Acetaminophen (APAP)-induced liver injury is a major cause of liver failure and involves multiple intracellular events, including mitochondrial damage, oxidative stress and inflammation. As an intrinsic inflammatory process, pyroptosis plays a crucial role in cellular lysis and pro-inflammatory cytokine release. Transient Receptor Potential Vanilloid 4 (TRPV4), described as a calcium-permeable cation channel, is associated with proinflammatory factors and pyroptosis. An increase of cytosolic Ca²⁺ plays a key role in the activation of the endosomal sorting complexes required for transport (ESCRT)-mediated membrane repair mechanism. Apigenin is a plant-derived flavone, which has potential preventive effects on the development of liver injury and has previously been reported as a TRP activator, activating TRPV4 channel. Thus, we speculated that novel mechanisms of alleviating APAP-induced hepatocyte pyroptosis and liver injury might be discovered through TRPV4 channel and ESCRT-mediated membrane repair. Our results showed that apigenin could indeed mitigate hepatocyte pyroptosis and liver injury induced by APAP. Further mechanism studies showed that apigenin could activate TRPV4 channel to induce calcium influx, while the increase of intracellular Ca²⁺ played a key role in the activation of ESCRT-mediated membrane repair mechanisms. In order to elucidate the relationship between TRPV4 channel, ESCRT-mediated membrane repair, and apigenin’s alleviation of APAP-induced pyroptosis and liver injury, we introduced the TRPV4 channel inhibitor HC067047, the calcium ion scavenger ethylenediaminetetraacetic acid (EDTA) and CHMP4B-siRNA to detect the indicators related to pyroptosis and liver injury. Altogether, the study revealed that apigenin could activate TRPV4 channel to induce calcium influx, leading to ECSRT-III expression and alleviating APAP-induced pyroptosis and liver injury. It provides new ideas and targets for using functional factors in food to improve APAP-induced liver injury.

Elaidic acid (EA) is a typical trans fatty acid (TFA) that emerges during the processing of various fatty foods. In this study, we found that EA induced renal injury with necroptosis. Pretreatment with a ROS inhibitor and a RIPK3 inhibitor alleviated EA-induced necroptosis. The data indicated that EA induced renal necroptosis through ROS/RIPK3/MLKL pathway. In mechanistic studies, we explored how EA induced ROS production. We found EA caused mitochondrial damage by testing MMP, MFN1, VDAC, and FIS1. Further, we also found EA suppressed mitophagy by testing the levels of LC3, p62, PINK1, Parkin, colocalization of LC3 and Mito-Tracker Red. Mitophagy is a process of selective degradation of damaged mitochondria. A large number of damaged mitochondria couldn’t be cleared by mitophagy in time, which increased ROS levels in renal cells. Pretreatment with a mitophagy activator decreased EA-induced ROS levels and mitochondrial damage. Taken together, our data identified that EA induced renal necroptosis by destroying mitochondria and inhibiting mitophagy, thereby activating the ROS/RIPK3/MLKL pathway.

Alcohol abuse has recently become a serious health concern worldwide, and the incidence of alcoholic liver disease (ALD) is rapidly increasing with high morbidity and mortality. Ferroptosis is a newly recognized form of regulated cell death caused by the iron-dependent accumulation of lipid peroxidation. Here we showed that the circadian clock protein BMAL1 in hepatocytes is both necessary and sufficient to protect against ALD by mitigating ferroptosis. Upon exposure to alcohol (5 % Lieber-DeCarli liquid alcohol diet for 10 days before binged alcohol with 5 g/kg body weight in vivo, 300 mmol/L for 12 h in vitro, respectively), the content of iron, reactive oxygen species (ROS) and malondialdehyde (MDA) was boosted significantly while glutathione (GSH) was decreased that mainly based on the downregulated protein expression of ferritin heavy chain (FTH), ferroportin (FPN), heme oxygenase1(HO-1) and anti-cystine/glutamate antiporter (SLC7A11), while these changes could be abolished by ferroptosis inhibitor Ferrostatin-1[Fer-1 (5 mg/kg body weight for 10 days in vivo, 10 μmol/L for 2 h in vitro, respectively)]. Further study indicated that the alcohol could activate the protein expression of brain and muscle arnt-like protein-1 (BMAL1) which exerts a protective effect against ferroptosis through promoting nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into nuclear and subsequently stimulating its downstream proteins FTH, FPN, glutathione peroxidase 4 activity (GPX4), HO-1, SLC7A11, while knockdown of BMAL1 and Nrf2 by RNA interference further downregulated the expression of these protein and thus promoting ferroptosis in response to alcohol. Collectively, our results unveiled that the protective action of BMAL1 during alcohol challenge depends on its ability to activate Nrf2-ARE antiferroptosis pathway and targeting hepatic BMAL1 to dampen hepatic ferroptosis signaling may have therapeutic potential for ALD.