Vibrio vulnificus cytolysin induces necroptosis in immortalized bone marrow-derived macrophage through RIP1/MLKL pathway

Objective　To investigate the effects of necroptosis induced by vibrio vulnificus cytolysin (VVc) on immortalized bone marrow-derived macrophage cell line (iBMDM) and its mechanism. Methods　The iBMDM were divided into five groups, control group, VVc(2μg/ml) group, VVc(4μg/ml) group, Necrostatin-1 (Nec-1)+VVc(2μg/ml) group, Nec-1+VVc(4μg/ml) group based on the concentration of VVc and whether the cells received receptor interacting protein 1 (RIP1). In the RIP1 inhibited groups, iBMDM was pretreated with Nec-1 (5μmol/L) before stimulation with VVc. Cells and culture supernatant were harvested at 2h after the challenge by cytolysin. Lactate dehydrogenase (LDH) activity in cell-culture supernatant following VVc stimulation was determined to evaluate cell death. The cell state was detected by flow cytometry with Annexin V/propidium iodide staining, and the expression of phosphorylated mixed lineage kinase domain-like (MLKL) and pyroptosis- and necroptosis-related proteins (caspase-1, caspase-11, gasdermin D) were detected by Western blotting. Results　The LDH level in the culture supernatant was increased in the groups stimulated by VVc and could also be inhibited by pretreatment with Nec-1 (P＜0.05). Compared with control and RIP1- inhibited groups, the VVc-treatment induced a high amount of the double positive staining cells in the VVc group (P＜0.05). In the RIP1 inhibited groups, Nec-1 pretreatment could significantly decrease the levels of pMLKL (P＜0.05), compared with VVc group. None of the cleaved pyroptosis-related protein was detected by Western blotting. Conclusion　Necroptosis induced by RIP1 may play an important role in VVc injury model of iBMDM, furthermore, Nec-1 could reduce the degree of injury after VVc challenge. DOI: 10.11855/j.issn.0577-7402.2018.05.11