| Part 1:Expression and distribution of ASICla and Endoplasmic reticulum stress in rat nucleus pulposusObjective:The main purpose of this part is to investigate expression and distribution of ASICla and Endoplasmic reticulum stress in rat nucleus pulposus.Methods:The IVD of Sprague-Dawley rats at different age were stained by Hematoxylin-Eosin staining and special staining to visualize the histocytological changes during the nature process of IVDD.Immunohistochemical staining was performed to evaluate the expression of ASICla and ER stress markers within normal and degenerated NP.The expression of ASICla and ER-stress markers were also quantified by qRT-PCR and western blotting analysis respectively.Results:In vivo,the expression of GRP78,p-eIF2a and XBP1 was downregulated while ASICla,CHOP and Caspase12 were up-reuglated within natural degeneration.In secondary degeneration,the expression of GRP78,p-eIF2a and XBP1 was downregulated while ASICla and CHOP were upregulated.Conclusion:ER stress is involved in rat nucleus pulposus degeneration and associates with nucleus pulposus function.ASICla is expressed in nucleus pulposus and is induced by disc degeneration.Part 2:Endoplasmic reticulum stress protect acid-induced injury of nucleus pulposus cellsObjective:The main purpose of this part is to establish mimic of acidic intervertebral disk environment,and to investigate the role ER stress played in acid-induced injury of nucleus pulposus cells.Methods:Normal rat NPCs were cultured in a monolayer culture with different pH to simulate acidic environment of nucleus pulposus.Transmission electron microscope was used to observe endoplasmic reticulum.4-PBA,a blockade of ER stress,was used to block ER stress.CCK-8 assay was used to evaluate the proliferation of NPCs.Cell apoptosis rate and cell cycle were quantified using flow cytometry analysis.SA-?-gal staining kit was used to observe aging cells.Acidity-induced changes in autophagy-,aging-and apoptosis-related markers were studied using Western blotting analysis.Results:Acute acid stimulus triggered ER stress and followed autophagy.And cell apoptosis and senescence were also increased,which can be enhanced by 4-PBA.NPCs showed its acid-resistence in mild acidic condition while severe acid stimulus promoted ER-stress spcific apoptosis pathway.Conclusion:ER stress attenuated acid-induced acute injury of nucleus pulposus cells and took part in NPCs' acid resistence.Part 3:ASICla partly mediated acid-induced endoplasmic reticulum stress and injury of nucleus pulposus cellsObjective:The main purpose of this part is to investigate the correlation between ASICla and ER stress.Methods:Normal rat NPCs were cultured in a monolayer culture with different pH to simulate acidic environment of nucleus pulposus.siRNA was used to block ASICla.CCK-8 assay and Ki67 was used to evaluate the proliferation of NPCs.Cell apoptosis rate was examined by annexin V/propidium iodide(PI)staining and was quantified using flow cytometry analysis.ASIC1a-mediated intracellular calcium was determined by Ca2+-imaging using Fura-2/AM.Expression of ASICla in acid stimulus was examined by western blotting.Acidity-induced changes in ER stress markers were studied using Western blotting analysis and qPCR.Results:ASICla was expressed in NPCs and could be up-regulated by acid stimulus.Acid stimulus increased intracellular calcium levels and cell apoptosis,and reduced cell viablity,which could be reversed by blocking of ASICla.4-PBA increased acid-induced apoptosis of NPCs in the presence or absence of siASICla.Acid-induced ER stress was also partly inhibited by blocking of ASICla,including GRP78,CHOP and caspase12,while splice of XBP1 and phosphorylation of eIF2a were barely affected.Conclusion:Under acid stimulus,activation of ASICla increased influx of Ca2+and promoted injury of NPCs and partly regulated ER stress,especially the pro-apoptotic markers.Blocking of ASICla attenuated acid-induced injury of NPCs.ASICla is a potential target for disc degeneration. |
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