Mechanism Of PGE2-EP4/p38 MAPK/CREB Axis Regulating Osteoclast Differentiation In TMJ OA In The Inflammatory Microenvironment

Objective:Temporomandibular joint osteoarthritis(TMJ OA)is a common disease of temporomandibular joint.The main pathological manifestations are progressive articular cartilage degeneration and subchondral bone changes.Clinically,it often causes pain in the temporomandibular joint area,joint noise,mandibular movement disorders,and even facial deformities,causing great physiological and psychological pressure to patients,and seriously affecting the quality of life of patients.However,so far,the pathological characteristics and molecular mechanism of TMJ OA remain unclear,and there is a lack of effective means to intervene in the disease course of TMJ OA.Most of the osteogenic class Ⅱ malocclusion is associated with reduced occlusal support height and inverse rotation of the mandible,resulting in joint compression and TMJ OA.The functional movement and biomechanical force of temporomandibular joint are closely related to occlusal height,and occlusal height reduction is considered to be a potential risk factor for TMJ OA.It is currently believed that cellular inflammatory cytokines play an important role in the pathological process of osteoarthritis(OA).Among them,prostaglandin E2(PGE2)is secreted by a variety of cells and has long been considered as a major inflammatory factor involved in OA and rheumatoid arthritis.In recent years,most studies on bone destruction in TMJ OA focused on osteoclast differentiation of monocyte macrophage system from peripheral blood in the bone marrow cavity,ignoring the important role of exogenous osteoclasts.In the occurrence and development of OA,PGE2 plays a very important role,including reducing collagen synthesis,chondrocyte apoptosis,matrix metalloproteinase synthesis,angiogenic factor synthesis,and vascular endothelial cell proliferation and apoptosis.The mechanism of PGE2 regulating osteoclast differentiation in TMJ OA has not been reported yet.This study aims to explore the signalling pathway that inflammatory factor PGE2 promotes intrasynovial osteoclast progenitor cells to osteoclast differentiation under the condition that occlusal height decrease induces TMJ OA,explore the deep molecular mechanism of inflammatory factor PGE2 regulating osteoclast differentiation,and clarify the mechanism of loss of occlusal support causing TMJ OA.It also provides the theoretical basis and new treatment ideas for the clinical treatment of TMJ OA.Methods:In this study,the rat TMJ OA model was established from the Angle of occlusal vertical distance reduction.Firstly,the destruction of the condyle and subchondral bone of the temporomandibular joint and the expression and localization of PGE2 in TMJ OA in rats were studied by Micro-CT,histomorphological observation,immunohistochemistry,immunofluorescence and other methods.Secondly,in vitro experiments,Western Blot,PCR and TRAP staining were used to further explore the deep mechanism of PGE2-EP4/p38 MAPK/CREB axis regulating osteoclast differentiation in the inflammatory microenvironment,aiming to determine the regulatory effect of PGE2 on osteoclast induction of osteoclast progenitor cells in synovium under inflammatory microenvironment.To explore the occurrence and development mechanism of TMJ OA.Results:1.Micro-CT results showed that BV/TV,BS/BV,Tb.N and BMD of the condylar process decreased in the experimental group.Tb.Sp and Tb.Th increased,and the differences were most obvious in the 6th week.Mankin histological score showed that the pathological score increased gradually with the decreased time of posterior occlusal height,and reached the maximum at the 6th week.2.Immunohistochemical staining showed that the expressions of MMP9,RANKL,TRAP and COX2 increased with the increase of modelling time,and the expressions were most obvious at the 6th week.Immunofluorescence chemical staining showed that PGE2 expression in the experimental group was mainly concentrated in the synovial tissue near the posterior band of the articular disc.The expression was most pronounced in week 6.3.The results of the CCK-8 cytotoxicity test showed that 50 μM and 100 μM PGE2 could promote the proliferation of RAW264.7 cells.Western-blot and q RT-PCR results showed that: The addition of 50 μM and 100 μM PGE2 can up-regulate the protein and m RNA expression levels of CTR,RANK,CTSK and TRAP.100 μM PGE2 has the most significant promoting effect on the protein and m RNA expression levels of TRAP and the marker genes of osteoclast differentiation4.TRAP staining results suggested that 100 μM PGE2 could significantly increase the formation of TRAP-positive prokaryotic cells induced by osteoclast inducers and promote osteoclast differentiation.5.In vitro experiments,Western-blot and q RT-PCR results showed that the expression levels of EP4 m RNA and protein increased gradually with the passage of time after adding 100 μM PGE2,and reached the highest level at 2 h.6.In vitro experiments,Western-blot results showed that the expression levels of EP4 receptor,p38 MAPK,p-p38 MAPK,CREB and p-CREB protein increased after the addition of PGE2 during osteoclast differentiation.After adding an EP4 inhibitor,the expression of the EP4 receptor,p38 MAPK and p-p38 MAPK protein decreased.The expression of p-p38 MAPK and p-CREB protein decreased significantly after the addition of the p38 MAPK inhibitorConclusions:1.The model of occlusal height reduction in rats was successfully constructed,and the decrease of occlusal vertical height could lead to the occurrence of TMJ OA.2.The expression of PGE2 increased in the condyle and synovial tissue of TMJ,and PGE2 played an important role in the bone destruction induced by subchondral bone and synovial tissue.3.100 μM PGE2 can induce osteoclast progenitor cells to differentiate into osteoclasts.4.PGE2 can regulate the osteoclastic differentiation of osteoclastic progenitor cells through EP4/p38 MAPK/CERB axis,and participate in the destruction of condylar surface cartilage and subchondral bone.