The Study Of COX-2/PGE₂Expression And Proliferative Regulation In Rheumatoid Arthritis Synoviocytes Under Mechanical Stimulation

Rheumatoid arthritis （RA） is a chronic, systemic autoimmune disease that mainlycharacterized by joint inflammation and synovial hyperplasia. Due to the complexetiology, the pathogenesis of RA has not yet been completely determined.Fibroblast-like synoviocytes （FLSs） is one of the most important components in thethickened synovial tissue in RA, which are in the long-term inflammatory activatedstate. RA FLSs can cause the adjacent cartilage and bone erosion and destruction via therapid proliferation, as well as the secretion of various inflammatory mediators andmatrix degrading enzymes, leading to joint dysfunction, even disability. The joint is oneof the most important pressured organs in the body, however, the influence ofmechanical factors on the development of RA disease is often neglected during thestudy of pathogenesis. Our previous study has found that the static mechanicalstimulation significantly decreased the expression of matrix metalloproteinases （MMPs）in RA FLSs. Based on these results, we further investigated the regulation role of thephysiological level （6%,1.0Hz） of cyclic mechanical stimulation on prostaglandin E₂（PGE₂） production and cell proliferation in human RA FLSs. The main experiments andresults are as follows:①In vitro primary culture of human FLSs and effects of cyclic mechanicalstimulation on cell morphology and cytoskeletonHuman FLSs were isolated from normal or RA synovial tissues obtained fromoperations via explant culture method to establish the synovial cell bank essential forthe subsequent experiments. Using the adjustable FX-4000T cellular mechanicalloading system, we treated the in vitro cultured normal and RA FLSs with6%equal-biaxial cyclic mechanical stimulation, following the observation of cellmorphology and cytoskeleton. The results showed that mechanical stimulation did notcause physical damage to the cells; the rearrangement of cytoskeleton of RA FLSs afterstretch demonstrated that the cells had felt mechanical stimulation and made responses,and then may influence the intracellular pathway regulation. These findings served asthe foundation for the subsequent experiments.②Effects of cyclic mechanical stimulation on PGE₂production and COX-2expression in RA FLSsPGE₂is one of the most important inflammatory mediators in RA synovium, which is mainly involved in joint cartilage and bone destruction. COX-2is its upper streamenzyme. After6%cyclic mechanical loading to RA FLSs in the stimulation of IL-1β,we detected the PGE₂levels in the culture medium and COX-2, MMP-2expression inthe cells, and further detected the expression of NF-κB inhibitors-IκB-α and IκB-β. Theresults showed that6%cyclic mechanical stimulation inhibited IL-1β induced MMP-1and COX-2expression, and reduced the production of PGE₂, which demonstrated thatthe physiological level of mechanical environment plays an positive regulation role torelieve RA synovitis;6%cyclic mechanical stimulation could prevent IL-1β inducedIκB-α and IκB-β protein degradation in the cytoplasma of RA FLSs, thereby maintainthe protein levels of IκB-α and IκB-β in the cytoplasma, which indicated thatmechanical stimulation may down regulate the inflammatory response of RA FLSs byblocking the NF-κB signal pathway.③Effects of cyclic mechanical stimulation on the proliferation of RAFLSsExcept for the sustained inflammatory response, another important reason thatcauses cartilage and bone erosion and joint destruction in RA is synovial hyperplasia.We carried out6%cyclic mechanical stimulation to RA FLSs for2h,6h and12hrespectively, and then detected cell proliferation with MTS and flow cytometry assay, aswell as the mRNA levels of cell cycle positive regulators CDK-2, cyclin D1, cyclin E1and negative regulator p27. The results showed that6%cyclic mechanical stimulationfor2h,6h and12h all significantly down regulated RA FLSs proliferation, whichdemonstrated that the physiological level of mechanical environment was beneficial torelieve RA synovial hyperplasia; under6%cyclic mechanical stimulation, the mRNAlevels of CDK-2and cyclin E1were obviously decreased, while p27increased andcyclin D1unchanged, indicating that mechanical stimulation may reduce RA FLSsproliferation via inhibiting the cell cycle transition from the G1phase to the S phase.④A comparative study of RA and normal FLSs in response to cyclic mechanicalstimulationTo further make sure the specific regulation role of mechanical factor to RA FLSs,and investigate whether the high level of PGE₂in RA synovial fluid is involved insynovial hyperplasia, we made a comparative study between RA and normal FLSs ontheir inflammatory and proliferative responses to mechanical stimulation, as well as theeffects of different concentrations of exogenous PGE₂on FLSs proliferation. The resultsshowed that mechanical stimulation only inhibited the proliferation of RA FLSs, but notnormal FLSs; cell apoptosis assay showed that the amount of apoptotic cells was not increased after stretch either in RA FLSs or in normal FLSs, indicating that mechanicalstimulation reduced RA FLSs proliferation was not depend on apoptosis. Mechanicalstimulation inhibited IL-1β induced COX-2expression and PGE₂production only in RAFLSs, but not in normal FLSs, indicating that the inflammatory responses of RA FLSsto mechanical stimulation may be correlated to its inflammatory activation propertiesthat differ from normal FLSs. Meanwhile, high concentrations （100,500ng/ml） ofexogenous PGE₂promoted RA FLSs proliferation, prompting that the high level ofPGE₂in RA synovial fluid may be involved in synovial hyperplasia. Furthermore, underthe environment of high level of PGE₂, the reduction role of mechanical stimulation toRA FLSs proliferation may not be obvious.⑤Effects of COX-2inhibitor on RA FLSs proliferation under cyclic mechanicalstimulationBased on the inhibition of COX-2expression and endogenous PGE₂production inRA FLSs by celecoxib, a selective COX-2inhibitor, we studied the effects ofphysiological level of mechanical stimulation on RA FLSs proliferation. The resultsshowed that in the condition of cell tolerance, the celecoxib itself did not affect RAFLSs proliferation; inhibition of COX-2expression and endogenous PGE₂productiondid not affect the reduction role of mechanical stimulation to RA FLSs proliferation,either. These results demonstrated that the reduction role of mechanical stimulation toRA FLSs proliferation was not played by inhibiting the expression of COX-2and itsdownstream PGE₂production.This study used biomechanical methods in the pathological study of RA, a kind ofautoimmune diseases, which provided a novel idea to establish its occurrence anddevelopment mechanism. Our results suggested that appropriate movements, whichmake the joints of RA patients subjected to certain mechanical stimulation, can play apositive role in the mitigation of RA disease in the aspect of both synovial inflammationand hyperplasia. This research is valuable for future clinical guidance for RA treatment.