Protocol to detect senescence-associated ß-galactosidase and immunoperoxidase activity in fresh-frozen murine tissues.

Double labeling to identify different markers in the same tissue section represents a useful tool either for in situ diagnosis or characterization of molecular associations. Here, we present a protocol to detect senescence-associated ß-galactosidase (SA-ßGal) and immunoperoxidase (IPO) activity in fresh-frozen murine tissues. We describe steps for tissue collection, solution preparation, SA-ßGal staining, IPO staining, hematoxylin counterstaining, microscopic observation, and signal quantification. This protocol can be used to detect in situ proteins alongside SA-ßGal activity. For complete details on the use and execution of this protocol, please refer to Pacheco-Rivera et al.1.

A model of alcoholic liver disease based on different hepatotoxics leading to liver cancer.

The worst-case scenario related to alcoholic liver disease (ALD) arises after a long period of exposure to the harmful effect of alcohol consumption along with other hepatotoxics. ALD encompasses a broad spectrum of liver-associated disorders, such as steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Based on the chronic administration of different hepatotoxics, including ethanol, sucrose, lipopolysaccharide, and low doses of diethylnitrosamine over a short period, here we aimed to develop a multiple hepatotoxic (MHT)-ALD model in the mouse that recapitulates the human ALD-associated disorders. We demonstrated that the MHT-ALD model induces ADH1A and NXN, an ethanol metabolizer and a redox-sensor enzyme, respectively; promotes steatosis associated with the induction of the lipid droplet forming FSP27, inflammation identified by the infiltration of hepatic neutrophils-positive to LY-6G marker, and the increase of MYD88 level, a protein involved in inflammatory response; and stimulates the early appearance of cellular senescence identified by the senescence markers SA-ß-gal activity and p-H2A.XSer139. It also induces fibrosis associated with increased desmin, a marker of hepatic stellate cells whose activation leads to the deposition of collagen fibers, accompanied by cell death and compensatory proliferation revealed by increased CASP3-mediated apoptosis, and KI67- and PCNA-proliferation markers, respectively. It also induces histopathological traits of malignancy and the level of the HCC marker, GSTP1. In conclusion, we provide a useful model for exploring the chronological ALD-associated alterations and stages, and addressing therapeutic approaches.

Nucleoredoxin Redox Interactions Are Sensitized by Aging and Potentiated by Chronic Alcohol Consumption in the Mouse Liver.

Aging is characterized by increased reactive species, leading to redox imbalance, oxidative damage, and senescence. The adverse effects of alcohol consumption potentiate aging-associated alterations, promoting several diseases, including liver diseases. Nucleoredoxin (NXN) is a redox-sensitive enzyme that targets reactive oxygen species and regulates key cellular processes through redox protein-protein interactions. Here, we determine the effect of chronic alcohol consumption on NXN-dependent redox interactions in the liver of aged mice. We found that chronic alcohol consumption preferentially promotes the localization of NXN either into or alongside senescent cells, declines its interacting capability, and worsens the altered interaction ratio of NXN with FLII, MYD88, CAMK2A, and PFK1 proteins induced by aging. In addition, carbonylated protein and cell proliferation increased, and the ratios of collagen I and collagen III were inverted. Thus, we demonstrate an emerging phenomenon associated with altered redox homeostasis during aging, as shown by the declining capability of NXN to interact with partner proteins, which is enhanced by chronic alcohol consumption in the mouse liver. This evidence opens an attractive window to elucidate the consequences of both aging and chronic alcohol consumption on the downstream signaling pathways regulated by NXN-dependent redox-sensitive interactions.

Identification of ABCC3 and its isoforms as potential biomarker in hepatocellular carcinoma.

Liver diseases preceding the occurrence of hepatocellular carcinoma (HCC) play a crucial role in the progression and establishment of HCC, a malignancy ranked as the third deadliest cancer worldwide. Late diagnosis, alongside ineffective treatment, leads patients to a poor survival rate. This scenario argues for seeking novel alternatives for detecting liver alterations preceding the early occurrence of HCC. Experimental studies have reported that ABCC3 protein increases within HCC tumors but not in adjacent tissue. Therefore, we analyzed ABCC3 expression in public databases and investigated the presence of ABCC3 and its isoforms in plasma, urine and its release in extracellular vesicles (EVs) cargo from patients bearing cirrhosis and HCC. The UALCAN and GEPIA databases were used to analyze the expression of ABCC3 in HCC. The results were validated in a case-control study including 41 individuals bearing cirrhosis and HCC, and the levels of ABCC3 in plasma and urine samples, as well as EVs, were analyzed by ELISA and western blot. Our data showed that ABCC3 expression was higher in HCC tissues than in normal tissues and correlated with HCC grade and stage. ABCC3 protein levels were highly increased in both plasma and urine and correlated with liver disease progression and severity. The isoforms MRP3A and MRP3B of ABCC3 were significantly increased in both EVs and plasma/urine of patients bearing HCC. ABCC3 expression gradually increases in HCC tissues, and its protein levels are increased in both plasma and urine of patients with cirrhosis and HCC. MRP3A and MRP3B isoforms have the potential to be prognostic biomarkers of HCC.

Serum α-SMA is a potential noninvasive biomarker of liver fibrosis.

The severity of fibrosis is central to the therapeutic course for patients with chronic liver disease; therefore, early detection of liver fibrosis is critical for timely therapeutic interventions. Liver biopsy is the gold standard for the diagnosis of liver fibrosis; however, it is contraindicated in several pathological conditions. Activated hepatic stellate cells (HSCs) are the main cells for fibrotic tissue synthesis, such as that of alpha-smooth muscle actin (α-SMA). This study aimed to determine whether serum α-SMA levels are a suitable noninvasive, sensitive, and reliable liver fibrosis marker. Fibrosis was induced in male Wistar rats via chronic CCl4 administration. Fibrosis was determined in the liver tissues by quantifying the hydroxyproline content and visualized using Masson's trichrome staining. Rats chronically administered CCl4 exhibited a progressive increment in the hepatic collagen content, as well as both hepatic and serum α-SMA levels in a time-dependent manner. Moreover, serum levels of α-SMA significantly correlated with hepatic α-SMA levels (p ≤ 0.001), as well as with the severity of liver fibrosis (p ≤ 0.001). These findings suggest that increased levels of serum α-SMA can be considered a potential reliable and noninvasive biomarker for early liver fibrosis.

NLRP3 inflammasome in hepatic diseases: A pharmacological target.

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway is mainly responsible for the activation and release of a cascade of proinflammatory mediators that contribute to the development of hepatic diseases. During alcoholic liver disease development, the NLRP3 inflammasome pathway contributes to the maturation of caspase-1, interleukin (IL)-1ß, and IL-18, which induce a robust inflammatory response, leading to fibrosis by inducing profibrogenic hepatic stellate cell (HSC) activation. Substantial evidence demonstrates that nonalcoholic fatty liver disease (NAFLD) progresses to nonalcoholic steatohepatitis (NASH) via NLRP3 inflammasome activation, ultimately leading to fibrosis and hepatocellular carcinoma (HCC). Activation of the NLRP3 inflammasome in NASH can be attributed to several factors, such as reactive oxygen species (ROS), gut dysbiosis, leaky gut, which allow triggers such as cardiolipin, cholesterol crystals, endoplasmic reticulum stress, and uric acid to reach the liver. Because inflammation triggers HSC activation, the NLRP3 inflammasome pathway performs a central function in fibrogenesis regardless of the etiology. Chronic hepatic activation of the NLRP3 inflammasome can ultimately lead to HCC; however, inflammation also plays a role in decreasing tumor growth. Some data indicate that NLRP3 inflammasome activation plays an important role in autoimmune hepatitis, but the evidence is scarce. Most researchers have reported that NLRP3 inflammasome activation is essential in liver injury induced by a variety of drugs and hepatotropic virus infection; however, few reports indicate that this pathway can play a beneficial role by inducing liver regeneration. Modulation of the NLRP3 inflammasome appears to be a suitable strategy to treat liver diseases.

Nicotinic acid attenuates experimental non-alcoholic steatohepatitis by inhibiting the NLRP3 inflammasome/pyroptosis pathway.

Non-alcoholic steatohepatitis (NASH) is a global public health concern that may progress into fibrosis, cirrhosis, and liver cancer, with limited curative treatment options. While the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is closely linked to NASH progression, nicotinic acid (NA), a vitamin used for the treatment of dyslipidemia, is an emerging pharmaceutical treatment for hepatic steatosis and fibrosis. Here, we investigated pharmacological effects of NA on experimental NASH and whether NLRP3 inflammasome/pyroptosis inhibition is an associated mechanism of action. Rats were fed a high-fat sucrose diet supplemented with cholesterol and a low dose of CCl4. NA significantly reduced inflammation by decreasing the protein levels of tumor necrosis factor-alpha and nuclear factor kappa B. Moreover, NA inhibited the formation of NLRP3- apoptosis-associated speck-like protein containing caspase recruitment domain-Caspase-1, decreasing interleukin-1beta, interleukin-18, and gasdermin D protein. In addition, NA reduced tumor growth factor-beta, alpha-smooth muscle actin, and hepatic levels of collagen-1, consequently decreasing extracellular matrix synthesis. Our results indicate that NA can inhibit NASH progression and encourage further basic and clinical studies on the use of NA for the treatment of human NASH.

Robotic versus open approach in total gastrectomy for gastric cancer: a comparative single-center study of perioperative outcomes.

The robotic approach to gastric cancer has been gaining interest in recent years; however, its benefit over the open procedure in total gastrectomy with D2 lymphadenectomy is still controversial. The aims of the study were to compare postoperative morbidity and mortality, hospital stay, and anatomopathological findings between the robotic and open approaches to oncologic total gastrectomy. We analyzed a prospectively collected database, which included patients who underwent total gastrectomy with D2 lymphadenectomy in our center using a robotic or an open approach between 2014 and 2021. Comparative analysis of clinicopathological, intraoperative, postoperative and anatomopathological variables between the robot-assisted group and the open group was performed. Thirty patients underwent total gastrectomy with D2 lymphadenectomy by a robotic approach and 48 patients by an open procedure. Both groups were comparable. The robot-assisted group presented a lower rate of Clavien-Dindo complications ≥ stage II (20 vs. 48%, p = 0.048), a shorter hospital stay (7 days vs. 9 days, p = 0.003) and had a higher total number of lymph nodes resected (22 nodes vs. 15 nodes, p = 0.001) compared to the open approach. Operative time was longer in the robotic group (325 min vs. 195 min, p < 0.001) compared to the open group. The robotic approach is associated with a longer surgical time, a lower rate of Clavien-Dindo complications ≥ stage II and a shorter hospital stay, and more lymph nodes were resected compared to the open approach.

Mechanisms of non-alcoholic fatty liver disease development in normal-weight individuals.

While non-alcoholic fatty liver disease (NAFLD) without inflammation or fibrosis is considered a relatively 'benign' disease, non-alcoholic steatohepatitis (NASH), by contrast, is characterized by marked inflammation in addition to lipid accumulation, and may include fibrosis, progression to cirrhosis and hepatocellular carcinoma. Obesity and type II diabetes are frequently associated with NAFLD/NASH; however, a significant number of lean individuals may develop these diseases. Little attention has been paid to the causes and mechanisms contributing to NAFLD development in normal-weight individuals. One of the main causes of NAFLD in normal-weight individuals is the accumulation of visceral and muscular fat and its interaction with the liver. Myosteatosis (triglyceride accumulation in the muscle) induces a loss of muscle by reducing blood flow and insulin diffusion, contributing to NAFLD. Normal-weight patients with NAFLD exhibit higher serum markers of liver damage and C-reactive protein levels, as well as more pronounced insulin resistance, compared to healthy controls. Notably, increased levels of C-reactive protein and insulin resistance are strongly correlated with the risk of developing NAFLD/NASH. Gut dysbiosis has also been associated with NAFLD/NASH progression in normal-weight individuals. More investigation is required to elucidate the mechanisms leading to NAFLD in normal-weight individuals.

The antioxidant and anti-inflammatory activities of caffeine effectively attenuate nonalcoholic steatohepatitis and thioacetamide-induced hepatic injury in male rats.

The antioxidant effect of caffeine, associated with its ability to upregulate the nuclear factor-E2-related factor-2 (Nrf2)-signaling pathway, was explored as a possible mechanism for the attenuation of liver damage. Nonalcoholic steatohepatitis (NASH) was induced in rats by the administration of a high-fat, high-sucrose, high-cholesterol diet (HFSCD) for 15 weeks. Liver damage was induced in rats by intraperitoneal administration of thioacetamide (TAA) for six weeks. Caffeine was administered orally at a daily dose of 50 mg/kg body weight during the period of NASH induction to evaluate its ability to prevent disease development. Meanwhile, rats received TAA for three weeks, after which 50 mg/kg caffeine was administered daily for three weeks with TAA to evaluate its capacity to interfere with the progression of hepatic injury. HFSCD administration induced hepatic steatosis, decreased Nrf2 levels, increased oxidative stress, induced the activation of nuclear factor-κB (NF-κB), and elevated proinflammatory cytokine levels, leading to hepatic damage. TAA administration produced similar effects, excluding steatosis. Caffeine increased Nrf2 levels; attenuated oxidative stress markers, including malondialdehyde and 4-hydroxynonenal; restored normal, reduced glutathione levels; and reduced NF-κB activation, inflammatory cytokine levels, and damage. Our findings suggest that caffeine may be useful in the treatment of human liver diseases.

Free radicals, antioxidants, nuclear factor-E2-related factor-2 and liver damage.

The liver performs various biochemical and molecular functions. Its location as a portal to blood arriving from the intestines makes it susceptible to several insults, leading to diverse pathologies, including alcoholic liver disease, viral infections, nonalcoholic steatohepatitis, and hepatocellular carcinoma, which are causes of death worldwide. Illuminating the molecular mechanism underlying hepatic injury will provide targets to develop new therapeutic strategies to fight liver maladies. In this regard, reactive oxygen species (ROS) are well-recognized mediators of liver damage. ROS induce nuclear factor-κB and the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 inflammasome, which are the main proinflammatory signaling pathways that upregulate several proinflammatory and profibrogenic mediators. Additionally, oxygen-derived free radicals induce hepatic stellate cell activation to produce exacerbated quantities of extracellular matrix proteins, leading to fibrosis, cirrhosis and eventually hepatocellular carcinoma. Exogenous and endogenous antioxidants counteract the harmful effects of ROS, preventing liver necroinflammation and fibrogenesis. Therefore, several researchers have demonstrated that the administration of antioxidants, mainly derived from plants, affords beneficial effects on the liver. Notably, nuclear factor-E2-related factor-2 (Nrf2) is a major factor against oxidative stress in the liver. Increasing evidence has demonstrated that Nrf2 plays an important role in liver necroinflammation and fibrogenesis via the induction of antioxidant response element genes. The use of Nrf2 inducers seems to be an interesting approach to prevent/attenuate hepatic disorders, particularly under conditions where ROS play a causative role.

Activation of the NLRP3 inflammasome by CCl4 exacerbates hepatopathogenic diet-induced experimental NASH.

INTRODUCTION AND OBJECTIVES: Administration of carbon tetrachloride (CCl4), along with an hepatopathogenic diet, is widely employed as a chemical inducer to replicate human nonalcoholic steatohepatitis (NASH) in rodents; however, the role of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome in this model remains unclear. We aimed to determine the relevance of NLRP3 inflammasome activation in the development of NASH induced by CCl4 along with an hepatopathogenic diet in male Wistar rats. MATERIALS AND METHODS: Animals were fed either a high fat, sucrose, and cholesterol diet (HFSCD) or a HFSCD plus intraperitoneal injections of low doses of CCl4 (400 mg/kg) once a week for 15 weeks. Liver steatosis, inflammation, fibrosis, and NLRP3 inflammasome activation were evaluated using biochemical, histological, ultrastructural, and immunofluorescence analyses, western blotting, and immunohistochemistry. RESULTS: Our experimental model reproduced several aspects of the human NASH pathophysiology. NLRP3 inflammasome activation was induced by the combined effect of HFSCD plus CCl4 and significantly increased levels of both proinflammatory and profibrogenic cytokines and collagen deposition in the liver; thus, NASH severity was higher in the HFSCD+CCl4 group than that in the HFSCD group, to which CCl4 was not administered. Hepatic stellate cells, the most profibrogenic cells, were activated by HFSCD plus CCl4, as indicated by elevated levels of α-smooth muscle actin. Thus, activation of the NLRP3 inflammasome, triggered by low doses of CCl4, exacerbates the severity of NASH. CONCLUSIONS: Our results indicate that NLRP3 inflammasome activation plays a key role and may be an important therapeutic target for NASH treatment.

Caffeine Inhibits NLRP3 Inflammasome Activation by Downregulating TLR4/MAPK/NF-κB Signaling Pathway in an Experimental NASH Model.

Caffeine elicits protective effects against liver diseases, such as NASH; however, its mechanism of action involving the pyrin domain-containing-3 (NLRP3) inflammasome signaling pathway remains to be elucidated. This study aimed to evaluate the effect of caffeine on the NLRP3 inflammasome signaling pathway in a rat model of NASH. NASH was induced by feeding rats a high-fat, -sucrose, and -cholesterol diet (HFSCD) for 15 weeks along with a weekly low dose (400 mg/kg, i.p.) of CCl4. Caffeine was administered at 50 mg/kg p.o. The effects of HFSCD+CCl4 and caffeine on the liver were evaluated using biochemical, ultrastructural, histological, and molecular biological approaches. The HFSCD+CCl4-treated rats showed fat accumulation in the liver, elevated levels of inflammatory mediators, NLRP3 inflammasome activation, antioxidant dysregulation, and liver fibrosis. Caffeine reduced necrosis, cholestasis, oxidative stress, and fibrosis. Caffeine exhibited anti-inflammatory effects by attenuating NLRP3 inflammasome activation. Moreover, caffeine prevented increases in toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) protein levels and mitigated the phosphorylation of mitogen-activated protein kinase (MAPK). Importantly, caffeine prevented the activation of hepatic stellate cells. This study is the first to report that caffeine ameliorates NASH by inhibiting NLRP3 inflammasome activation through the suppression of the TLR4/MAPK/NF-κB signaling pathway.

Is Nucleoredoxin a Master Regulator of Cellular Redox Homeostasis? Its Implication in Different Pathologies.

Nucleoredoxin (NXN), an oxidoreductase enzyme, contributes to cellular redox homeostasis by regulating different signaling pathways in a redox-dependent manner. By interacting with seven proteins so far, namely disheveled (DVL), protein phosphatase 2A (PP2A), phosphofructokinase-1 (PFK1), translocation protein SEC63 homolog (SEC63), myeloid differentiation primary response gene-88 (MYD88), flightless-I (FLII), and calcium/calmodulin-dependent protein kinase II type alpha (CAMK2A), NXN is involved in the regulation of several key cellular processes, including proliferation, organogenesis, cell cycle progression, glycolysis, innate immunity and inflammation, motility, contraction, protein transport into the endoplasmic reticulum, neuronal plasticity, among others; as a result, NXN has been implicated in different pathologies, such as cancer, alcoholic and polycystic liver disease, liver fibrogenesis, obesity, Robinow syndrome, diabetes mellitus, Alzheimer's disease, and retinitis pigmentosa. Together, this evidence places NXN as a strong candidate to be a master redox regulator of cell physiology and as the hub of different redox-sensitive signaling pathways and associated pathologies. This review summarizes and discusses the current insights on NXN-dependent redox regulation and its implication in different pathologies.

Caffeine mitigates experimental nonalcoholic steatohepatitis and the progression of thioacetamide-induced liver fibrosis by blocking the MAPK and TGF-ß/Smad3 signaling pathways.

INTRODUCTION AND OBJECTIVES: Caffeine consumption is associated with beneficial effects on hepatic disorders. The objectives of this study were to evaluate the antifibrotic effects of caffeine on experimental nonalcoholic steatohepatitis (NASH) induced with a high-fat, high-sucrose, high-cholesterol diet (HFSCD), as well as to evaluate the ability of caffeine to prevent the progression of experimental liver fibrosis induced by the administration of thioacetamide (TAA) in rats and explore the mechanisms of action. METHODS: NASH and fibrosis were induced in rats by the administration of an HFSCD for 15 weeks, and liver fibrosis was induced by intraperitoneal administration of 200 mg/kg TAA 3 times per week, for 6 weeks. Caffeine was administered at a dose of 50 mg/kg body weight. The effects of diet, TAA, and caffeine on fibrosis were evaluated by biochemical and histological examinations. The profibrotic pathways were analyzed by western blotting and immunohistochemistry. RESULTS: Rats exhibited liver fibrosis after HFSCD feeding and the administration of TAA. Caffeine could reduce the hepatic level of collagen and the fibrotic area in the liver. Caffeine prevented the progression of liver fibrosis by decreasing transforming growth factor-beta (TGF-ß), connective tissue growth factor (CTGF), and alpha-smooth muscle actin (α-SMA) expression and by inhibiting the activation of mitogen-activated protein kinases (MAPKs) and Smad3 phosphorylation. CONCLUSIONS: Caffeine attenuates NASH and the progression of liver fibrosis due to its antifibrotic effects and modulating the MAPK and TGF-ß pathways. Therefore, caffeine could be a suitable candidate for treating liver diseases associated with fibrosis.

Days of symptoms and days of hospital admission before surgery do not influence the results of cholecystectomy in moderate acute calculous cholecystitis-Cholecystectomy remains the best treatment.

BACKGROUND AND AIMS: early cholecystectomy is the gold-standard treatment for acute calculous cholecystitis (ACC), although many surgeons still prefer delayed cholecystectomy for grade II to avoid surgical complications. The aim of this study was to analyze the postoperative morbidity and mortality of Tokyo Guidelines grade-II ACC as treated with cholecystectomy, taking into account the days of symptoms and days since hospital admission. MATERIALS AND METHODS: a unicenter, retrospective study was performed based on a prospective database. Patients with grade-II ACC treated with cholecystectomy were selected. Patients were analyzed according to days of symptoms (DS) and days of hospital admission (DHA) until cholecystectomy. Patients were subdivided into 3 groups: < 3 days, 3-5 days, and > 5 days. Univariate and multivariate analyses were performed for morbidity and mortality. Categorical variables were compared using the Chi-squared or Fischer's exact test. Continuous variables were compared using the Mann-Whitney U-test. The level of statistical significance was set at p < 0.05. RESULTS: a total of 998 patients with ACC diagnosis were included; 567 with grade-II ACC and 368 treated with cholecystectomy. Nearly 90 % were treated laparoscopically and 48.1 % underwent surgery the same day of emergency admission. With regard to DS and DHA, there were no statistical differences for severe postoperative complications, although a greater number of complications were detected in the > 5 DS group (p: 0.32) and > 5 DHA group (p: 0.00). Statistically significant differences were found in DS for mortality (p: 0.04). Postoperative length of stay was longer for > 5 DHA group cholecystectomies (p > 0.05). There were no differences with regard to hospital readmission. CONCLUSION: with regard to DS or DHA until cholecystectomy, there were no statistically significant differences related to severe postoperative complications, length of stay, or mortality.

Models of nonalcoholic steatohepatitis potentiated by chemical inducers leading to hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the most common primary liver cancer, arises after a long period of exposure to etiological factors. Nonalcoholic steatohepatitis (NASH) is ranked as the main risk factor for developing HCC; hence, experimental models of NASH leading to HCC have become key tools both to investigate the molecular mechanisms underlying the pathophysiology and to evaluate new putative drugs for treating chronic liver diseases in humans. Animal models of NASH induced by a high-fat diet (HFD) plus chemical inducers, such as the NASH-HCC (STAM), high-fat diet/diethylnitrosamine (HFD/DEN), choline-deficient high-fat diet/DEN (CDHFD/DEN), and Western diet/carbon tetrachloride (WD/CCl4) models, are promising because they exacerbate liver damage and significantly shorten the experimental time. In this review, we critically summarize and discuss the ability of these models to recapitulate the liver alterations that precede and lead to HCC progression, as well as the impact of the diet in promoting liver injury progression. We also emphasize the strengths and weaknesses of the models' ability to closely mimic the stages of liver injury development that occur in humans. Based on the molecular mechanisms induced by the currently available NASH models leading to HCC, we argue that although several NASH models have importantly contributed to describing the disease chronology, the progress in emulating the progression from NASH to HCC has been partial. Thus, the development of novel NASH/HCC models remains an unmet need.