| Low back pain(LBP)caused by Intervertebral Disc Degeneration(IDD)is a common and serious public health problem.Although conservative treatment and surgical treatment are the mainstream treatment methods at present,the high recurrence rate and more complications caused by current treatment prompt the urgent need to seek new drugs and treatment methods.Therefore,it is particularly important to further clarify the pathogenesis of IDD.This study aims to preliminarily explore the mechanism of the release of pro-inflammatory factors by P2Y2 receptor(P2Y2RS)and YES-associated Protein(YAP)in the intervertebral disc nucleus pulposus tissue,which could be a foundation research for our comprehensive understanding of intervertebral disc degeneration by YAP-mediated of activation of P2Y2Rs,Part I Expression Changes of P2Y2 Receptors and YAP in the Nucleus Pulposus Tissues of Human Degenerative Intervertebral DiscObjectiveThe expression of P2Y2 receptors(P2Y2Rs)and YAP in intervertebral disc degeneration(IDD)was studied by using the nucleus pulposus tissue of human degenerative disc.MethodsAccording to Pfirrman classification,the nucleus pulposus tissues of human degenerative disc were selected and divided into light intervertebral disc degeneration(LIDD)group,moderate intervertebral disc degeneration(MIDD)group and severe intervertebral disc degeneration(SIDD)group.Two-label immunohistochemistry technique(IF)and confocal microscopy were used to detect the expression of P2Y2RS and YAP in the nucleus pulposus tissues of human degenerative disc.The expression of P2Y2RS and YAP in the nucleus pulposus tissues with different degree of IDD was determined by Western blot.Results(1)P2Y2Rs and YAP were co-expressed in the nucleus pulposus tissue of MIDD group using the immunofluorescence histography(IF).(2)Compared with the LIDD group,the protein expressions of P2Y2Rs and YAP in the MIDD group and SIDD group were significantly increased(p<0.01 and p<0.001 n=6).Compared with MIDD group,the protein expressions of P2Y2Rs and YAP in the MIDD group were no significant changes(p>0.05,n=6).ConclusionWestern blot and IF results showed that P2Y2Rs and YAP were co-expressed in the nucleus pulposus of intervertebral disc,and the protein expressions of P2Y2Rs and YAP were significantly increased in the MIDD and SIDD,suggesting that they may be involved in the pathogenesis of IDD.Part II Activation of P2Y2Rs is involved in the pathogenesis of intervertebral disc degeneration by promoting YAP expression and activation in the Nucleus Pulposus Cells of Human Degenerative Intervertebral DiscObjectiveTo explore the mechanism of P2Y2Rs and YAP for the intervertebral disc degeneration in the nucleus pulposus cells of human degenerative intervertebral disc.Methods(1)To investigate the co-expression of P2Y2Rs and YAP.One generation of the nucleus pulposus cells(NPCs)of human degenerative intervertebral disc were selected and pretreated for 72h.The specific pretreatment was as follows,DMEM/F-12 medium containing 10%FBS was used for 60h,and then DMEM/F-12 medium containing 1%FBS was used for 12h to starve cells.Two-label immunohistochemistry technique(IF)and confocal microscopy were used to detect the expression and localization of P2Y2Rs and YAP in human intervertebral disc NPCs.(2)To explore the expression changes of YAP when P2Y2Rs were activated.NPCs were incubated with 10μM,100μM,500μM,1000μM UTP for 24h,respectively.Fluorescence immunohistochemistry(IF)was used to observe the expression of YAP in the cytoplasm and nucleus of NPCs.One generation pretreated NPCs were divided into three groups(Control group,UTP group and UTP+AR-C118925XX group),and the expression of YAP was detected by Western-blot.(3)To investigate the effect of P2Y2Rs activation on the apoptosis of NPCs.TUNEL was used to detect the apoptosis of NPCs after 24h of UTP incubation with different concentrations(10μM,100μM,500μM,1000μM).(4)To investigate the content of inflammatory cytokines in the cultured medium of NPCs after UTP incubation.ELISA was used to determine the contents of IL-1β,IL-6 and TNF-α in the cultured medium of NPCs after UTP incubation for 24 hours at different concentrations(10μM,100μM,500μM,1000μM).The pretreated NPCs were divided into three groups(Control group,UTP group and UTP+AR-C118925XX group),and the contents of IL-1β,IL-6 and TNF-α were determined in the cultured medium of NPCs.(5)To investigate the effects of P2Y2Rs activation on the extracellular matrix(ECM).IL-1β was used to stimulate Nucleus Pulposus Cells(NPCs)to establish a cell model of intervertebral disc degeneration.In the inflammatory environment,YAP agonist Lysophosphatidic acid(LPA),YAP antagonist Verteporfin(Ver)and P2Y2Rs antagonist(AR-C 118925XX),were administered,respectively.Then the expression changes of Extracellular matrix(ECM)were detected.Results(1)After the nucleus pulposus cells were successfully cultured,the IF results showed that P2Y2Rs and YAP were co-expressed.P2Y2Rs was mainly expressed in the cell membrane,while YAP was expressed in both cytoplasm and nucleus.(2)The IF results showed that the nuclear expression of YAP was increased when UTP was greater than or equal to 100μM(p<0.01,n=4).Compared with control group,the protein expression of YAP was significantly increased in UTP group(p<0.05,n=3).On the contrary,the expression of YAP was significantly decreased when inhibition of P2Y2Rs(p<0.05,n=3).These results indicated that the activation of P2Y2Rs increased the expression and promoted the activation of YAP in NPCs.(3)The TUNEL test showed that apoptosis began to occur 500μM UTP incubation,and increased after 100μM UTP incubation.These results indicated that the apoptosis of NPCs was promoted when P2Y2Rs were activated.(4)ELISA results showed that the contents of IL-1β,IL-6 and TNF-α in the cultured medium were significantly increased when UTP concentration was at 1000μM or above(P<0.001,n=3).Notably,there was a statistically significant increase in IL-6 in the cultured medium of NPCs incubated with the lowest dose of UTP(10μM)(P<0.01,n=3),suggesting that activation of P2Y2Rs by UTP at a low dose can easily induce IL-6 release from nucleus pulposus cells.The one generation pretreated NPCs were divided into three groups(Control group,UTP group and UTP+AR-C118925XX group),and the contents of IL-1β,IL-6 and TNF-α in the cultured medium of cells were significantly decreased by inhibiting P2Y2 receptors(P<0.01,n=3).(5)In inflammatory environment,the expression of Aggrecan(AGC)was significantly decreased when inhibition of P2Y2Rs.The expression of Extracellular matrix(ECM)in NPCs was decreased by either excitation or inhibition of YAP.However,the decrease of aggrecan(AGC)was the most obvious after YAP activated.Conclusion(1)Activation of P2Y2Rs is involved in the pathogenesis of IDD through YAP by promoting cell apoptosis,increasing the secretion of IL-1β,IL-6 and TNF-α,and decreasing the content of AGC in ECM.(2)Activation of P2Y2Rs by a concentration of 100μM UTP did not induce NPCs apoptosis,but increased the release of inflammatory cytokines IL-1β and IL-6.Furthermore,activation of P2Y2Rs by a concentration of 1000μM UTP could increase the release of inflammatory cytokines IL-1β and IL-6,and TNF-α,induce the apoptosis of NPCs,and partially reduce ECM.Above all elucidate that the mechanism of P2Y2Rs and YAP for the intervertebral disc degeneration. |
