The Effect Of Piezo1 On Cartilage Degradation In Malocclusion Induced Temporomandibular Osteoarthritis

Objective:As a severe subtype of temporomandibular disorders,temporomandibular joint osteoarthritis(TMJOA)is fairly common among the general population.TMJOA is characterized by the loss and destruction of articular cartilage matrix,synovitis,subchondral bone remodeling and osteophyte formation.Condylar cartilage is highly sensitive to mechanical loading,and malocclusion is considered to be a potential pathological factor leading to TMJOA.Therefore,exploring the underneath mechanism of chondrocyte mechanotransduction may have therapeutic prospects for controlling cartilage degeneration in the early stages of TMJOA.To date,a novel mechanically activated cation channel,Piezo1,has been identified to mechanotransduct mechanical cues and regulate stress-drive cellular biological responses with unclear mechanism.In this study,we intended to explore the mechanism of Piezo1 on stress-induced cartilage matrix metabolism by establishing a malocclusion-induced TMJOA model in vivo combined with the fluid flow shear stress model in vitro.We tried to shed light on the development of target treatments to prevent early-stage OA.Methods:1.The TMJOA model was established by unilateral anterior crossbite(UAC),condylar cartilage of rats at 2,4,and 8 weeks were harvested,and the cartilage matrix degeneration was observed by HE staining,Safranin O staining,and immunohistochemical staining.The expression and distribution of Piezo1 in rat condylar cartilage tissue were detected by immunohistochemistry and immunofluorescence staining.2.In vitro,ATDC5 chondrocyte-like cell line was loaded with 24dyn/cm2 fluid flow shear stress(FFSS)using the Flexcell mechanical loading device,and the effect of mechanical treatment on Piezo1 was detected by Real-time PCR,Western Blot and Fluo-4AM.3.The Piezo1 silencing model was established in vitro,and then 24 dyn/cm2 fluid flow shear stress was applied on ATDC5 cells for 4 h.Immunofluorescence staining,real-time PCR and western blot were used to detect the regulation effect of Piezo1 on the nuclear translocation of the downstream molecule YAP protein under FFSS,and the expression changes of LATS1,p-LATS1,YAP,p-YAP and mob1 in Hippo pathway were detected by Western Blot.In vivo,the changes of YAP subcellular localization during the progression of TMJOA were observed by immunofluorescence staining.4.ATDC5 cells were treated with 24dyn/cm2 fluid flow shear stress for 4h in vitro,and metabolism-related genes were detected by Real-time PCR.The ATDC5 cells were treated with YAP inhibitor Verteporfin for 2 h and then loaded with 24dyn/cm2 fluid flow shear stress.The expressions of MMP13 and ADAMTS5 were detected by Real-time PCR and Western Blot.5.The UAC-TMJ injection model was established in vivo.Safranin O staining and immunohistochemical staining were used to observe the therapeutic effect of intra-articular injection of Piezo1 inhibitor on TMJOA cartilage matrix loss.All experiments were repeated 3 times,and the experimental results were expressed as mean±standard deviation.P<0.05 was statistically significant.Results:1.Under the stimulation of UAC,the condylar cartilage had a degenerative phenotype,the cartilage thickness and the expressions of proteoglycan and Aggrecan were decreased.Immunohistochemical staining results showed that Piezo1 was expressed in the whole layers of condylar cartilage,and the expression distribution of Piezo1 changed with the progression of UAC.2.The expression of Piezo1 in ATDC5 cell line was up-regulated under the treatment of fluid flow shear stress in vitro,and the intracellular calcium ion concentration increased in a Piezo1-dependent manner under FFSS treatment.3.Fluid flow shear stress treatment can lead to Piezo 1-dependent nuclear translocation of YAP protein in ATDC5 cells,and the expression of YAP target genes are up-regulated.Piezo1 affects the nuclear activation of YAP by regulating phosphorylation of LATS and YAP under fluid flow shear stress.It was also observed in rat condylar cartilage tissue that UAC promoted the translocation of YAP protein into the nucleus in condylar chondrocytes.4.The expressions of matrix-degrading enzymes MMP13 and ADAMTS5 were up-regulated in a Piezo1-dependent manner under FFSS treatment in vitro.Also,UAC stimulation resulted in up-regulation of MMP13 and ADAMTS5 in condylar cartilage tissue in vivo.Inhibition of YAP in vitro reduced FFSS-induced activation of MMP13 and ADAMTS5.5.Temporomandibular joint injection of Piezol inhibitor can reduce the loss of cartilage matrix proteoglycan caused by UAC stimulation,reduce the expression of matrix degrading enzymes MMP13 and ADAMTS5 in condylar cartilage,and alleviate the loss of cartilage matrix caused by malocclusion.Conclusion:Abnormal occlusion can lead to the cartilage degradation in TMJ condylar cartilage.Piezo1 widely expressed in whole layers of condylar cartilage and mechanical loading promoted both expression and function of Piezo1.Piezo1 can respond to mechanical stimulation and regulate YAP translocation by inhibiting the phosphorylation of LATS1 and YAP,which leads to the degradation of cartilage matrix.Piezo1 inhibitor can alleviate the loss of cartilage matrix caused by abnormal occlusion.