| Intervertebral disc degeneration(IDD)remains the major pathogenesis of chronic pain in the lower neck and back and has become a leading cause of spine-related disability worldwide.As a complicated and multifactorial chronic disease in clinic practice,IDD imposes a substantial socio-economic and health burden on the affected families and society at large due to the high morbidity and the limitation of current treatment modalities.IDD is mainly attributed to the excessive apoptosis and decomposition of the extracellular matrix(ECM)in nucleus pulposus(NP)cells.Meanwhile,inflammatory factors secreted by NP cells accelerate the pathophysiological process of IDD.In this regard,searching for effective drugs that have the ability to suppress the apoptosis,ECM degradation,and inflammation in NP cells may be a feasible solution in preventing and treating IDD.Tyrosol is a polyphenolic compound that exhibits anti-osteoporotic,cardioprotective,anti-oxidant,anti-apoptotic,and anti-inflammatory effects.The objective of our study was to explore the effects of tyrosol on apoptosis,inflammation,and ECM degradation in interleukin(IL)-1?-induced human nucleus pulposus cells(HNPCs)and to preliminarily analyze the corresponding mechanism.Part 1 Tyrosol suppressed apoptosis,inflammation,and ECM degradation in IL-1?-stimulated HNPCsObjective:To explore the effects of tyrosol on IDD progression in interleukin(IL)-1?-stimulated HNPCs.Methods:1.HNPCs were cultured in vitro.To establish an in vitro IDD model,HNPCs were stimulated with 10 ng/mL recombinant human IL-1? for 48 h.For cell treatment,HNPCs were treated with or without 10 ?M tyrosol in the presence or absence of 10 ng/mL IL-1? for 48 h.2.Cell viability was detected by cell counting kit-8(CCK-8).Apoptosis was detected by flow cytometry analysis.Caspase 3/7 activity was also measured to assess apoptosis using the Caspase-Glo(?)3/7 detection kit.The production of tumor necrosis factor-?(TNF-?),IL-6,nitric oxide(NO),and prostaglandin E2(PGE2)was examined to evaluate inflammation.The mRNA expression of TNF-? and IL-6 was detected with qRT-PCR.The protein concentrations of TNF-?,IL-6,and PGE2 in the supernatant of treated HNPCs were measured using the corresponding commercially available enzyme-linked immunosorbent assay(ELISA)kits.NO production was determined using a Griess reagent-based colorimetric assay.The mRNA levels of ECM catabolic genes,include matrix metalloproteinase-3/9/13(MMP-3/9/13),and ECM anabolic genes,include collagen type ?,SRY-related high mobility group box 9(SOX-9),and aggrecan was measured using qRT-PCR.Results:1.The CCK-8 assay suggested that tyrosol at certain doses(range 2.5 to 20 ?M)showed no cytotoxic effect on HNPCs,but the stimulation with IL-1?dose-dependently reduced the viability of HNPCs.IL-1?-induced decrease of the viability of HNPCs was significantly recuperated by addition of tyrosol in a concentration-dependent manner(P<0.05);2.The flow cytometry analysis and caspase-3/7 activity assay suggested the apoptotic rate and caspase-3/7 activity were enhanced in response to IL-1?treatment in HNPCs,which was counteracted following the addition of tyrosol.3.IL-1?-stimulated HNPCs showed increased mRNA expression of TNF-? and IL-6,while these phenomena were antagonized after the treatment with tyrosol(P<0.05 compared with IL-1? group).4.Meanwhile,IL-1? stimulation augmented the production of NO and PGE2 in HNPCs.However,the increase of NO and PGE2 was significantly inhibited by the treatment with tyrosol.5.The mRNA levels of ECM catabolic genes MMP-3,MMP-9,and MMP-13 were upregulated following stimulation with IL-1?,but the tyrosol treatment attenuated the effects of IL-1?.6.The mRNA levels of ECM anabolic genes collagen II,SOX-9,and aggrecan were downregulated in HNPCs following treatment with IL-1?,while treatment with tyrosol reversed these changes.Conclusions:Tyrosol resisted IL-1?-induced viability reduction,apoptosis,inflammation and ECM degradation in HNPCs.Part 2 Tyrosol suppressed apoptosis,inflammation,and ECM degradation in IL-1?-stimulated HNPCs through activation of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathwayObjective:To investigate the role of PI3K/Akt pathway in mediating the protective effects of tyrosol against IL-1?-induced apoptosis,inflammation,and ECM degradation in HNPCs.Methods:1.HNPCs were cultured in vitro and were treated with 10 ng/mL IL-1?with or without 10 ?M tyrosol in the presence or absence of 10 ?M PI3K inhibitor LY294002 for 48 h.2.Cell viability was detected with cell counting kit-8(CCK-8).The levels of protein kinase B(Akt),phosphorylated Akt(p-Akt),collagen type II,SOX-9,and aggrecan were determined with western blot.Apoptosis was detected using flow cytometry analysis and caspase 3/7 activity assay.The mRNA expression of TNF-?,IL-6,MMP-3/9/13,collagen type II,SOX-9,and aggrecan were detected with qRT-PCR.The protein concentrations of TNF-?,IL-6,and PGE2 in the supernatant of treated HNPCs were measured using ELISA kits.NO production was determined using a Griess reagent-based colorimetric assay.Results:1.Treatment with tyrosol elevated p-Akt expression and exposure to IL-1? declined p-Akt expression in HNPCs.Compared with the IL-1? group,Akt phosphorylation was increased in tyrosol+IL-1? group.However,tyrosol-induced increase of p-Akt in IL-1?-stimulated HNPCs was attenuated after reintroduction with LY.2.LY abolished the suppressive effect of tyrosol on IL-1?-induced increment of apoptotic rate and caspase-3/7 activity in HNPCs.Tyrosol-induced inhibition of TNF-? and IL-6 mRNA expression and NO and PGE2 secretion in IL-1?-stimulated HNPCs were relieved in response to LY.The LY treatment alleviated the effects of tyrosol on IL-1?-induced increase of MMP-3,MMP-9,and MMP-13 expression and reduction of collagen II,SOX-9,and aggrecan expression in HNPCs.Conclusions:1.Tyrosol activated the PI3K/Akt pathway in IL-1?-stimulated HNPCs.2.Tyrosol suppressed IL-1?-induced apoptosis,inflammation,and ECM degradation in HNPCs by activation of PI3K/Akt pathway.Part 3 Upregulation of Sirtl by tyrosol suppressed apoptosis and inflammation and regulated ECM remodeling in IL-1?-stimulated HNPCs through activation of PI3K/Akt pathway.Objective:To further clarify whether the effects of tyrosol on IL-1?-induced apoptosis,inflammation,and ECM degradation were mediated by Sirtl in HNPCs or not.Methods:1.For Sirtl knockdown,HNPCs were transfected with siRNA targeting Sirtl(si-Sirtl)or its negative control(si-con)using Lipofectamine 2000.Transfected HNPCs were divided into four groups:si-con group,si-con+IL-1? group,si-con+IL-1?+tyrosol group,si-Sirtl+IL-1?+tyrosol group.2.The protein levels of Sirtl,Akt,p-Akt,collagen type II,SOX-9,and aggrecan were determined using western blot.Apoptosis was detected using the flow cytometry analysis and caspase 3/7 activity assay.The mRNA expression of TNF-?,IL-6,MMP-3/9/13,collagen type II,SOX-9,and aggrecan were detected with qRT-PCR.The protein concentrations of TNF-?,IL-6,and PGE2 in the supernatant of treated HNPCs were measured using ELISA kits.NO production was determined using a Griess reagent-based colorimetric assay.Results:1.Sirtl expression was restrained after IL-1? stimulation in HNPCs,while such effect was abolished by treatment with tyrosol.Sirtl knockdown-induced decrease of Akt phosphorylation in HNPCs treated with or without IL-1?.2.Apoptosis assay showed that the inhibitory effects of tyrosol on IL-1?-induced increase of apoptosis and caspase-3/7 activity in HNPCs were ameliorated following Sirtl knockdown.Moreover,tyrosol-induced repression of MMP-3,MMP-9,and MMP-13 mRNA expression and increment of collagen II,SOX-9,and aggrecan mRNA and protein expression in HNPCs stimulated with IL-1? were overturned in response to introduction with si-Sirtl.Furthermore,si-Sirtl transfection also abolished the suppressive effects of tyrosol on IL-1?-induced increase of the mRNA levels and concentrations of TNF-? and IL-6,as well as concentrations of NO and PGE2 in HNPCs.Conclusions:1.Sirtl was upregulated by tyrosol treatment.2.Sirtl knockdown inactivated the PI3K/Akt pathway in IL-1?-stimulated HNPCs.3.Tyrosol-induced inhibition on apoptosis,inflammation,and ECM degradation in IL-1?-stimulated HNPCs was recuperated by Sirtl downregulation.4.Upregulation of Sirtl by tyrosol suppresses apoptosis and inflammation and regulates extracellular matrix remodeling in IL-1?-stimulated HNPCs through activation of PI3K/Akt pathway. |
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