Interleukin-6 Induces Extracellular Matrix Degradation And Angiogenesis In Osteoarthritis Vitro Models Of Temporomandibular Joint Via Estrogen-related Receptor ?

Objectives:Matrix metalloproteinase-9(MMP9)and vascular endothelial growth factor-a(VEGFA)produced by chondrocytes is an essential effector of cartilage destruction during the pathogenesis of temporomandibular joint osteoarthritis(TMJOA).Cartilage vascularization of TMJOA is also inseparable from the up-regulation of VEGFA.Inflammation and vascularization in osteoarthritis(OA)often have a mutually reinforcing effect.Interleukin-6(IL6)is one of the most critical inflammatory factors in the progression of TMJOA.The objective of this study is to verify the related mechanism of estrogen-related receptor ?(ERR?),a key transcription factor downstream of IL6-ERK1/2 signaling pathway,in the process of extracellular matrix degradation and vascularization of rat condylar cartilage.The effect of IL6-MAPK/ERK-ERRy-MMP9/VEGFA axis on the development of rat TMJOA was discussed in detail.Methods:Three-week-old wistar rats were dissected after injection,and the condylar cartilage was removed from the bilateral temporomandibular joint.The condylar cartilage was cut into 1 mm3 tissue blocks,and the primary condylar chondrocytes were obtained by type ? collagenase digestion.The cartilage culture results were identified by observing the morphology of the cells and the Alcian blue staining method.The first generation of condylar chondrocytes(P1 generation)were randomly divided into two large groups:a concentration gradient group and a time gradient group.The concentration gradient components were 5 groups:treated with IL6 at a concentration of 0 ng/ml,5 ng/ml,10 ng/ml,20 ng/ml,40 ng/ml for 12 hours.Time gradient fractions were stimulated in groups of 5 ng/ml IL6 for 0.5 h,1 h,3 h,6 h,12 h,24 h and 48 h.mRNA expression changes of ERR?,ERR?,HIF1?,MMP9/13,VEGFA,COL2 and AGG were detected by real-time quantitative polymerase chain.reaction(RT-qPCR).Determine the optimal stimulation concentration and stimulation time for the modeled IL6 used.The chondrocyte-associated phenotype protein after IL6 stimulation was identified by immunofluorescence staining to determine whether the modeling was successful or not.The ERK-specific inhibitor U0126 was added to the chondrocytes of the TMJOA model in vitro,and the expression of downstream related transcription factors ERRy,ERRa,HIF-la and downstream related phenotype proteins MMP9 and VEGFA were detected by Western blotting.P1 cells were transfected with ERR?-cDNA plasmid and ERRy-siRNA,and the expression of downstream MMP9,VEGFA,COL2 and AGG were observed by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting.The ERR?-specific inhibitors GSK5182 and U0126 were added to the chondrocytes of the TMJOA model in vitro,and the expression changes of MMP9,VEGFA and COL2 were detected by double-label immunofluorescence staining,alixin blue staining and Western blotting.P1 chondrocytes were divided into two groups:control group(0 ng/ml,IL6)and OA group(20 ng/ml,IL6).Formaldehyde was fixed after stimulation for 12 hours,and cells were collected after glycine neutralization.It was confirmed by chromatin immunoprecipitation(ChIP)whether ERRy directly regulated the expression of MMP9 and VEGFA.The angiogenesis inhibition experiments were divided into four groups:DMSO group,IL6+DMSO group,IL6+U0126 group,IL6+GSK5182 group(inhibitor diluted with DMSO).The effects of four groups of reagents on angiogenesis were verified by chick embryo chorioallantoic membrane(CAM)experiments.Results:(1)After the rat temporomandibular joint condylar chondrocytes were successfully extracted and cultured,the normal in vitro chondrocytes showed a regular small round shape.After 1 day of incubation,most of the chondrocytes adhered successfully.The morphology of the cells was polygonal or star-shaped after microscopic observation.After 4-5 days of chondrocyte culture,the cells were observed to be arranged in a "paving stone".After one passage,the growth rate of adherent cells was accelerated;the cytoplasm of the condylar chondrocytes was light blue by Alcian blue staining,and the dedifferentiated state of chondrocytes cultured from the third generation(P3)began to dedifferentiate.The cells are quadrilateral and the cytoplasm is deeply stained,extending in a fibrous shape,and the proliferative ability is decreased.RT-qPCR results showed that ERR?,ERR?,HIF1?,MMP9/13,VEGFA in condylar chondrocytes with changes in IL6 concentration and stimulation time.The expressions of COL2 and AGG were changed to different extents.When the concentration of IL6 was 20 ng/ml,the expression of COL2 and AGG in condylar chondrocytes decreased to the maximum,while MMP9/13 The expression of VEGFA was up-regulated to the maximum.The expression of ERRty in condylar chondrocytes induced by IL6(20 ng/mL)was significantly up-regulated;while the expression of ERRa and HIF-la was not statistically significant.By simultaneously labeling MMP9 and COL2 immunofluorescence results,IL6(20 ng/mL)stimulation for 12h can construct TMJOA in vitro model.(2)After U0126 inhibited the phosphorylation of ERK,Western results showed that the expression of ERRy and MMP9 and VEGFA were down-regulated,and there was no statistical difference in the expression of ERRa and HIF1?.Western blotting of ERR?-cDNA plasmid transfected P1 chondrocytes showed that MMP9 and VEGFA expression were up-regulated and COL2 and AGG expression were decreased.ERRysiRNA significantly inhibited the inflammation of TMJOA in vitro chondrocytes,and Western blot showed that MMP9 and VEGFA expression were down-regulated.,COL2,AGG expression was slightly restored.After GSK5182 and U0126 inhibitors were added to TMJOA in vitro model chondrocytes,immunofluorescence results and Western blot showed that MMP3/9,VEGFA expression was significantly inhibited,COL2 and AGG expression were up-regulated;The cytoplasm of most cells in the OA group was dark blue,while the cytoplasm of most chondrocytes in the control and inhibitor groups was pale blue.Our nucleotide sequence analysis identified a number of putative ERR response elements(ERRE,TCAAGGTCA)40 in the promoter regions of Mmp9 and Vegfa.ChIP assays revealed that ERRy was bound to ERRE#2(-65 to-48)in the Mmp9 promoter,and to ERRE#2(-107 to-92)in the Vegfa promoter.These findings suggest that MMP9 and VEGFA are direct target genes of ERRy in mouse articular chondrocytes.Conclusions:As an orphan nuclear receptor,ERRy is an important transcription factor for the development of inflammation.In this study,the pathway of activation of downstream P-ERK and up-regulation of MMP9 and VEGFA by IL6 binding receptor gp 130 was improved from epigenetic level,and ERRy is the "missing loop" on this pathway.These findings suggest that MMP9 and VEGFA are direct target genes of ERRy in mouse articular chondrocytes.The specific inhibitors GSK5182 and U0126 have a certain therapeutic effect on TMJOA and can inhibit angiogenesis.Inhibition of angiogenesis is currently a major direction in osteoarthritis research and tumor research.The role of GSK5182 as a new drug in tumors needs further investigation.