Influences Of Oxygen Tension And Desferoxamine On Behavior Of Rat Nucleus Pulposus Cells

Backgrounds and ObjectivesCells of intervertebral disc reside in a hypoxia condition that are removed from theblood supply. Both survival and extracellular matrix metablism of nucleus pulposus cellsare manipulated by Hypoxia.Progenitor cells exsit in the hypoxia intervertebral disc,andproliferation of progenitor cells is regulated by the hypoxia-sensetive Notch signalingpathway.1.To observe the roles of hypoxia on the proliferation and extracellular matrixmetablism of nucleus pulposus cells and whether Notch signaling pathway takes a role inthe proliferation process of nucleus pulposus cells;2.To invest the influences of hypoxia mimic desferoxamine on the proliferation andextracellular matrix metablism of nucleus pulposus cells;3.To compare the expression levels of Notch target genes and hypoxia induciblefactor under both hypoxia and hypoxia mimic desferoxamine,and whether HIF and Notchsignaling pathway play a role in intervertebral disc cell proliferation process.Methods:1.Male SD Rats(Wt:200~220g) were euthanized with Pentobarbital. The lumbarspinal columns were removed under aseptic conditions.The gel-like nucleus nucleus wasseparated from the annulus fibrosus and treated with0.2%II collagenase for2~3hoursunder37℃.The digested cells were maintainded in DMEM/F12and20%FBSsupplemented with antibiotics in a humidified atmosphere containing5%CO2at37℃.When confluent,the cells were lifted using trypsin(0.25%) EDTA(1mM) solutionand sub-cultured in10cm dishes.The third passage of nucleus pulposus cells were used inthe experiments. Rat nucleus pulposus cells were cultured in vitro under hypoxia(1%O2、5%CO2、94%N2) or normoxia(21%O2、5%CO2) condition or with Notch signalinginhibitor(4uM L685458)for8~24hours. The proliferation and cell cycle of nucleuspulposus cells were examined by CCK-8assay and flow cytometry. The expression levelof extracellular matrix were tested by quantitative real-time polymerase chain reaction(qRT-PCR).2.Nucleus pulposus cells were administrated to different concontrations ofdesferoxamine (25uM、50uM、100uM、200uM) for8~24hours, the proliferation and cell cycle of nucleus pulposus cells were examined by CCK-8assay and flow cytometry.Theexpression level of extracellular matrix were tested by qRT-PCR..3.Rat nucleus pulposus cells were cultured in vitro under hypoxia(1%O2、5%CO2、94%N2) or normoxia(21%O2、5%CO2) or with50uM desferoxamine for8~48hours. Theexpression level of Notch signaling target genes and HIF-α were tested by qRT-PCR andWestern-blot.Results:1.CCK-8assay and flow cytometry indicated that the viability and percentage of Sphase cells of nucleus pulposus cells under hypoxia was increased when compared withunder normoxia, while decreased with L685458whether under hypoxia or normoxia. Theexpression level of COL2A1、Aggrecan、Sox-9、COL1A1mRNA were increased underhypoxia when compared with under normoxia.2.CCK-8assay and flow cytometry indicated that the viability and percentage of Sphase cells of nucleus pulposus cells with25uM or50uM DFO were increased whencompared with control group，while decreased with200uM DFO.There was no significantdifference between100uM DFO and control group as to the viability and percentage of Sphase cells of nucleus pulposus cells.When nucleus pulposus cells were cultured with50uM DFO for8~24hours, the expression level of COL2A1、Aggrecan mRNA wereincreased,while Sox-9mRNA was decreased when compared with control group.3.The expression level of Hes1、Hes5、Hes7、HIF-1α mRNA were increased andHey1、Hey2mRNA were decreased under hypoxia when compared with under normoxia.The expression level of Hes1、Hes7、HIF-1αprotein were increased under hypoxia whencompared with under normoxia. No significant differences existed as to Hes5andHIF-2αexpression level between hypoxia and normoxia group. The expression level ofHes1、Hes7、Hey1mRNA were decreased with50uM DFO when compared with controlgroup. The expression level of Hes7protein was decreased with50uM DFO whencompared control group. No significant differences existed as to Hes5expression levelbetween with DFO and control group The expression level of HIF-1α、HIF-2αwereincreased both at mRNA and protein levels with50uM DFO when compared with controlgroup.Conclusion:1.Hypoxia promotes the proliferation process and increases the expression ofextracellular matrix components of nucleus pulposus cell. 2.Hypoxia promotes expression of Notch signaling target genes to maintain nucleuspulposus cells proliferation,and those genes may offer a therapeutic target for manipulatingthe proliferation process of nucleus pulposus cells.3.Proper concentration of DFO promotes the proliferation process and increases theexpression of extracellular matrix components of nucleus pulposus cell. DFO may adjustthe proliferation process of nucleus pulposus cells through a HIF-1α/HIF-2α dependentmanner.DFO may become a substantial therapeutic drug for lowering down the process ofintervertebral disc degeneration.