Necrostatin-1 protects C2C12 myotubes from CoCl2-induced hypoxia

Necrostatin-1 (Nec-1) is a selective and potent allosteric inhibitor of necroptosis by specifically inhibiting the activity of receptor-interacting protein (RIP) 1 kinase. The aim of the present study was to determine the effect of Nec-1 on an anoxia model comprising mouse skeletal C2C12 myotubes. In the present study, a hypoxic mimetic reagent, cobalt chloride (CoCl2), was used to induce hypoxia in C2C12 myotubes. The cytotoxic effects of CoCl2-induced hypoxia were determined by a Cell Counting kit-8 assay and flow cytometry. Transmission electron microscopy (TEM) was used to characterize the morphological characteristics of dead cells at the ultrastructural level. To clarify the signaling pathways in CoCl2-mediated cell death, the expression levels of RIP1, RIP3, extracellular signal-regulated kinase (ERK)1/2, hypoxia-inducible factor (HIF)-1 and B cell lymphoma-2 adenovirus E1B 19-kDa interacting protein 3 (BNIP3) were investigated by western blotting. Oxidative stress was determined using 2,7-dichlorofluorescin diacetate to measure intracellular reactive oxygen species (ROS) and the fluorescent dye JC-1 was used to measure mitochondrial membrane potential (m). The results showed that the ratios of apoptotic and necrotic C2C12 cells were increased following CoCl2 treatment, typical necroptotic morphological characteristics were able to observe by TEM, whereas Nec-1 exhibited a protective effect against CoCl2-induced oxidative stress. Treatment with Nec-1 significantly decreased the levels of RIP1, p-ERK1/2, HIF-1, BNIP3 and ROS induced by CoCl2, and promoted C2C12 differentiation. Nec-1 reversed the CoCl2-induced decrease in mitochondrial membrane potential. Together, these findings suggested that Nec-1 protected C2C12 myotubes under conditions of CoCl2-induced hypoxia.