Andrographolide prevents necroptosis by suppressing the generation of reactive oxygen species

Abstract

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 present study, we investigate whether Andro and its derivatives can suppress necroptosis. Our results demonstrate that Andro notably inhibits necroptosis in the in vitro cellular models induced by either lipopolysaccharide (LPS) plus IDN-6556 or a combination of TNF-α, LCL-161 (Smac mimetic) and IDN-6556. In these cellular models, Andro inhibits the phosphorylation of receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like pseudokinase (MLKL), as well as the formation of necrosomes. Specifically, Andro reduces the levels of intracellular reactive oxygen species (ROS) and mitochondrial superoxide (mtROS), preserves the mitochondrial membrane potential during necroptotic induction, and activates the antioxidant transcription factor nuclear factor E2-related factor 2 (Nrf2). Upon necroptotic stimulation, some mitochondrial proteins, such as Bcl-2 and Bak, oligomerize and co-localize with RIPK1, RIPK3, and phosphorylated MLKL (p-MLKL) in necrosomes. However, this process of necrosome formation can be prevented by Andro. In contrast, derivatives, including dehydroandrographolide, neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, and 14-deoxyandrographolide, have 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.

1. Introduction

Andrographis paniculata, a Chinese traditional medicine, is widely used to treat inflammatory diseases, including gastroenteritis, tracheitis, lung abscess, cholecystitis, and oropharyngeal swelling/pain. Andrographolide (Andro), a diterpene lactone compound isolated from this plant, has demonstrated therapeutic efficacy against a number of inflammatory conditions, such as pneumonia [1], colitis [2,3], myocarditis [4], and rheumatoid arthritis [5].

Previous studies have revealed multiple mechanisms underlying the anti-inflammatory effects of Andro. First, Andro covalently modifies cysteine residues within the nuclear factor (NF)-κB p50 subunit to suppress NF-κB signaling [6,7]. Second, Andro can activate nuclear factor E2-related factor 2 (Nrf2) [8]. Third, Andro can inhibit inflammasome activation, thus preventing pyroptosis [9,10]. Despite these advances, more investigations are warranted for a comprehensive understanding of its anti-inflammatory mechanisms.

Regulated cell death (RCD) is a key event in inflammatory organ injury. Among the various forms of RCD, necroptosis critically influences the modulation of physiological and pathological processes, causing a range of human diseases, including ischemic brain injury, immune system diseases, and cancer [11]. This lytic cell death is characterized by disruption of the cell membrane, resulting in the release of cellular components and inflammatory mediators, consequently triggering inflammation. Unlike other forms of RCD, necroptosis is independent of cysteinyl aspartate-specific proteinases (caspases) but is controlled by the receptor-interacting protein kinase 1 (RIPK1)/RIPK3/mixed lineage kinase domain-like pseudokinase (MLKL) signaling pathway. Mechanistically, phosphorylated RIPK1 recruits and phosphorylates RIPK3 [12,13], thus forming a necrosome complex that subsequently phosphorylates/activates MLKL. Activated MLKL translocates to the plasma membrane and oligomerizes to form membrane-disrupting pores [14], which can be inhibited by necrostatin-1 (Nec-1), a specific RIPK1 inhibitor [15]. This necroptotic signaling pathway can be initiated by the b