| Intervertebral disc degeneration(IVDD)is an age-related degenerative disease.IVDD severely affects the living standards of patients and increases the economic burden of society.The intervertebral disc is composed of three parts: nucleus pulposus(NP),annulus fibrosus(AF)and cartilage endplate(CEP).Intervertebral disc degeneration(IVDD)is closely related to the senescence of nucleus pulposus cells.As the aging of NP cells increases,the accumulation of damaged mitochondria in the cells increases,and the number of NP cells decreases,resulting in a decline in the ability to synthesize extracellular matrix(ECM).Increased secretion of inflammatory factors eventually leads to cell death and degeneration.Heme oxygenase(HO)-1 is the rate-limiting enzyme for heme degradation,and has antioxidant,anti-inflammatory,anti-apoptotic,and anti-aging effects.It has been proven to protect or delay the progression of disease in many diseases.Our previous studies have confirmed that HO-1 can inhibit the nucleus pulposus cells apoptosis.However,the role of HO-1 in NP cell senescence and autophagy is still unclear.Therefore,this subject intends toelaborate from the following three aspects: HO-1 and cell senescence phenotype expression in human intervertebral disc nucleus pulposus cells.HO-1 inhibits the senescence of nucleus pulposus cells by promoting autophagy in the mitochondrial pathway.HO-1 induced autophagy inhibits the degeneration of nucleus pulposus cells.PART 1: EXPRESSION OF HO-1 AND CELL AGING PHENOTYPE IN HUMAN NUCLEUS PULPOSUS CELLSObjective: To explore the interaction between HO-1 and cell senescence in human intervertebral disc nucleus pulposus cells.Methods: The normal and degenerated human intervertebral disc nucleus pulposus tissues were obtained from patients.western blot and immunohistochemistry experiments were used to observe the expression of HO-1 and cell aging phenotypes.The degenerated human intervertebral disc nucleus pulposus cells were cultured in vitro,and the P1,P3 and P6 generations of the nucleus pulposus cells were taken for experiments.The expression of HO-1 and p16 and p21 of nucleus pulposus cells of each generation were detected by cellular immunofluorescence and western blot.SA-β-gal staining was used to detect the senescence of nucleus pulposus cells.Results: Immunohistochemistry of human nucleus pulposus tissue showed that compared with the normal group,the number of HO-1 positivecells in the degenerated group was significantly reduced,while the number of p21 and p16 positive cells in the degenerated group was significantly higher than that in the normal group(P <0.05).western blot results of human nucleus pulposus tissue showed that the expression of HO-1decreased and the expression of p16 and p21 increased in the degenerated group(P <0.05).Immunofluorescence and western blot experiments of nucleus pulposus cells showed that with continuous passage of nucleus pulposus cells,HO-1 expression level decreased,but SA-β-gal staining level and p16 and p21 expression levels were significantly increased(P<0.05).Conclusion: With the degeneration of the intervertebral disc,the senescence of nucleus pulposus cells increases,but the expression of HO-1decreases.PART 2: HO-1 INHIBITS SENESCENCE OF NUCLEUS PULPOSUS CELLS BY PROMOTING AUTOPHAGY IN THE MITOCHONDRIAL PATHWAYObjective: To explore the role of HO-1 in the senescence of nucleus pulposus cells and its specific molecular mechanism.Methods: Lentiviral overexpression of HO-1 was used to transfect nucleus pulposus cells,and the expression of senescence of nucleus pulposus cells in different treatment groups was detected by cell cycle detection,SA-β-gal staining,and western blot experiments.Transmission electron microscopy,GFP-LC3 adenovirus transfection and western blot experiments were used to detect the level of autophagy in nucleus pulposus cells.JC-1 staining,intracellular ROS staining and transmission electron microscopy were used to detect the mitochondrial function of nucleus pulposus cells.Results: In the nucleus pulposus cells overexpressing HO-1transfected with lentivirus,the experimental results showed that the ratio of cells in the G1 phase of the nucleus pulposus cell overexpressing HO-1 was significantly lower(P <0.05),and the expression levels of SA-β-gal staining and the expression of senescence-related proteins p16 and p21 were significantly reduced(P <0.05).Detection of mitochondrial function of nucleus pulposus cells revealed that mitochondrial membrane potential in cells overexpressing HO-1 was significantly increased compared with the control group,and the content of reactive oxygen species ROS in the cells was significantly reduced.Electron microscopic observation showed that compared with the control group,the nucleus pulposus cells overexpressing HO-1 showed less swollen mitochondria and cristae-deficient mitochondria.These suggests that HO-1 can protect the mitochondrial functional damage of senescent nucleus pulposus cells.For further study,it was shown that overexpression of HO-1 can significantly promote mitochondrial autophagy of nucleus pulposus cells,significantly increase the expression of LC3-II protein,and decrease the expression of P62,and promote the transport of Parkin protein from the cytoplasm to mitochondria.GFP-LC3 and Mito-Tracker co-transfection laser confocal results further confirmed that the number of green fluorescent spots in the cells increased significantly after overexpression of HO-1 lentivirus,and promoted the increase of the co-localization area of GFP-LC3 and Mito-Tracker.Electron microscopy results showed that the nucleus pulposus cells overexpressing HO-1 had a typical double membrane autophagosome,and fragmented mitochondrial structures appeared in the autophagosome.Therefore,these results suggest that HO-1 overexpression promotes mitochondrial autophagy in nucleus pulposus cells.To further understand the relationship between autophagy and the anti-aging effect of HO-1 in nucleus pulposus cells,the autophagy inhibitor 3-MA was used to treat nucleus pulposus cells,and it was found that after 3-MA treatment,the nucleus pulposus cell senescence proteins p16,p21 and p53 proteins expression levels and SA-β-gal staining level increased significantly,and the mitochondrial membrane potential of cells decreased significantly.Conclusion: HO-1 inhibits the senescence of nucleus pulposus cells,and may promote autophagy through the mitochondrial pathway.PART Ⅲ: HO-1 INDUCED AUTOPHAGY INHIBITS DISC DEGENERATIONObjective: To further explore the effect of HO-1 induced autophagy on disc degeneration.Methods: The experiment was divided into control group,LV-NC group,LV-HO-1 group and LV-HO-1 + 3-MA group.Cellular immunofluorescence,western blot and RT-PCR were used to detect nucleus pulposus extracellular matrix type II collagen(COL2)and proteoglycan(AGG),as well as the expression of key ECM metabolic enzymes MMP-3and MMP-13.Finally,LV-HO-1 was injected into the rabbit IDD animal model to explore its actual effect on delaying disc degeneration.Results: After nucleus pulposus cells were transfected with HO-1lentivirus,the cell immunofluorescence,western blot and PCR experiments showed that the expression levels of type II collagen(COL2)and proteoglycan(AGG)were significantly increased,while MMP-3 and MMP-13 expression decreased significantly.After treatment with autophagy inhibitor 3-MA,the expression levels of type II collagen(COL2)and proteoglycan(AGG)in nucleus pulposus cells were significantly reduced,while the expressions of MMP-3 and MMP-13 were significantly increased.It is suggested that HO-1 induced autophagy plays a protective role in disc degeneration.Finally,in vivo experiments the rabbit disc degeneration model showed that HO-1 can promote the autophagy level of nucleus pulposus cells,and at the same time relieve the senescence of the disc,thereby inhibiting the degeneration of the disc.Conclusion: HO-1 induced autophagy inhibits degeneration of nucleus pulposus cells... |
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