The Regulation And Its Mechanism Of Endoplasmic Reticulum Stress In TNF-alpha-Induced Intervertebral Disc Degeneration

Part 1: The role of TNF-? in the process of rat intervertebral disc degenerationObjective: In vivo,different doses of exogenous TNF-? were injected into the rat tail intervertebral discs.After one-month,magnetic resonance imaging(MRI)scan was performed to detect the imaging findings of intervertebral discs.HE staining was used to observe the composition and morphology of intervertebral discs.Toluidine blue staining was used to observe the expression of proteoglycans.Immunohistochemistry was used to observe the expression of type ? collagen.Defining the role of TNF-? in the process of intervertebral disc degeneration.Results: After one month of exogenous TNF-? injection,T2 value calculated from T2-mapping,the intervertebral disc height and nucleus pulposus volume were reduced.HE staining showed that the distribution of cells and extracellular matrix in the nucleus pulposus of the normal intervertebral disc was homogeneous.And the annulus fibrosus was layered distribution and the structure was clear.the number of nucleus pulposus cells was significantly reduced,unevenly distributed,and the extracellular matrix was disordered;the layer structure of annulus fibrosus was disordered and torn,and the nucleus pulposus matrix and cells embed in the region of annulus fibrosus.In addition,the results of immunohistochemistry and toluidine blue staining showed that the number of cells in the region of nucleus pulposus was reduced,and the expression of proteoglycan and type ? collagen was significantly decreased.Conclusion: TNF-? can induce apoptosis of nucleus pulposus cells and accelerate the degradation of extracellular matrix,which is an important early pathogenetic factor in intervertebral disc degeneration.Part 2: Endoplasmic reticulum stress affects the biological activity of nucleus pulposus cells in inflammatory microenvironmentObjective: Simulating the microenvironment of intervertebral disc inflammation in vitro,clarifying the effect of TNF-? stimulation on nucleus pulposus cells,exploring the activation of endoplasmic reticulum stress in the inflammatory microenvironment and its role in the biological changes of nucleus pulposus cells induced by TNF-?.Methods: In vitro,a gradient concentration of TNF-? treat rat nucleus pulposus cells at different time points to simulate the inflammatory microenvironment of intervertebral disc,and establish a TNF-?-induced nucleus pulposus cell injury model.Cell viability was detected by trypan blue staining,apoptosis was analyzed by flow cytometry and Western blot.Cell proliferation was detected by CCK-8 and cell cycle was analyzed by flow cytometry.q RT-PCR and Western blot were used to measure the expression of cyclin related protein.The expression of extracellular matrix degradation enzymes was measured through q RT-PCR.After endoplasmic reticulum stress was blocked by the specific blocker 4PBA.q RT-PCR,Western blot and cellular immunofluorescence were used to detect the activation of endoplasmic reticulum stress.Apoptosis was analyzed by flow cytometry and Western blot.Cell proliferation was detected by CCK-8,cell cycle was analyzed by flow cytometry,cyclin related protein expression was detected by Western blot,and extracellular matrix degradation enzymes were detected by q RT-PCR.Results: Acute TNF-? stimulation reduced the viability and proliferation of NPCs and caused an increase in apoptosis in a concentration-dependent manner.However,as time goes by,the viable nucleus pulposus cells showed a biological response of accelerated proliferation.Under TNF-? stimulation,the expression of endoplasmic reticulum stress and unfolded protein response(UPR)markers in nucleus pulposus cells was up-regulated.Endoplasmic reticulum stress inhibitor 4PBA weaken TNF-?-activated endoplasmic reticulum stress and significantly aggravated TNF-?-induced apoptosis of NPCs,inhibited cell proliferation,increased extracellular matrix degradation enzyme expression,and aggravated extracellular matrix degradation.Conclusion: Acute TNF-? stimulation can induce endoplasmic reticulum stress.Endoplasmic reticulum stress plays an important role of anti-apoptosis,promote cell proliferation and reduce the degradation of extracellular matrix in TNF-? stimulation.Part 3: Role of UPR-coupled autophagy in TNF-?-induced biological changes in nucleus pulposus cellsObjective: The main purpose of this part is to clarify the activation of UPR pathway and autophagy in nucleus pulposus stimulated by TNF-?,and explore the coupling relationship between autophagy and endoplasmic reticulum stress-related UPR.And investigate the role of autophagy in TNF-?-induced biological changes in nucleus pulposus cells.Methods: At different time points of TNF-? stimulation of nucleus pulposus cells,we took q RT-PCR,Western blot and immunofluorescence to measure the activation of UPR related PERK/e IF2? pathway in nucleus pulposus cells,and the expression of autophagy markers LC3 B,Beclin-1,ATG5.Specific si RNAs were constructed to interfere with PERK/e IF2? pathway and autophagy,Western blot and immunofluorescence were used to evaluate the activation of signaling pathway after si RNA interference.Trypan blue staining and TUNEL staining were used to evaluate the effect of autophagy on nucleus pulposus cell viability.Flow cytometry and Western blot was used to measure the apoptosis of nucleus pulposus cells.q RTPCR was used to detect the expression of extracellular matrix degradation enzymes.Results:(1)Acute TNF-? stimulation induced PERK/e IF2? pathway in nucleus pulposus cells,and up-regulated the expression of autophagy markers LC3 B,Beclin-1 and ATG5.(2)Silence of PERK/e IF2? pathway,autophagy markers,LC3 B,Beclin-1,ATG5 were down-regulated,which indicated that downstream autophagy activation is mediated by PERK/e IF2?.(3)The results of trypan blue staining,TUNEL staining,flow cytometry and Western blot showed that either silence PERK/e IF2? pathway or autophagy could significantly increase the apoptosis of nucleus pulposus cells in TNF-?.(4)Silence of autophagy signal can increase the expression of MMP3,MMP13 and ADAMTS4 induced by TNF-? significantly.Conclusion: TNF-? can activate the downstream autophagy signaling while activating the PERK/e IF2? pathway of the UPR response of nucleus pulposus cells.Blocking autophagy can significantly increase the TNF-?-induced nucleus pulposus cells apoptosis and the expression of extracellular matrix degradation enzymes,which indicate that PERK/e IF2? signaling can reduce cell apoptosis and inhibit the degradation of extracellular matrix by activating autophagy.Part 4: Role of UPR-mediated NF-?B activation in TNF-?-induced biological changes in nucleus pulposus cellsObjective: To elucidate the activation of UPR pathway and NF-?B signaling and their relationship in nucleus pulposus stimulated by TNF-?,and clarify their role in the biological changes of nucleus pulposus cells induced by TNF-?.Methods: At different time points of TNF-?,we took q RT-PCR,Western blot and immunofluorescence to measure the activation of IRE1/XBP1 pathway of UPR and NF-?Bp65 signaling in nucleus pulposus cells.Specific si RNAs were used to block the IRE1/XBP1 pathway and NF-?B-p65,respectively.Silencing efficiency were verified by by immunofluorescence and Western blot,and the coupling relationship between XBP1 and NF-?B signaling was determined.The proliferation of NPCs was detected by CCK-8 assay.We subjected the proliferating relevant antigen Ki67 to immunofluorescence staining and conducted cell cycle analysis by flow cytometry.The expression of cyclin related protein was detected by Western blot.The effects of NF-?B signaling on the vitality and proliferation of nucleus pulposus cells was comprehensively evaluated.Results:(1)Acute TNF-? stimulation activated IRE1/XBP1 pathway in nucleus pulposus cells and promoted NF-?B-p65 phosphorylation and nuclear translocation.(2)Interference of IRE1/XBP1 pathway significantly inhibited p65 phosphorylation and nuclear translocation,which indicate that the IRE1/XBP1 pathway can activate downstream NF-?B signaling.(3)Either knockdown XBP1 or NF-?B signaling could weaken cell proliferation by CCK-8 analysis and Ki67 immunofluorescence.Cell cycle analysis showed that the proportion of cells in the S phase is significantly reduced.The expression of cyclin related protein was significantly reduced after interference by Western blot analyses.These results indicate that both XBP1 and NF-?B signaling can promote the proliferation of nucleus pulposus cells in TNF-? stimulation.Conclusion: TNF-? stimulation can activate IRE1/XBP1 pathway of UPR and the downstream NF-?B signaling in nucleus pulposus cells,result in increased phosphorylation of p65 and nuclear translocation.Endoplasmic reticulum stress promotes proliferation of nucleus pulposus cells in the inflammatory microenvironment by activating the IRE1/XBP1-mediated NF-?B signaling.