Abstract：Necrostatin-1 (Nec-1) is a small molecule inhibitor of necroptosis which specifically target the receptor-interacting protein 1 (RIP1) kinase, and has been extensively used in disease models to investigate the role of RIP1 in cell death and inflammation. RIP1 is a crucial adaptor kinase involved in the activation of NF-κB, production of reactive oxygen species (ROS) and phosphorylation of mitogen activated protein kinases (MAPKs), which all play important roles in apoptotic signaling. It has been proved that the vaccine strain Mycobacterium bovis BCG can elicit robust protective immunity and induce apoptosis of macrophages. However, the effect of Nec-1 on the apoptosis of macrophages induced by BCG remains unknown. In this study, we investigated the role of Nec-1 on apoptosis induced by BCG in a macrophage cell line RAW264.7.Cell viability was assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The cell apoptosis rate was detected by Annexin V-FITC/PI double staining. Mitochondrial membrane potential (MMP) was assessed by Jc-1 dye. Caspase-3 activity in the cell extract sampled was measured by Caspase-3 Colorimetric Assay Kit. Furthermore, the mRNA level of RIP1, RIP3, Bax and Bcl-2 were tested with RT-PCR, and the expression level of Bax, Bcl-2, Caspase-3, RIP1 and RIP3 were analyzed by western blotting. The results showed that Nec-1 significantly increase MMP and reduce apoptosis rate of RAW264.7 infected with BCG. RT-PCR and western blotting confirmed that the mRNA and protein expression of Bcl-2 was increased, while mRNA and protein levels of RIP1, RIP3 and BAX was decreased. We also found that expression and activity of caspase-3 was decreased. Taken together, these findings indicated that Nec-1 could inhibit BCG-induced macrophage apoptosis by increasing the level of Bcl-2 and decreasing expression of the pro-apoptotic protein BAX, RIP1 and RIP3. This finding may thus provide an insight into the underlying mechanism of alveolar macrophage cell death in response to mycobacterial infection.
