Moderate Mechanical Stimulation Regulates Inflammation And Metabolism Of Annulus Fibrosus Through YAP-mediated Suppression Of NF-?B Signaling Pathway

Intervertebral disc degeneration(IDD)is a major cause of low back pain,which accounts for disability worldwide.The inflammatory response caused by aberrant mechanical loading is one of the main factors leading to annulus fibrosus(AF)degradation and intervertebral disc degeneration.Moreover,there is evidence that moderate mechanical stimulation can regulate anabolic and anti-inflammatory activities of AF cells(AFCs).However,how mechanical loading is sensed by cells and is ultimately linked to activity of AF inflammation remains poorly understood.Recently,YAP is found as a mechanical sensitive transcription factor in mechanotransduction systems,by which cells sense diverse types of biomechanical stimuli and translate them into biochemical signals controlling cell behaviors.Here we report the regulation mechanism of mechanical stimulation on the inflammatory responses of AFCs through YAP/NF-?B signaling pathway.In this study,three aspects were carried out.Firstly,the effects of mechanical stimulation on AFCs were investigated by cyclic tensile strain(CTS)with amplitude of 5%and 12%,and then the inflammatory model of AFCs induced by IL-1? was used to further verify the anti-inflammatory effect of moderate mechanical stimulation.Finally,we next used YAP inhibitor verteporfin(VP)to investigate whether YAP is involved in the regulation of NF-?B signaling pathway by moderate mechanical stimulation.In the first part of this study,the effects of mechanical stimulation on cell metabolism of AFCs were investigated.AFCs were extracted and cultured from AF tissue of SD rats.After plated on the fibronectin-coated silicon chambers made from polydimethylsiloxane(PDMS),AFCs were subjected to cyclic tensile strain(CTS,5%or 12%at 0.5 Hz)for 24 h.Cell morphology and proliferation were observed by F-actin staining and EdU assay,respectively.The expression of anabolic genes(Aggrecan,Col-I and Col-II),catabolic genes(Adamts5,MMP3 and MMP13)and pro-inflammatory genes(IL-1?,COX-2 and iNOS)were analyzed by RT-qPCR and Western blot.The results showed that AFCs under static conditions displayed a flat polygonal shape and were randomly orientated and distributed.In contrast,AFCs in 5%CTS group tended to spread along the loading direction,whereas cells with 12%CTS exhibited a more elongated cell shape and aligned perpendicular to the loading direction.EdU staining showed that 5%CTS promoted cell proliferation,while 12%CTS inhibited cell proliferation.The results of RT-qPCR showed that 12%CTS significantly down-regulated mRNA expression of anabolic genes(Aggrecan,Col-I and Col-II),but up-regulated that of catabolic genes(Adamts5,MMP3 and MMP13)and pro-inflammatory genes(IL-1?,COX-2 and iNOS).In contrast,5%CTS promoted mRNA expression of anabolic genes(Aggrecan,Col-? and Col-?),but did not exert any significant effect on transcription of catabolic and pro-inflammatory genes.Consistent with RT-qPCR results,Western blot analyses further showed that 12%CTS increased protein levels of matrix degrading enzymes(MMP3 and MMP13)and pro-inflammatory cytokines(iNOS and COX-2),whereas 5%CTS promoted expression of aggrecan protein,an important component of extracellular matrix of AFCs.In the second part of this study,the effects of mechanical stimulation on inflammatory response of AFCs were investigated,intended to confirm 5%CTS to be a moderate mechanical stimulation.We combined inflammatory model induced by IL-1? with 5%and 12%CTS,respectively.EdU staining was used to detect cell proliferation,and RT-qPCR was used to evaluate the expression of anabolic genes(Aggrecan,Col-? and Col-?),catabolic genes(Adamts5,MMP3 and MMP13)and pro-inflammatory genes(IL-1?,COX-2 and iNOS).The results showed that 5%CTS nearly blocked IL-1?-induced expression of pro-inflammatory(IL-1?,COX-2 and iNOS)and catabolic genes(Adamts5,MMP3 and MMP13),while at the same time significantly alleviated its inhibitory effect on expression of anabolic genes(Aggrecan,Col-I and Col-?).In contrast,12%CTS enhanced the expression of pro-inflammatory(IL-1?,COX-2 and iNOS)and catabolic genes(Adamts5,MMP3 and MMP13)induced by IL-1?,and further decreased the expression of anabolic genes(Aggrecan,Col-? and Col-?).EdU staining showed that IL-1? treatment significantly reduced the percentage of proliferating AFCs,which was largely restored by 5%CTS.In contrast,12%CTS inhibited cell proliferation to the similar levels in IL-1?-treated or-untreated AFCs.In the third part of this study,the regulation mechanism of mechanical stimulation on the inflammatory responses of AFCs through YAP/NF-?B signaling pathway was investigated.We combined inflammatory model induced by IL-1? with 5%CTS,and YAP inhibitor verteporfin(VP)was added to the entire course of mechanical loading.EdU staining was used to detect cell proliferation,and RT-qPCR was used to assess the expression of anabolic genes(Aggrecan,Col-? and Col-?),catabolic genes(Adamts5,MMP3 and MMP13)and pro-inflammatory genes(IL-1?,COX-2 and iNOS).The protein levels of YAP,p-YAP,p65 and p-p65 were evaluated by Western blot,and p65 nuclear localization by immunofluorescent staining.The results showed that 5%CTS inhibited IL-1?-induced activation of NF-?B signaling pathway,but the process was attenuated in the presence of YAP inhibitor.At the same time,VP relieved inhibitory effect of 5%CTS on IL-1?-induced expression of pro-inflammatory genes(IL-1?,COX-2 and iNOS)and catabolic genes(Adamts5,MMP3 and MMP13).In addition,inhibition of YAP expression by VP treatment reversed the stimulatory impact of 5%CTS on the expression levels of anabolic genes(Aggrecan,Col-? and Col-?),as well as the percentage of EdU-positive cells in IL-1?-treated cells.In summary,our data have demonstrated that moderate and excessive mechanical stimulation induced the distinct biological consequences.Moderate CTS,similar to that experienced by AFCs in our spine,suppresses inflammatory response via the inhibiton of NF-?B signaling by mechanically activated YAP.This work identifies force as a parameter that alters AFCs function,and demonstrates a physiological role for YAP and mechanoregulation in inflammation.Such a finding may provide the necessary information to consider rational design of therapeutic exercise,and contribute to the understanding of exercise rehabilitation on the spine and other diseases.Evenmore,targeting YAP-mediated inactivation of NF-?B signaling pathway is a promising target therapy for IDD.