The Effects Of Autophagy On The Process Of Compression-induced Nucleus Pulposus Cell Injury And Correlated Mechanisms

Part Ⅰ Autophagy was involved in the process of compression-induced IVD degenerationObjectiveTo determine whether autophagy contributes to the pathogenesis of degenerative disc disease or retards the intervertebral disc (IVD) degeneration, and further analyze the mechanism of the compression-induced rat NP cell injury or death.MethodsRat nucleus pulposus (NP) cells were isolated from the thoracolumbar IVD of3-month-old Sprague Dawley rats. Second generation rat NP cells were used for all experiments. Rat NP cells were placed on cell culture plates or culture dishes, and subjected to a1Mpa mechanical stress for0h,12h,24h,36h or48h. The autophagosome and autophagy-related markers were used to explore the role of autophagy in rat NP cells under compressive stress, which were measured directly by electronic microscopy, real-time fluorescent quantitative PCR, western blot, and indirectly by analyzing the impact of pharmacological inhibitors of autophagy such as3MA and chloroquine. Cell viability was determined by the Cell Counting Kit-8(CCK8).ResultsCompression treatment of rat NP cells initiated morphological changes. By conducting an ultrastructural study using TEM, the typical autophagic features of rat NP cells were detected after compression stimulus for36h or48h. the CCK8assay indicated compression with1MPa for48h remarkably reduced cell viability, and caused injury to rat NP cells. The autophagy inhibitor3-MA significantly triggered cell death compared with the control groups at12h,24h and36h. P values of each treatment group were0.049,0.021and0.002, respectively. The number of rat NP cells at48h after compression with3-MA treatment was markedly elevated compared with compression treatment only groups, and the P value was0.039. The genes of LC3B and Beclin-1expression were dramatically elevated after compression treatment of rat NP cells12h,24h,36h,48h(P<0.05). LC3B-Ⅱ and Beclin-1protein expression were dramatically elevated after compression treatment of rat NP cells12h,24h,36h,48h(P<0.05), And the conversion of LC3B-I to LC3B-Ⅱ after compression treatment was detected by western blotting.ConclusionsAutophagy was involved in the process of compression-induced IVD degeneration. Enhanced degradation of damaged components of NP cells by autophagy may be a crucial survival response against mechanical overload, and extensive autophagy may trigger autophagic cell death. Regulating autophagy may retard IVD degeneration. Part Ⅱ The relationship between autophagy and apotosis in compression-induced IVD cell degenerationObjectiveTo determine the relationship between autophagy and apotosis in compression-induced IVD cell degeneration.MethodsRat nucleus pulposus (NP) cells were isolated from the thoracolumbar IVD of3-month-old Sprague Dawley rats. Second generation rat NP cells were used for all experiments. Annexin-V/propidium iodide (Pl)-fluorescein labeling was conducted to detect the apotosis level of Rat nucleus pulposus (NP) which were exposed to1MPa mechanical stress for Oh,12h,24h,36h and48h. To analyze the relationship between compression-induced autophagy and apoptosis,3-MA was applied to inhibit autophagy and observe apoptosis. The presence of autophagic vacuoles, as a marker of autophagy, was detected by the fluorescent dye monodansylcadaverine (MDC). Intracellular MDC fluorescence levels were measured by an Olympus Fluoview FV100laser scanning confocal microscope. And the positive cell number was quantified based on the results of flow cytometry. Moreover, Z-VAD-FMK was applied to inhibit apoptosis and observe autophagy.ResultsThe results showed that the autophagic vacuoles distinctly increased in a time-dependent manner, which indicated that the positive cell number gradually elevated over time.Compression treatment of rat NP cells initiated morphological changes. There was a statistically significant decrease in autophagic vacuole formation as indicated by a decrease of the positive cells after treatment with long exposure time or the use of3-MA.3-MA markedly decreased the number of autophagosomes at24,36and48h as compared with the control groups, and P values of each treatment group were0.004,0.002and0.003, respectively. Annexin-V/PI-fluorescein staining was used to detect the levels of apoptosis after compression exposure for12h,24h,36h and48h, and the rate of apoptosis were4.93±0.15,14.93±0.25,31.83±0.72,35.46±0.85respectively. When pretreated with3-MA, the percentage of annexin-V-positive cells markedly increased compared with cells that were not treated with3-MA at12,24,36and48h. P values of each treatment group were0.006,0.019,0.007and0.021, respectively. When pretreated with Z-VAD-FMK, the positive cell number markedly increased compared with that of control groups at12,24,36and48h. P values of each treatment group were0.006,0.019,0.007and0.021, respectively. ConclusionsBoth autophagy and apoptosis were involved in the process of compression-induced IVD degeneration. Compression-induced autophagy was closely related to apoptosis. Inhibition of autophagy could upregulate compression-induced apoptosis, and inhibition of apoptosis could upregulate compression-induced autophagy in rat NP cells. PartⅢ Autophagy is mediated by reactive oxygen species in nucleus pulposus cells exposed to compressionObjectiveTo investigate the possible relationship between compression-induced autophagy and intracellular reactive oxygen species (ROS) in Nucleus pulposus (NP) cells in vitro, and further analyze the mechanism of the compression-induced rat NP cell autophagy.MethodsRat nucleus pulposus (NP) cells were isolated from the thoracolumbar IVD of3-month-old Sprague Dawley rats. Second generation rat NP cells were used for all experiments. Rat NP cells were placed on cell culture plates or culture dishes, and subjected to a1Mpa mechanical stress for0h,12h,24h,36h or48h. ROS were measured to determine whether these factors are responsible for the development of compression-induced autophagy. ROS generation in rat NP cells after compression treatment in the presence or absence of N-acetylcysteine (NAC, Sigma) or3-MA was revealed using10μM2’-7’-dihydrodichlorofluoroscein diacetate (H2DCF-DA). To quantify the ROS levels, the cellular mean fluorescence intensity (MFI) was analyzed using a flow cytometer after the cells were stained with5mmol/L H2DCF-DA. We also applied the ROS scavenger NAC to suppress the generation of ROS, and tested the effect of the autophagy inhibitor3-MA to investigate the role of autophagy in the process of ROS generation. The rat NP cells were pretreated with NAC or3-MA for1h and then exposed to1MPa compression. The autophagosome and autophagy-related markers were used to explore the role of autophagy in rat NP cells under compressive stress, which were measured directly by western blot, and indirectly by analyzing the impact of pharmacological inhibitors of autophagy such as3MA and NAC. JNK, pJNK, PI3K, AKT, pAKT were measured directly by western blot..ResultsThe results indicated treatment with1Mpa compression increased the formation of ROS relative to normal culture after12h exposure in rat NP cells, and the P value was0.006. The concentration of ROS increased in a time-dependent manner, which was significantly attenuated by the ROS scavenger, NAC at12,24, and36h. P values of each treatment group were0.0008,0.019, and0.011respectively. The autophagy inhibitor3-MA markedly elevated the level of ROS during48h of compression treatment, and the P value was0.0025(Fig.7). Furthermore, the expression of LC3B-Ⅱ and beclin-1also significantly decreased compared with cells that were not treated with NAC at24,36, and48h (Fig.8), P values were0.019,0.019, and0.010respectively in LC3B-Ⅱ expression groups, and P values were0.045,0.017, and0.035in beclin-1expression groups. The expression of PI3Kand AKT also significantly decreased and pJNK significantly increased compared with cells that were not treated with NAC at24,36, and48h..ConclusionsCompression-induced autophagy was closely related to intracellular ROS production in the process of compression-induced rat NP cell injury and death. Autophagy could eliminate intracellular ROS to protect rat NP cells from compression stress. ROS play a pivotal role in the induction of autophagy by elevating expression of the JNK protein and inhibiting PI3K-AKT-mTOR signaling pathways.