Lu, N.; Li, Q.; Duan, L.-h.; Xu, R.; Li, Y.-p.; Shi, F.-l.; Zhou, Z.-y.; Gan, Y.-q.; Hu, B.; Li, J.-h.; He, X.-h.; Ouyang, D.-y.; Zha, Q.-b.

Andrographolide (Andro), a natural product extracted from the Chinese traditional medicine herb Andrographis paniculata, has been applied for the treatment of diverse inflammatory diseases. However, its effects on necroptosis, a lytic form of cell death implicated in various inflammatory diseases, remain uncharacterized. In the current research, we investigate whether Andro and its derivatives can suppress necroptosis. The results demonstrate that Andro notably inhibits the necroptosis in in vitro cellular models induced by either lipopolysaccharide (LPS) plus IDN-6556 or a combination of TNF-, LCL-161 (Smac mimetic) and IDN-6556. In these cellular models, Andro exhibits inhibitory effects on the phosphorylation of receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), as well as on the formation of necrosomes. Specifically, Andro reduces intracellular reactive oxygen species (ROS) and mitochondrial superoxide (mtROS), preserves mitochondrial membrane potential during necroptotic induction, and activates the antioxidant transcription factor nuclear factor E2-related factor 2 (Nrf2). Upon the necroptotic stimulation, some mitochondrial proteins such as Bcl-2 and Bak oligomerize and colocalize with RIPK1, RIPK3, and phosphorylated MLKL (p-MLKL) in necrosomes. However, such a process of necrosome formation can be prevented by Andro. In contrast, derivatives including dehydroandrographolide, neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, and 14-deoxyandrographolide show no anti-necroptotic effects and fail to upregulate Nrf2. Collectively, our findings demonstrate that Andro specifically inhibits the RIPK1/RIPK3/MLKL signaling axis to suppress necroptosis, highlighting its therapeutic potential against necroptosis-related disorders.