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Background & Aims: Expression of P21, encoded by the CDKN1A gene, has been associated with fibrosis progression in steatotic liver disease (SLD); however, the underlying mechanisms remain unknown. In the present study, we investigated the function of CDKN1A in SLD. Methods: CDKN1A expression levels were evaluated in different patient cohorts with SLD, fibrosis, and advanced chronic liver disease (ACLD). Cdkn1a-/- and Cdkn1a+/+ mice were fed with either a Western diet (WD), a Lieber-DeCarli (LdC) diet plus multiple EtOH (ethanol) binges, or a DuAL diet (metabolic dysfunction-associated fatty liver disease and alcohol-related liver). Primary hepatocytes were isolated and functional assays performed. Results: A significant increase in CDKN1A expression was observed in patients with steatohepatitis and fibrosis (with a positive correlation with both NAFLD Activity Score and fibrosis staging scores), cirrhosis and ACLD. Cdkn1a+/+ mice, fed a DuAL diet exhibited liver injury and cell death increased reactive oxygen species (ROS), and markers of senescence (γH2AX, β-GAL, Cdkn1a/p53) contributing to steatosis and inflammation. In contrast, Cdkn1a-/- mutant mice showed a significant decrease in senescence-associated markers as well as in markers of liver injury, hepatic steatosis and an increase in fatty acid oxidation and reduction in free fatty acid uptake as well as de novo lipogenesis. Mechanistically, activation of the AMPK-SIRT3 was observed in Cdkn1a-deleted animals. Conclusions: Cdkn1a deletion protected against preclinical SLD by promoting fatty acid oxidation and preventing free fatty acid uptake and de novo lipogenesis via the AMPK-SIRT3 axis. CDKN1A expression was found to be directly correlated with increased severity of NAFLD Activity Score and fibrosis in patients with SLD. CDKN1A could be a potential theragnostic target for the treatment of metabolic dysregulation in patients with SLD, with and without alcohol consumption. Impact and implications:: Expression of p21, encoded by the CDKN1A gene, has been associated with fibrosis progression in steatotic liver disease (SLD), but the molecular mechanisms remain elusive. Interestingly, in this study we found that Cdkn1a deletion protected against preclinical SLD by promoting fatty acid oxidation and preventing free fatty acid uptake and de novo lipogenesis, via the AMPK-SIRT3 axis. Translationally, Cdkn1a expression was found to be directly correlated with increased severity of NAFLD Activity Score (NAS) and fibrosis in SLD patients, and therefore, CDKN1A might be used potential theragnostic target for the treatment of metabolically induced SLD, with and without alcohol consumption.

Abstract Acetaminophen (APAP) hepatotoxicity induces endoplasmic reticulum (ER) stress which triggers the unfolded protein response (UPR) in hepatocytes. However, the mechanisms underlying ER stress remain poorly understood, thus reducing the options for exploring new pharmacological therapies for patients with hyperacute liver injury. Eight-to-twelve-week-old C57BL/6J Xbp1-floxed (Xbp1 f/f ) and hepatocyte-specific knockout Xbp1 mice (Xbp1 ∆hepa ) were challenged with either high dose APAP [500 mg/kg] and sacrificed at early (1–2 h) and late (24 h) stages of hepatotoxicity. Histopathological examination of livers, immunofluorescence and immunohistochemistry, Western blot, real time (RT)-qPCR studies and transmission electron microscopy (TEM) were performed. Pharmacological inhibition of XBP1 using pre-treatment with STF-083010 [STF, 75 mg/kg] and autophagy induction with Rapamycin [RAPA, 8 mg/kg] or blockade with Chloroquine [CQ, 60 mg/kg] was also undertaken in vivo. Cytoplasmic expression of XBP1 coincided with severity of human and murine hyperacute liver injury. Transcriptional and translational activation of the UPR and sustained activation of JNK1/2 were major events in APAP hepatotoxicity, both in a human hepatocytic cell line and in a preclinical model. Xbp1 ∆hepa livers showed decreased UPR and JNK1/2 activation but enhanced autophagy in response to high dose APAP. Additionally, blockade of XBP1 splicing by STF, mitigated APAP-induced liver injury and without non-specific off-target effects (e.g., CYP2E1 activity). Furthermore, enhanced autophagy might be responsible for modulating CYP2E1 activity in Xbp1 ∆hepa animals. Genetic and pharmacological inhibition of Xbp1 specifically in hepatocytes ameliorated APAP-induced liver injury by enhancing autophagy and decreasing CYP2E1 expression. These findings provide the basis for the therapeutic restoration of ER stress and/or induction of autophagy in patients with hyperacute liver injury.

Individuals exhibiting an intermediate alcohol drinking pattern in conjunction with signs of metabolic risk present clinical features of both alcohol‐associated and metabolic‐associated fatty liver diseases. However, such combination remains an unexplored area of great interest, given the increasing number of patients affected. In the present study, we aimed to develop a preclinical DUAL (alcohol‐associated liver disease plus metabolic‐associated fatty liver disease) model in mice. C57BL/6 mice received 10% vol/vol alcohol in sweetened drinking water in combination with a Western diet for 10, 23, and 52 weeks (DUAL model). Animals fed with DUAL diet elicited a significant increase in body mass index accompanied by a pronounced hypertrophy of adipocytes, hypercholesterolemia, and hyperglycemia. Significant liver damage was characterized by elevated plasma alanine aminotransferase and lactate dehydrogenase levels, extensive hepatomegaly, hepatocyte enlargement, ballooning, steatosis, hepatic cell death, and compensatory proliferation. Notably, DUAL animals developed lobular inflammation and advanced hepatic fibrosis. Sequentially, bridging cirrhotic changes were frequently observed after 12 months. Bulk RNA‐sequencing analysis indicated that dysregulated molecular pathways in DUAL mice were similar to those of patients with steatohepatitis. Conclusion: Our DUAL model is characterized by obesity, glucose intolerance, liver damage, prominent steatohepatitis and fibrosis, as well as inflammation and fibrosis in white adipose tissue. Altogether, the DUAL model mimics all histological, metabolic, and transcriptomic gene signatures of human advanced steatohepatitis, and therefore serves as a preclinical tool for the development of therapeutic targets.

Abstract Patients with liver diseases are at high risk for the development of acute respiratory distress syndrome (ARDS). The liver is an important organ that regulates a complex network of mediators and modulates organ interactions during inflammatory disorders. Liver function is increasingly recognized as a critical determinant of the pathogenesis and resolution of ARDS, significantly influencing the prognosis of these patients. The liver plays a central role in the synthesis of proteins, metabolism of toxins and drugs, and in the modulation of immunity and host defense. However, the tools for assessing liver function are limited in the clinical setting, and patients with liver diseases are frequently excluded from clinical studies of ARDS. Therefore, the mechanisms by which the liver participates in the pathogenesis of acute lung injury are not totally understood. Several functions of the liver, including endotoxin and bacterial clearance, release and clearance of pro-inflammatory cytokines and eicosanoids, and synthesis of acute-phase proteins can modulate lung injury in the setting of sepsis and other severe inflammatory diseases. In this review, we summarized clinical and experimental support for the notion that the liver critically regulates systemic and pulmonary responses following inflammatory insults. Although promoting inflammation can be detrimental in the context of acute lung injury, the liver response to an inflammatory insult is also pro-defense and pro-survival. A better understanding of the liver–lung axis will provide valuable insights into new diagnostic targets and therapeutic strategies for clinical intervention in patients with or at risk for ARDS.

Binge drinking, i.e., heavy episodic drinking in a short time, has recently become an alarming societal problem with negative health impact. However, the harmful effects of acute alcohol injury in the gut-liver axis remain elusive. Hence, we focused on the physiological and pathological changes and the underlying mechanisms of experimental binge drinking in the context of the gut-liver axis. Eight-week-old mice with a C57BL/6 background received a single dose (p.o.) of ethanol (EtOH) [6 g/kg b.w.] as a preclinical model of acute alcohol injury. Controls received a single dose of PBS. Mice were sacrificed 8 h later. In parallel, HepaRGs and Caco-2 cells, human cell lines of differentiated hepatocytes and intestinal epithelial cells intestinal epithelial cells (IECs), respectively, were challenged in the presence or absence of EtOH [0–100 mM]. Extracellular vesicles (EVs) isolated by ultracentrifugation from culture media of IECs were added to hepatocyte cell cultures. Increased intestinal permeability, loss of zonula occludens-1 (ZO-1) and MUCIN-2 expression, and alterations in microbiota—increased Lactobacillus and decreased Lachnospiraceae species—were found in the large intestine of mice exposed to EtOH. Increased TUNEL-positive cells, infiltration of CD11b-positive immune cells, pro-inflammatory cytokines (e.g., tlr4, tnf, il1β), and markers of lipid accumulation (Oil Red O, srbep1) were evident in livers of mice exposed to EtOH, particularly in females. In vitro experiments indicated that EVs released by IECs in response to ethanol exerted a deleterious effect on hepatocyte viability and lipid accumulation. Overall, our data identified a novel mechanism responsible for driving hepatic injury in the gut-liver axis, opening novel avenues for therapy.

The selection of the anesthetic regime is a crucial component in many experimental animal studies. In rodent models of liver disease, the combination of ketamine and diazepam (KD), generally by the intramuscular (i.m.) route, has traditionally been the anesthesia of choice for the evaluation of systemic and hepatic hemodynamics but it presents several problems. Here, we compared the performance of inhalational sevoflurane (Sevo) against the KD combination as the anesthesia used for hemodynamic studies involving the measurement of portal pressure in normal rats (Ctrl) and rats with non-cirrhotic portal hypertension induced by partial portal vein ligation (PPVL). Compared with Ctrl rats, rats with PPVL presented characteristic alterations that were not influenced by the anesthetic regime, which included liver atrophy, splenomegaly, increased plasma fibrinogen, decreased alkaline phosphatase and glycemia, and frequent ascites. The use of the KD combination presented several disadvantages compared with the inhalational anesthesia with sevoflurane, including considerable mortality, a higher need of dose adjustments to maintain an optimal depth of anesthesia, increases of heart rate, and alteration of blood biochemical parameters such as the concentration of aspartate aminotransferase, lactate, and lactic dehydrogenase. Rats anesthetized with sevoflurane, on the other hand, presented lower respiratory rates. Importantly, the anesthetic regime did not influence the measurement of portal pressure either in Ctrl or PPVL rats, with the increase of portal pressure being similar in Sevo- and KD- anesthetized groups of PPVL rats compared with their respective control groups. Overall, our results suggest that anesthesia with sevoflurane is preferable to the combination of KD for performing systemic and hepatic hemodynamic studies in rats with non-cirrhotic portal hypertension.