Acute liver injury (ALI) is a severe consequence of sudden and massive hepatocyte injury, with a frequently fatal outcome (Stravitz and Lee, 2019). Mitochondria are important targets for drug-induced hepatotoxicity caused by a wide variety of drugs and chemicals (Ma et al., 2020). Recent evidence indicates that abnormal mitochondrial function triggers the onset of various liver diseases and contributes to ALI caused by infection, toxin, and drug abuse (Barbier-Torres et al., 2017; Fromenty and Roden, 2023). Mitochondrial perturbations also affect the recovery of hepatic function (van et al., 2016), and improving mitochondrial function has been a hot research area for exploring potential therapeutics for various liver disorders, including ALI.

In recent years, the research on the application of traditional Chinese medicine for the treatment of liver injuries has gained significant prominence (Deng et al., 2024; Li et al., 2023). Dendrobium nobile Lindl. (DNL), (Orchidaceae), originally a natural precious Chinese medicine (Guo et al., 2020), is included in the Pharmacopoeia of China (2020 Edition). In the ancient book “Shen Nong's Herbal Classic”, DNL is regarded as a significant medicinal herb for preventing and treating liver and gallbladder diseases. Dendrobium nobile Lindl. alkaloids (DNLA) are the main active components of DNL (Wang et al., 2010). Studies have reported that DNLA possesses various biological activities such as antioxidant, anti-inflammatory, autophagy flux activation, metabolism regulation, and mitochondrial function regulation (Fu et al., 2023). Increasing evident has shown that DNLA can exert significant hepatoprotective effects through antioxidation and improvement of hepatic lipid homeostasis (Li et al., 2019; Nie et al., 2018). Previous studies revealed that DNLA could protect the carbon tetrachloride (CCl4)-induced ALI by improving oxidative stress and protecting mitochondrial function (Li et al., 2019; Zhou et al., 2020). However, the exact molecular signaling pathway linking mitochondrial dysfunction to liver injury has yet to be further characterized to fully understand the molecular mechanism by which DNLA confers liver protection.

Cellular stress and cell death induced by various toxicants or pathogenic factors play a key role in all acute and chronic liver diseases (Schwabe and Luedde, 2018). Regulated cell death (RCD) is a fundamental biological process, with necroptosis being one of the best-studied forms of RCD (Tang et al., 2019). Necroptosis typically occurs in a specific context where apoptotic signaling is inhibited (Dillon et al., 2014; Oberst et al., 2011). The classical necroptosis pathway relies on receptor interacting protein kinase 3 (RIPK3)-mediated phosphorylation of mixed lineage kinase domain-like protein (MLKL) (Wegner et al., 2017). Recent studies have found that the activation and regulation of the RIPK1/RIPK3/MLKL signaling pathway is closely related to mitochondrial dysfunction in ALI models (Chu et al., 2021; Wang et al., 2021). After drugs or toxins enter the human body, they attack mitochondria, affecting energy synthesis and electron transfer in the respiratory chain, thereby producing a large amount of mitochondrial reactive oxygen species (mtROS). The increase in mtROS levels further activates inflammatory factors or their interactions, ultimately leading to hepatocyte damage (Chen et al., 2015; Zhang, I.W. et al., 2021; Zhao et al., 2020). In addition, studies have revealed that reactive oxygen species (ROS) levels may be a key factor in the process of necroptosis, increasing the tendency of cells to undergo necroptosis (Bock and Tait, 2020; Rius-Perez et al., 2022; Zhang et al., 2017). Especially in ALI models, it has been shown that the activation and regulation of necroptosis signaling pathways are closely related to mitochondrial dysfunction, particularly mtROS (Zhang, I.W. et al., 2021; Zhao et al., 2020). However, the exact mode of action by which mtROS promotes necroptosis and the RIPK1 activation remains not fully elucidated in the course of necroptosis. It was recently reported that DNLA is protective in CCl4-induced liver injury and improves mitochondrial functions (Zhang, Y. et al., 2021; Zhou et al., 2020). However, whether DNLA may confer liver protection by regulating the phosphorylation of RIPK1 and RIPK1/RIPK2/RIPK3 signaling, thus inhibiting necroptosis, remains unknown.

Therefore, the present study aimed to determine whether the hepatoprotective effect conferred by DNLA is mediated through reduction of mtROS, thereby inhibiting RIPK1/RIPK2/RIPK3 signaling cascade and inhibiting necroptosis.