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

Part ⅠResearch on the effects of necroptosis in the process of compression-induced rat nucleus pulposus cells deathObjective:Research on whether necroptosis contributes to compression-induced rat nucleus pulposus (NP) cells death as well as discuss both the possible mechanisms of its occurrence and its relativity to autophagy.Methods:Rat NP cells from thoracolumbar intervertebral discs were extracted, cultivated and identified. The second generation rat NP cells were used throughout all the experiments. The cells were cultured with 1.0 MPa mechanical compression for Oh,12h,24h,36h,48h respectively. Morphological characteristics were examined using microscope, the cell ultrastructural change was observed by using transmission electron microscopy (TEM). The cell viability was determined by counting kit-8 (CCK-8). Flow cytometry was performed to get quantitative measurement of propidium dide (PI) positive rate. Calcein-AM/PI staining was applied to observe the NP cells death intuitively through laser scanning confocal microscope (LSM). The gene and protein expression of necroptosis key molecules (RIPK1, RIPK3) were measured through real-time PCR and Western blot. Additionally, both the influence of necroptosis inhibitor Nec-1 and (SiRNA-RIPKl, SiRNA-RIPK3) techonology on NP cells mortality as well as immunohistochemistry test to detect the necroptosis key protein RIPK1 and RIPK3 in normal and degenerative intervertebral disc were all performed in the experiment. Finally, MDC staining was applied to observe the quantitative percentage of cells with autophagic vacuoles through flow cytometry when pretreated with Nec-1 or not. Western blot was cairred out to investigate the protein expression of autophagy key molecules LC3B, Beclin-1 before and after Nec-1 treatment.Results:The longer cultured with mechanical compression, the more cell death was observed through light microscope. Necrotic ultrastructure change was detected by TEM. CCK-8 results indicated that the cell activity declined obviously (p<0.05). Flow cytometry results showed that the cell mortality increased gradually (p<0.01). Nec-1 obviously inhibit compression-induced NP cells death, the cells viability decrease (p<0.05) as well as necrotic ultrastructure changes. The gene and protein expression of necroptosis key molecules RIPK1 and RIPK3 increased gradually at first until reached the peak and then decreased. The experimental groups protein and gene expression were higher than the control group (0h) (p<0.01) at all the time points. SiRNA-RIPK3 could reduce compression-induced NP cells death, while SiRNA-RIPK1 received the opposite effects (p<0.01). The immunohistochemistry test results indicated that the protein expressions of RIPK1 and RIPK3 in degenerative intervertebral discs are higher than normal ones, which was consistent with the results in vitro. Furthermore, Nec-1 reduced not only the MDC positive rate, but also the protein expression level of LC3B, Beclin-1 significantly (p<0.01).Conclusion:Combined in vivo and in vitro study, RIPK1/RIPK3 may play an important role in the process of compression-induced NP cells necroptosis. RIPK1 may play a double-sided effects in this process. On one hand, over-expression of RIPK1 promoted the occurrence of necroptosis, on the other hand, its moderate expression may contribute to cell survival. Autophagy may be the downstream pathway of necroptosis. This results may provide a new strategy for reducing compression-induced NP cells death and ultimately retard or prevent intervertebral disc degeneration process.Part ⅡResearch on the relativity of both mitochondrial function disorder and oxidative stress to compression-induced rat NP cells necroptosisObjective:To discuss the relativity between both mitochondrial function disorder and oxidative stress to compression-induced rat NP cells necroptosis, and ultimately discuss the role of RIPK1 in the process.Methods:Rat nucleus pulposus cells from thoracolumbar intervertebral discs were extracted, cultivated and identified. The second generation rat NP cells were used throughout all the experiments. Cells were cultured with 1.0 MPa mechanical compression for 24h,36h before and after RIPK1 kinase inhibitor Nec-1 treatment. Flow cytometry and LSM were applied to detect the mitochondrial membrane potential after fluorescent probe JC-1 staining. The mitochondrial permeability transition pore (PTP) change was determined by MPTP fluorence kit. To detect ROS level, flow cytometry and LSM were performed after fluorescent probe H2DCF-DA staining. Necrotic ultrastructure change was detected by TEM. To evaluate ATP level changes, fluorence microplate reader was applied. Detecting the absorbance to indirectly reflect the activity of MDA and SOD through enzyme labeling instrument was also performed. Ultimately, the gene and protein expression of RIPK1 was quantified by real-time PCR and Western blot after antioxidant N-acetyl-L-cysteine (NAC) was added.Results:In the 24h and 36h treatment group:mitochondrial membrane potential reduced obviously (p<0.01), mitochondrial membrane pore opening increased evidently (p<0.01). TEM observation showed that mitochondrial cristae disappeare, a palpable phenomenon of mitochondrial cavitation and ATP severe depletion (p<0.01), which indicated mitochondrial function was damaged severely. Nec-1 could obviously improve the above compression induced mitochondrial dysfunction. With the extension of pressure culture time, the oxidative stress indicator ROS and MDA level both increased gradually, while antioxidative indicator SOD activity reduced evidently, which indicated that oxidative stress level increased gradually under pressure condition (p<0.01). Nec-1 could significantly reduce ROS, MDA activity and increase SOD activity (p<0.01), which demonstrated that Nec-1 significantly decreased the level of oxidative stress. Meanwhile, antioxidant NAC had no effect on compression induced RIPK1 protein and gene expression level (p<0.001).Conclusion:Nec-1 could significantly reduce compression-induced mitochondrial function disorder and oxidative stress. Based on that antioxidant NAC had no effect on compression induced RIPK1 protein and gene expression, we speculate that RIPK1-mediated mitochondrial function disorder and oxidative stress may play an important role in necroptosis. The protective strategies aiming to regulate compression-induced mitochondrial function disorder and oxidative stress may exert a beneficial effect in NP cells death process, and ultimately retard IVD degeneration.