondrocyte ECM degradation induced by fluoride qRT-PCR results showed that with the increase of fluoride concentration,the expression of COL2A1 mRNA increased at first and then decreased,ACAN mRNA levels in 15mg/L and 20mg/L NaF exposure groups were significantly higher than that in the control group（P<0.05）,and MMP13 mRNA level was up-regulated.The levels of MST1,LATS1 and YAP1 mRNA were increased with the increase of fluoride concentration,among which,the YAP1 mRNA levels in the 15 mg/L and 20 mg/L NaF exposure groups increased more significantly than that in control group（P<0.05）.Western blot results showed that compared with the control group,the protein levels of COL2A1 and ACAN in 20mg/L NaF exposure group were significantly decreased,while the protein levels of MMP13 in 15mg/L and 20mg/L NaF exposure groups were significantly increased（P<0.05）;The protein levels of p-MST1/2,pLATS1/2 and p-YAP1 in 15mg/L and 20mg/L NaF exposure groups were significantly lower than those in the control group,while the protein level of YAP1 in 20mg/L NaF exposure group was significantly higher than that in the control group（P<0.05）.Through cell immunofluorescence experiments,it was observed that the expression of COL2A1 and YAP1 protein in the cytoplasm decreased with the increase of fluoride exposure,and the expression of YAP1 protein in the nucleus increased.Conclusions Fluoride exposure inhibited the proliferation of ATDC5 cells,induced oxidative stress and degradation of ECM.In this process,the activity of Hippo pathway is inhibited,and YAP1 protein may be activated as a protective factor.