Hydrogen peroxide induces programmed necrosis in rat nucleus pulposus cells through the RIP1/RIP3-PARP-AIF pathway

This study aimed to systematically investigate whether programmed necrosis contributes to H₂ O₂ -induced nucleus pulposus (NP) cells death and to further explore the underlying mechanism involved. Rat NP cells were subjected to different concentrations of H₂ O₂ for various time periods. The cell viability was measured using a cell counting kit-8, and the death rate was detected by Hoechst 33258/propidium iodide (PI) staining. The programmed necrosis-related molecules receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), poly (ADP-ribose) polymerase (PARP), and apoptosis inducing factor (AIF) were determined by real-time polymerase chain reaction and Western blotting, respectively. The morphologic and ultrastructural changes were examined by phasecontrast microscopy and transmission electron microscopy (TEM). In addition, the necroptosis inhibitor Necrostatin-1 (Nec-1), the PARP inhibitor diphenyl-benzoquinone (DPQ) and small interfering RNA (siRNA) technology were used to indirectly evaluate programmed necrosis. Our results indicated that H₂ O₂ induced necrotic morphologic and ultrastructural changes and an elevated PI positive rate in NP cells; these effects were mediated by the upregulation of RIP1 and RIP3, hyperactivation of PARP, and translocation of AIF from mitochondria to nucleus. Additionally, NP cells necrosis was significantly attenuated by Nec-1, DPQ pretreatment and knockdown of RIP3 and AIF, while knockdown of RIP1 produced the opposite effects. In conclusion, these results suggested that under oxidative stress, RIP1/RIP3-mediated programmed necrosis, executed through the PARP-AIF pathway, played an important role in NP cell death. Protective strategies aiming to regulate programmed necrosis may exert a beneficial effect for NP cells survival, and ultimately retard intervertebral disc (IVD) degeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1269-1282, 2018.