The Role And The Mechanism Of Autophagy In The Intervertebral Disc Degeneration

Part I The expression and function of autophgy in rat intervertebral disc tissue and nucleus pulposus cells Objective: To determine whether autophagy happens in rat intervertebral disc in vivo and in rat nucleus pulposus cells in vitro, and the effect of autophagy in the intervertebral disc degeneration and the survival of nucleus pulposus cells. Method: 1. Experimental intervertebral disc degeneration was induced by making lumber dynamic instability in rats, and was evaluated by X ray, hematoxylin and eosin staining, Safranine O/fast green staining and Picro-sirius red staining after surgery for 9 month. The expressions of autophagic marker proteins(LC3 and Becllin-l) in normol and degenerative disc were determined by immunohistochemical and Western Blot study. The apoptosis index was examined by TUNEL staining. 2. Rat nucleus pulposus cells were isolated and cultured in vitro. Serum deprivation was applicated to modulate the state of insufficient blood supplying in intervertebral disc in vivo. The autophagy level was evaluated by autophagosome examination under transmission electronic microscope, lysosomes activity examination by AO, Lyso-Tracker Red, and MDC staining, autophagy related proteins: Beclin-1 and LC3 examination by Western Blot, and autophagy incidence quantification by flow cytometry. The apoptosis incidence was evaluated by flow cytometry and apoptotic marker proteins(caspase-3, Bax, Bcl-2) were detected by Western Blot. Wortmannin(an autophagy inhibitor) or Rapamycin(an autophagy inducer) was used respectively to determine the interaction between autophagy and apoptosis. Result: 1. The X-ray of degenerative model rat showed the straight of lumbar curvature, endplate calcification and fusion, and the narrowing of disc space. Histomorphology manifested the narrowing of disc space, the disorganization of annulus fibrosus, the decreased expression of aggrecan and collagen II, and the emergence of cartilage lacuna under endplate. The LC3 and Beclin-1 expressions were significantly lower in the degenerative discs, while the apoptosis index was increased significantly in the degenerative discs. 2. The basal level of autophgy in the nucleus pulposus cells cultured in serum medium was low, while the autophagy level increased obviously after serum deprivation for 12 hours, peaked at 48 hours and then declined gradually. The apoptosis of nucleus pulposus cells wasn’t induced obviously until serum deprivation for 24 hours, and then the apoptosis incidence increased with the time of serum deprivation. When autophagy was suppressed by Wortmannin or further stimulated by Rapamycin, the apoptosis incidence both increased. Conclusion: 1. Lumber dynamic instability in rats for 9 month could cause sever disc degeneration. The autophagy level was lower while the apoptosis index was higher in severe degenerativeintervertebral disc in vivo. The reduction or decompensation of auophagy level might be one of the causes of cell apoptosis and even disc degeneration. 2. Autophagy also took place in rat nucleus pulposus cells in vitro, and serum deprivation could further increase the autophagy activity of the cells. Autophagy had dual roles on the survival of nucleus pulposus cells in serum starvation: a pro-survival role of appropriate autophagy activity and a pro-death role of excessive autophagy.Part II The effect of hypoxia on the autophagy and apoptosis in rat nucleus pulposus cells Objective: To explore the effect of hypoxia on the level of autophagy in rat nucleus pulposus cells and the role of autophagy in the survival of nucleus pulposus cells under hypoxia. Method: Rat nucleus pulposus cells were cultured under normoxic(20%O2) or hypoxic(2%O2) environment, with serum supplying or serum deprivation. Immunofluorescence, lysosomal activity assessment, flow cytometry and Western Blot were performed to determine the effect of hypoxia to the level of autophagy. NAC, a reactive oxygen species(ROS) blocker, was used to determine the mechanism of autophagy regulation by hypoxia. Apoptosis incidence of nucleus pulposus cells under hypoxia or normoxia was measured after serum deprivation for 48 hours, and was also measured in the condition of insufficient autophagy by Wortmannin or excessive autophagy by Rapamycin respectively, to explore the role of autophagy in the survival of nucleus pulposus cells under hypoxia. Result: Hypoxia down-regulated the autophagy in nucleus pulposus cells, no matter cultured with or without serum. ROS production was also significantly lower in the cells under hypoxia than in those under normoxia. NAC inhibited the ROS production and reduced the autophagy incidence significantly. And when ROS was inhibited by NAC, the autophagy incidence in the cells under normoxia was almost the same as in those under hypoxia. The apoptosis of the nucleus pulposus cells with serum deprivation for 48 hours was lower under hypoxia than under nomoxia. Wortmannin significantly decreased the autophagy incidence while significantly increased the apoptosis incidence in both the cells under normoxia and under hypoxia. Rapamycin led to the further increasing of both the autophagy incidence and the apoptosis incidence in the cells in serum deprivation. However, both the autophagy incidence and the apoptosis incidence in the cells were lower under hypoxia than under normoxia. Conclusion: Hypoxia couldn’t induce autophagy in nucleus pulposus cells, but actually down-regulated their autophagy activity through restricting ROS production. Hypoxia favored the survival of NP cells in serum starvation by down-regulating the excessive autophagy activity.Part Ⅲ The effect of oxidative stress on the autophagy and apoptosis in rat nucleus pulposus cells Objective: To explore the responses of autophagy and apoptosis and their interactions in the nucleous pulposus cells under oxidative stress. Method: Rat nucleus pulposus cells were isolated and cultured in vitro. H2O2 was used to modulate the oxidative stress state. Apoptosis and autophagy was examined after Nucleous pulposus cells cells were treated with H2O2. Caspase-9 activity, mitochondrial membrane potential, and the protein expression of Bax and Bcl-2 were examined to explore the mechanism of apoptosis. The activation of MAPKs(ERK, JNK, p38) was measured, and relevant specific inhibitors were used to explore the mechanism of autophagy induction. The interaction between autophagy and apoptosis was determined by examining the apoptosis level after autophagy inhibiton. Result: H2O2 could definitely cause death of the nucleus pulposus cells and this cytotoxicity was dependent on its dose and acting time. Almost half of the cells would be dead when they were insulted by 400μM H2O2 for 24 hours. Flow cytometric analysis and Hoechst 33258 staining showed H2O2 induced apoptotic cell death in nucleus pulposus cells. H2O2 activated both caspase-3 and caspase-9, decreased the mitochondrial membrane potential, and promoted the protein expressions of Bax and Bcl-2 in nucleus pulposus cells. Meanwhile, H2O2 induced autophagy in the nucleus pulposus cells, evidenced by the accumulation of GFP-LC3 punctate green fluorescence and the increased of LC3-II/ I. H2O2 activated multiple members of MAPK (ERK, JNK, p38) in nucleus pulposus cells, while only the ERK specific inhibitor(U0126) could inhibited the induction of LC3-II by H2O2. Suppression of autophagy by bafilomycin A1 or U0126 reduced the apoptosis incidence significantly and increased mitochondrial membrane potential. Conclusion: H2O2 induced autophagy through the ERK signaling pathway and induced apoptosis through the mitochondrial pathway in the nucleus pulposus cells. Oxidative stress-induced apoptosis was partly dependent on the autophagy induction, autophagy inhibition could down-regulate the mitochondrial-mediated apoptosis in the nucleus pulposus cells under oxidative stress.