Inhibition of ZFP281/ZNF281-RIPK1/RIPK3/MLKL signaling in hepatocytes by pterostilbene relieves hepatic lipometabolic disorder and inflammation in non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is the most common cause of chronic liver diseases with its pathophysiological mechanism poorly understood. In this work, serological, histological, molecular biological, biochemical, and immunological methods were applied to explore the pathological significance and action of zinc finger protein 281 (ZFP281 in mouse, ZNF281 in human) and targeted strategies. We reported that ZFP281/ZNF281 abundance in hepatocytes was positively correlated with the progression of NASH. Hepatocyte-specific knockdown of Zfp281 prevented mice from NASH diet-induced liver injury, steatosis, inflammation, and fibrosis. Consistently, the metabolic syndromes in NASH mice, characterized by obesity, hyperglycemia, insulin resistance, and hyperlipidemia, were also relieved by hepatocyte-specific Zfp281 deficiency. Mechanistically, incremental ZNF281 led to the upregulation of proinflammatory signaling, receptor-interacting protein kinase 1 (RIPK1)/RIPK3/mixed lineage kinase domain like pseudokinase (MLKL) axis in hepatocytes bearing free fatty acid stress. Activated MLKL translocated to the mitochondrial membrane, disrupting mitochondrial fatty acid β-oxidation and facilitating lipid accumulation in hepatocytes exposed to free fatty acid stimulation; also, MLKL in activated form orientated to the plasma membrane, triggering the lytic death mode in hepatocytes and launching hepatocellular proinflammatory responses. Moreover, we screened a ZFP281 inhibitor, pterostilbene, and demonstrated that pterostilbene, by inhibiting ZFP281 elevation in NASH livers, reduced hepatocyte injury, steatosis, inflammatory responses and fibrogenesis. In conclusion, this work proposes that induction of ZFP281/ZNF281-RIPK1/RIPK3/MLKL signaling disrupts fatty acid metabolism, promoting lipid accumulation, and triggers proinflammatory cell death, accelerating hepatic necroinflammation. Our work identifies ZFP281/ZNF281 as a promising target as well as pterostilbene as a potential strategy for NASH managing.

Lu C ，Zhang Y ，Miao J ，Wei W ，Wang Y ，Han Y ，Li Y ，Tong Y ，Wang T ，Bao X ... -  《-》

GTPBP8 mitigates nonalcoholic steatohepatitis (NASH) by depressing hepatic oxidative stress and mitochondrial dysfunction via PGC-1α signaling.

Nonalcoholic steatohepatitis (NASH) is emerging as a major cause of liver transplantation and hepatocellular carcinoma (HCC). Regrettably, its pathological mechanisms are still not fully comprehended. GTP-binding protein 8 (GTPBP8), belonging to the GTP-binding protein superfamily, assumes a crucial role in RNA metabolism, cell proliferation, differentiation, and signal transduction. Its aberrant expression is associated with oxidative stress and mitochondrial dysfunctions. Nevertheless, its specific functions and mechanisms of action, particularly in NASH, remain elusive. In our current study, we initially discovered that human hepatocytes L02 displayed evident mitochondrial respiratory anomaly, mitochondrial damage, and dysfunction upon treatment with palmitic acids and oleic acids (PO), accompanied by significantly reduced GTPBP8 expression levels through RNA-Seq, RT-qPCR, western blotting, and immunofluorescence assays. We then demonstrated that GTPBP8 overexpression mediated by adenovirus vector (Ad-GTPBP8) markedly attenuate lipid accumulation, inflammatory response, and mitochondrial impair and dysfunction in hepatocytes stimulated by PO. Conversely, adenovirus vector-mediated GTPBP8 knockdown (Ad-shGTPBP8) significantly accelerated lipid deposition, inflammation and mitochondrial damage in PO-treated hepatocytes in vitro. Furthermore, we constructed an in vivo NASH murine model by giving a 16-week high fat high cholesterol diet (HFHC) diet to hepatocyte specific GTPBP8-knockout (GTPBP8HKO) mice. We firstly found that HFHC feeding led to metabolic disorder in mice, including high body weight, blood glucose and insulin levels, and liver dysfunctions, which were accelerated in these NASH mice with GTPBP8 deficiency in hepatocytes. Consistently, GTPBP8HKO remarkably exacerbated the progression of NASH phenotypes induced by HFHC, as proved by the anabatic lipid accumulation, inflammation, fibrosis and reactive oxygen species (ROS) production in liver tissues, which could be largely attributed to the severe mitochondrial damage and dysfunction. Mechanistically, we further identified that GTPBP8 interacted with peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in hepatocytes. Importantly, the hepaprotective effects of GTPBP8 against mitochondrial dysfunction, oxidative stress and inflammation was largely dependent on PGC-1α expression. Collectively, GTPBP8 may exert a protective role in the progression of NASH, and targeting the GTPBP8/PGC-1α axis may represent a potential strategy for NASH treatment by improving mitochondrial functions.

Meng D ，Chang M ，Dai X ，Kuang Q ，Wang G ... -  《-》

Short-term exposure to low doses of aflatoxin B1 aggravates nonalcoholic steatohepatitis by TLR4-mediated necroptosis.

Aflatoxin B1 (AFB1), a worldwide mycotoxin found in food and foodstuffs, is a potent hepatotoxin in humans and animals. Non-alcoholic fatty liver disease (NAFLD), a widespread disease, could progress from simple steatosis to non-alcoholic steatohepatitis (NASH), hepatic cirrhosis, and even hepatocellular carcinoma (HCC). To date, little is known concerning the relationship between AFB1 and the progression of NAFLD. The effects of low doses of AFB1 on the development of NASH and their mechanism were investigated in vivo and in vitro. The results in vivo showed that AFB1 at 20 and 40 μg/kg.bw aggravated CDAHFD-induced NASH in mice as demonstrated by increasing the serum and liver lipid accumulation, liver inflammation and injury. The results in vitro showed that AFB1 at 1.0 μM aggravated FFA-induced lipid accumulation, inflammation and cell damage in HepG2 cells. In addition, RNA-seq indicated that necroptosis, Toll like receptor signaling and TNF-α signaling showed a significant change in KEGG pathway enrichment in AFB1 at 40 μg/kg.bw. AFB1 significantly upregulated the mRNA and protein levels of TLR4, RIPK3, p-RIPK3, MLKL and p-MLKL, and increased TUNEL positive cells. Also, immunofluorescence results showed that TUNEL, TLR4, TNF-α had co-localized with RIPK3, respectively. Necroptosis inhibitor (GSK-872) attenuated the aggravating effects of AFB1 on NASH. Knockout of TLR4 inhibited necroptosis and rescued the aggravating effects of AFB1 on NASH. These data indicate that low dose of AFB1 aggravated NASH via TLR4-mediated necroptosis. This suggests that low dose of AFB1 is potentially harmful to animals and humans, as they exacerbate NASH.

Zeng J ，Chen L ，Tang J ，Xue J ，Wang Q ，Feng X ，Chen X ，Huang K ，Gan F ... -  《-》

Inhibition of receptor-interacting protein kinase 1 improves experimental non-alcoholic fatty liver disease.

In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD. C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlkl in fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in β-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity. The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD. There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.

Majdi A ，Aoudjehane L ，Ratziu V ，Islam T ，Afonso MB ，Conti F ，Mestiri T ，Lagouge M ，Foufelle F ，Ballenghien F ，Ledent T ，Moldes M ，Cadoret A ，Fouassier L ，Delaunay JL ，Aït-Slimane T ，Courtois G ，Fève B ，Scatton O ，Prip-Buus C ，Rodrigues CMP ，Housset C ，Gautheron J ... -  《-》

Cinnabarinic acid protects against metabolic dysfunction-associated steatohepatitis by activating aryl hydrocarbon receptor-dependent AMPK signaling.

Patil NY ，Rus I ，Ampadu F ，Abu Shukair HM ，Bonvicino S ，Brush RS ，Eaton E ，Agbaga MP ，Oh TG ，Friedman JE ，Joshi AD ... -  《-》