The Role And Mechanism Of Oxidative Stress-induced NF-κB-mediated Apoptosis In Intervertebral Disc Degeneration

Intervertebral disc degeneration(IVDD) is the premise and foundation pathological process of spinal degeneration series, which is the root cause of most diseases of the spine. It is associated with clinical syndromes, including cervical and lumbar radiculopathy, cervical myelopathy, and discogenic low back pain, etc. Currently treatment for disc degeneration degenerative diseases include surgical and non-surgical treatment, but the majority of treatment does not change the development of the disease itself, and thus can not be treated and prevented the source of disc degeneration. Scholars investigated the mechanism of disc degeneration from molecular biology, biochemistry, biomechanics, immunology, but its exact mechanism is inconclusive.The reduced active cells, the reduction of extracellular matrix synthesis and its composition changes is the main disc degeneration disc pathology. Excessive apoptosis of intervertebral disc cells is the main cause of reduced cells. Research showed that there was a relationship between nuclear transcription factor NF-κB and cell apoptosis in disc degeneration, and inhibiting NF-κB signaling pathway can effectively alleviate the process of disc degeneration. But the NF-κB signaling pathway in disc degeneration is how actived. But the NF-κB signaling pathway in disc degeneration is how active. NF-κB signaling pathway is closely associated with abnormal activation of oxidative stress, and studies have confirmed the existence of oxidative damage in the intervertebral disc degeneration, disc cells thus activate NF-κB signaling pathway, and its possible mechanism of oxidative damage. Based on the above analysis, we speculate that the disc degeneration may be the way for: oxidative damage induced NF-κB signaling pathways abnormally activated, thereby increasing its transcriptional activity, triggering apoptotic signal, causing a large number of apoptotic cells in the nucleus, leading to disc degeneration changed. To confirm the above conclusions, we designed the following experiments:1. The clinical specimens were collected, and malondialdehyde(MDA) and myeloperoxidase(MPO) was measured.2. The clinical specimens were collected, and the active of NF-κB was detected by EMSA.3. The clinical specimens were collected, CHOP and Caspase-3 was detected by Western Blot and RT-PCR.4. To establish in vitro experiment model, nucleus pulposus cells were treated with different concentrations of H2O2 for 24 h. And CCK-8 was used for detect the cell viability.5. The nucleus pulposus cells were treated with H2O2 for 24 h, and P65,P50 were detected by Western Blot.6. To explore the relationship between NF-κB and oxidative stress, nucleus pulposus cells were pretreated with NAC, and then exposed with 200 μmol/L H2O2. Then the markers P65, P50 of NF-κB were investigated by Western Blot and RT-PCR.7. To explore the relationship between NF-κB and nucleus pulposus cells apoptosis, nucleus pulposus cells were pretreated with BAY 11-7082, which is the inhibitor of NF-κB, and then exposed with 200 μmol/L H2O2.Western Blot and RT-PCR were used to investigate the expression of apoptosis proteins CHOP and Caspase-3The main results are as follow:1. The levels of MDA and MPO was significantly increased in intervertebral disc degeneration. The results indicated that oxidative stress played an important role in intervertebral disc degeneration.2. Intervertebral disc degeneration, the NF-κB was actived.3. Western Blot and RT-PCR showed that the protein CHOP and Caspase-3 were increased in intervertebral disc degeneration.4. The cell viability was significantly reduced in a dose-dependent manner, and cell viability in 200μmol/L H2O2 for 24 h was about 56.24±4.36 %, and cells changed topolyonal morphology. In 200μmol/L of H2O2 for 24 h was chosen for further experiments in this study.5. Western Blot and RT-PCR showed that NAC could inhibit the activity of NF-κB. The results indicated that NF-κB may be triggered by oxidative stress in intervertebral disc degeneration.6. Western Blot and RT-PCR showed that NF-κB inhibitor BAY 11-7082 could reduce the expression of CHOP and Caspase-3. The results indicated that NF-κB could take part in intervertebral disc degeneration through inducing apoptosis.In summary, our study demonstrated that the mechanism of intervertebral disc degeneration is the activity of NF-κB, which was triggered by oxidative stress, and could induce apoptosis.