Inflammatory Stimulation Mediates Nucleus Pulposus Cell Necroptosis Through Mitochondrial Function Disfunction and Oxidative Stress Pathway

Background: The mutual activation between nucleus pulposus (NP) cells death and inflammation is an important pathogenic factor of intervertebral disc degeneration. Whether inflammation mediates NP cells necroptosis, and its relationship with mitochondrial dysfunc-tion and oxidative stress remains unclear. Methods: In this study, 50 ng/mL of TNF-alpha and 20 ng/mL of IL-1 beta were used to co-treatment with rat NP cells for 0, 24, 48, 72 hours, then Western blot and RT-PCR techniques were utilized to evaluate the expression level of necroptosis-associated target molecules, such as RIPK1, RIPK3 and MLKL. The results established that with prolongation of TNF-alpha and IL-1 beta treatment time, the expression level of necroptosis-associated molecules gradually increased. The 48 hours of TNF-alpha and IL-1 beta treatment was selected throughout the following experiments. The RIPK1 specific inhibitor necrostatin-1 (Nec-1), RIPK3 in-hibitor GSK872, MLKL inhibitor necrosulfonamide (NSA) and small interfering RNA (siRNA) technology were employed. Results: Under the treatment of TNF-alpha or IL-1 beta, administration of Nec-1, GSK872 or NSA notably reduced NP cells death and up-regulated NP cells viability. Consistently, SiRNA-mediated knockdown of RIPK3 (SiRIPK3) or MLKL (SiMLKL) promoted the survival of NP cells. However, SiRIPK1 aggravated NP cells death. Furthermore, after 48 hours of TNF-alpha and IL-1 beta treatment, the mitochondrial membrane potential decreased, opening of mitochondrial permeability transition pore enhanced, and oxidative stress level notably elevated. The Nec-1, GSK872 or NSA treatment largely restored the normal mitochondrial function and down-regulated oxidative stress. Conclusions: In summary, RIPK1/RIPK3/MLKL-mediated necroptosis play an important role in NP cells death during inflammatory irritation, which might be closely related to mitochondrial dysfunction and up-regulation of oxidative stress.