Acetaminophen overdose-induced acute liver injury can be alleviated by static magnetic field.

Acetaminophen (APAP), the most frequently used mild analgesic and antipyretic drug worldwide, is implicated in causing 46% of all acute liver failures in the USA and between 40% and 70% in Europe. The predominant pharmacological intervention approved for mitigating such overdose is the antioxidant N-acetylcysteine (NAC); however, its efficacy is limited in cases of advanced liver injury or when administered at a late stage. In the current study, we discovered that treatment with a moderate intensity static magnetic field (SMF) notably reduced the mortality rate in mice subjected to high-dose APAP from 40% to 0%, proving effective at both the initial liver injury stage and the subsequent recovery stage. During the early phase of liver injury, SMF markedly reduced APAP-induced oxidative stress, free radicals, and liver damage, resulting in a reduction in multiple oxidative stress markers and an increase in the antioxidant glutathione (GSH). During the later stage of liver recovery, application of vertically downward SMF increased DNA synthesis and hepatocyte proliferation. Moreover, the combination of NAC and SMF significantly mitigated liver damage induced by high-dose APAP and increased liver recovery, even 24 h post overdose, when the effectiveness of NAC alone substantially declines. Overall, this study provides a non-invasive non-pharmaceutical tool that offers dual benefits in the injury and repair stages following APAP overdose. Of note, this tool can work as an alternative to or in combination with NAC to prevent or minimize liver damage induced by APAP, and potentially other toxic overdoses.

Assessing the impact of COVID-19 era drug combinations on hepatic functionality: A thorough investigation in adult Danio rerio.

Current and thorough information on the ecotoxicological consequences of pharmaceuticals is accessible globally. However, there remains a substantial gap in knowledge concerning the potentially toxic effects of COVID-19 used drugs, individually and combined, on aquatic organisms. Given the factors above, our investigation assumes pivotal importance in elucidating whether or not paracetamol, dexamethasone, metformin, and their tertiary mixtures might prompt histological impairment, oxidative stress, and apoptosis in the liver of zebrafish. The findings indicated that all treatments, except paracetamol, augmented the antioxidant activity of superoxide dismutase (SOD) and catalase (CAD), along with elevating the levels of oxidative biomarkers such as lipid peroxidation (LPX), hydroperoxides (HPC), and protein carbonyl content (PCC). Paracetamol prompted a reduction in the activities SOD and CAT and exhibited the most pronounced toxic response when compared to the other treatments. The gene expression patterns paralleled those of oxidative stress, with all treatments demonstrating overexpression of bax, bcl2, and p53. The above suggested a probable apoptotic response in the liver of the fish. Nevertheless, our histological examinations revealed that none of the treatments induced an apoptotic or inflammatory response in the hepatocytes. Instead, the observed tissue alterations encompassed leukocyte infiltration, sinusoidal dilatation, pyknosis, fatty degeneration, diffuse congestion, and vacuolization. In summary, the hepatic toxicity elicited by COVID-19 drugs in zebrafish was less pronounced than anticipated. This attenuation could be attributed to metformin's antioxidant and hormetic effects.

The p21+ perinecrotic hepatocytes produce the chemokine CXCL14 after a severe acetaminophen overdose promoting hepatocyte injury and delaying regeneration.

Fifty percent of all acute liver failure (ALF) cases in the United States are due to acetaminophen (APAP) overdose. Assessment of canonical features of liver injury, such as plasma alanine aminotransferase activities are poor predictors of acute liver failure (ALF), suggesting the involvement of additional mechanisms independent of hepatocyte death. Previous work demonstrated a severe overdose of APAP results in impaired regeneration, the induction of senescence by p21, and increased mortality. We hypothesized that a discrete population of p21+ hepatocytes acquired a secretory phenotype that directly impedes liver recovery after a severe APAP overdose. Leveraging in-house human APAP explant liver and publicly available single-nuclei RNAseq data, we identified a subpopulation of p21+ hepatocytes enriched in a unique secretome of factors, such as CXCL14. Spatial transcriptomics in the mouse model of APAP overdose confirmed the presence of a p21+ hepatocyte population that directly surrounded the necrotic areas. In both male and female mice, we found a dose-dependent induction of p21 and persistent circulating levels of the p21-specific constituent, CXCL14, in the plasma after a severe APAP overdose. In parallel experiments, we targeted either the putative senescent hepatocytes with the senolytic drugs, dasatinib and quercetin, or CXCL14 with a neutralizing antibody. We found that targeting CXCL14 greatly enhanced liver recovery after APAP-induced liver injury, while targeting senescent hepatocytes had no effect. These data support the conclusion that the sustained induction of p21 in hepatocytes with persistent CXCL14 secretion are critical mechanistic events leading to ALF in mice and human patients.

Protective Effect of Black Seed and Lettuce Oils Against Paracetamol-Induced Hepatotoxicity in Rats.

<b>Background and Objective:</b> The liver is one of the organs that play an essential role in the human body, including supporting metabolism, immune functions, digestive system, detoxification, storage of vitamins and other functions. This investigation aimed to study the protective effects of black seed and lettuce oil against hepatotoxicity as induced by paracetamol in experimental rats. <b>Materials and Methods:</b> Twenty male Sprague-Dawley albino rats weighing 150±5 g were divided randomly into four groups (5 rats each) and distributed as follows; 1st group was controlled negative (C -ve group), 2nd group controlled positive (orally administered with 500 mg/kg b.wt., paracetamol), 3rd and 4th groups were orally administered with black seed oil and lettuce oil at a dose of 1 mL/kg b.wt., each) as a preventive dose. All rats were sacrificed and blood was collected for biochemical analysis and then statistically analyzed. <b>Results:</b> The rat administered with black seed and lettuce oils enhanced body weight gain, food intake and feed efficiency ratio. Moreover, exhibited a significant reduction in the liver enzymes AST, ALT, ALP and TBIL. Meanwhile, black seed and lettuce oils significantly improved kidney functions, lipid profiles and some immune biomarkers including creatine kinase (CK), Creatine Kinase-MB (CK-MB) and Lactate Dehydrogenase (LDH). <b>Conclusion:</b> This study revealed that the oils of black seed (<i>Nigella sativa</i>) and lettuce (<i>Lactuca sativa</i>) have a protective role in improving body weight gain, food intake, feed efficiency ratio, liver enzymes, kidney functions, lipid profiles and some immune biomarkers against paracetamol-induced hepatotoxicity in experimental rats.

Acetaminophen overdose causes a breach of the blood-bile barrier in mice but not in rats.

Acetaminophen (APAP) is known to cause a breach of the blood-bile barrier in mice that, via a mechanism called futile bile acid (BA) cycling, increases BA concentrations in hepatocytes above cytotoxic thresholds. Here, we compared this mechanism in mice and rats, because both species differ massively in their susceptibility to APAP and compared the results to available human data. Dose and time-dependent APAP experiments were performed in male C57BL6/N mice and Wistar rats. The time course of BA concentrations in liver tissue and in blood was analyzed by MALDI-MSI and LC-MS/MS. APAP and its derivatives were measured in the blood by LC-MS. APAP-induced liver damage was analyzed by histopathology, immunohistochemistry, and by clinical chemistry. In mice, a transient increase of BA in blood and in peri-central hepatocytes preceded hepatocyte death. The BA increase coincided with oxidative stress in liver tissue and a compromised morphology of bile canaliculi and immunohistochemically visualized tight junction proteins. Rats showed a reduced metabolic activation of APAP compared to mice. However, even at very high doses that caused cell death of hepatocytes, no increase of BA concentrations was observed neither in liver tissue nor in the blood. Correspondingly, no oxidative stress was detectable, and the morphology of bile canaliculi and tight junction proteins remained unaltered. In conclusion, different mechanisms cause cell death in rats and mice, whereby oxidative stress and a breach of the blood-bile barrier are seen only in mice. Since transient cholestasis also occurs in human patients with APAP overdose, mice are a clinically relevant species to study APAP hepatotoxicity but not rats.

Comparative analysis of gene expression between mice and humans in acetaminophen-induced liver injury by integrating bioinformatics analysis.

OBJECTIVE: Mice are routinely utilized as animal models of drug-induced liver injury (DILI), however, there are significant differences in the pathogenesis between mice and humans. This study aimed to compare gene expression between humans and mice in acetaminophen (APAP)-induced liver injury (AILI), and investigate the similarities and differences in biological processes between the two species. METHODS: A pair of public datasets (GSE218879 and GSE120652) obtained from GEO were analyzed using "Limma" package in R language, and differentially expressed genes (DEGs) were identified, including co-expressed DEGs (co-DEGs) and specific-expressed DEGS (specific-DEGs). Analysis of Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed analyses for specific-DEGs and co-DEGs. The co-DEGs were also used to construct transcription factor (TF)-gene network, gene-miRNA interactions network and protein-protein interaction (PPI) network for analyzing hub genes. RESULTS: Mouse samples contained 1052 up-regulated genes and 1064 down-regulated genes, while human samples contained 1156 up-regulated genes and 1557 down-regulated genes. After taking the intersection between the DEGs, only 154 co-down-regulated and 89 co-up-regulated DEGs were identified, with a proportion of less than 10%. It was suggested that significant differences in gene expression between mice and humans in drug-induced liver injury. Mouse-specific-DEGs predominantly engaged in processes related to apoptosis and endoplasmic reticulum stress, while human-specific-DEGs were concentrated around catabolic process. Analysis of co-regulated genes reveals showed that they were mainly enriched in biosynthetic and metabolism-related processes. Then a PPI network which contains 189 nodes and 380 edges was constructed from the co-DEGs and two modules were obtained by Mcode. We screened out 10 hub genes by three algorithms of Degree, MCC and MNC, including CYP7A1, LSS, SREBF1, FASN, CD44, SPP1, ITGAV, ANXA5, LGALS3 and PDGFRA. Besides, TFs such as FOXC1, HINFP, NFKB1, miRNAs like mir-744-5p, mir-335-5p, mir-149-3p, mir-218-5p, mir-10a-5p may be the key regulatory factors of hub genes. CONCLUSIONS: The DEGs of AILI mice models and those of patients were compared, and common biological processes were identified. The signaling pathways and hub genes in co-expression were identified between mice and humans through a series of bioinformatics analyses, which may be more valuable to reveal molecular mechanisms of AILI.

Magnesium hydride protects against acetaminophen-induced acute kidney injury by inhibiting TXNIP/NLRP3/nf-κb pathway.

Acetaminophen (APAP)-induced acute kidney injury (APAP-AKI) has turned into one of reasons for clinic obtained renal insufficiency. Magnesium hydride (MgH2), as a solid-state hydrogen source, might be potentially applied in clinical practice. The current study aimed to investigate the protective effect of MgH2 against APAP-AKI. The results showed that MgH2 improved renal function and histological injury in mice of APAP-AKI. MgH2 also had protective effects on APAP-induced cytotoxicity in HK-2 cells. In addition, the increased level of reactive oxygen species (ROS) and expressions of inflammatory cytokines (TNF-α and IL-1ß) and pro-apoptotic factors (Bad, Bax, Caspase3, and CytC) induced by APAP were downregulated with MgH2 treatment. Furthermore, the expressions of molecules related to TXNIP/NLRP3/NF-κB pathway (TXNIP, NLRP3, NF-κB p65 and p-NF-κB p65) in renal tissues and HK-2 cells were enhanced by APAP overdose, which were reduced by MgH2 administration. Collectively, this study indicated that MgH2 protects against APAP-AKI by alleviating oxidative stress, inflammation and apoptosis via inhibition of TXNIP/NLRP3/NF-κB signaling pathway.

Deficiency of ASGR1 promotes liver injury by increasing GP73-mediated hepatic endoplasmic reticulum stress.

Liver injury is a core pathological process in the majority of liver diseases, yet the genetic factors predisposing individuals to its initiation and progression remain poorly understood. Here we show that asialoglycoprotein receptor 1 (ASGR1), a lectin specifically expressed in the liver, is downregulated in patients with liver fibrosis or cirrhosis and male mice with liver injury. ASGR1 deficiency exacerbates while its overexpression mitigates acetaminophen-induced acute and CCl4-induced chronic liver injuries in male mice. Mechanistically, ASGR1 binds to an endoplasmic reticulum stress mediator GP73 and facilitates its lysosomal degradation. ASGR1 depletion increases circulating GP73 levels and promotes the interaction between GP73 and BIP to activate endoplasmic reticulum stress, leading to liver injury. Neutralization of GP73 not only attenuates ASGR1 deficiency-induced liver injuries but also improves survival in mice received a lethal dose of acetaminophen. Collectively, these findings identify ASGR1 as a potential genetic determinant of susceptibility to liver injury and propose it as a therapeutic target for the treatment of liver injury.

Inhibitory Effect of Acetaminophen on Ocular Pigmentation and its Relationship with Thyroxine in Zebrafish Embryos.

Acetaminophen (N-acetyl-p-aminophenol; APAP) is one of the most widely used analgesics. To examine the toxicity of APAP, we used zebrafish embryos as model animals to detect the effect of APAP on the thyroid system of zebrafish embryos. The zebrafish embryos were exposed to APAP from 4 h post fertilization (4 hpf) until observation. The experimental results showed that APAP caused pericardial edema and decreased pigmentation in the zebrafish embryos or larvae. The APAP treatment caused a decrease in the expression of tpo and thrß in the zebrafish at 36 and 72 hpf. The transcriptomic analysis found that APAP affected retinol metabolism, the metabolism of xenobiotics by cytochrome P450, and the tyrosine metabolism pathway. The harmful effect of APAP on zebrafish embryos might be due to its disrupting effect on the functional regulation of the thyroid hormone system.

The Modulation of Phospho-Extracellular Signal-Regulated Kinase and Phospho-Protein Kinase B Signaling Pathways plus Activity of Macrophage-Stimulating Protein Contribute to the Protective Effect of Stachydrine on Acetaminophen-Induced Liver Injury.

Stachydrine, a prominent bioactive alkaloid derived from Leonurus heterophyllus, is a significant herb in traditional medicine. It has been noted for its anti-inflammatory and antioxidant characteristics. Consequently, we conducted a study of its hepatoprotective effect and the fundamental mechanisms involved in acetaminophen (APAP)-induced liver injury, utilizing a mouse model. Mice were intraperitoneally administered a hepatotoxic dose of APAP (300 mg/kg). Thirty minutes after APAP administration, mice were treated with different concentrations of stachydrine (0, 2.5, 5, and 10 mg/kg). Animals were sacrificed 16 h after APAP injection for serum and liver tissue assays. APAP overdose significantly elevated the serum alanine transferase levels, hepatic pro-inflammatory cytokines, malondialdehyde activity, phospho-extracellular signal-regulated kinase (ERK), phospho-protein kinase B (AKT), and macrophage-stimulating protein expression. Stachydrine treatment significantly decreased these parameters in mice with APAP-induced liver damage. Our results suggest that stachydrine may be a promising beneficial target in the prevention of APAP-induced liver damage through attenuation of the inflammatory response, inhibition of the ERK and AKT pathways, and expression of macrophage-stimulating proteins.

Deficiency of purinergic P2X4 receptor alleviates experimental autoimmune hepatitis in mice.

Purinergic P2X4 receptor (P2X4R) has been shown to have immunomodulatory properties in infection, inflammation, and organ damage including liver regeneration and fibrosis. However, the mechanisms and pathophysiology associated with P2X4R during acute liver injury remain unknown. We used P2X4R-/- mice to explore the role of P2X4R in three different models of acute liver injury caused by concanavalin A (ConA), carbon tetrachloride, and acetaminophen. ConA treatment results in an increased expression of P2X4R in the liver of mice, which was positively correlated with higher levels of aspartate aminotransferase and alanine aminotransferase in the serum. However, P2X4R gene ablation significantly reduced the severity of acute hepatitis in mice caused by ConA, but not by carbon tetrachloride or acetaminophen. The protective benefits against immune-mediated acute hepatitis were achieved via modulating inflammation (Interleukin (IL)-1ß, IL-6, IL-17A, interferon-γ, tumor necrosis factor-α), oxidative stress (malondialdehyde, superoxide dismutase, glutathione peroxidase, and catalase), apoptosis markers (Bax, Bcl-2, and Caspase-3), autophagy biomarkers (LC3, Beclin-1, and p62), and nucleotide oligomerization domain-likereceptorprotein 3(NLRP3) inflammasome-activated pyroptosis markers (NLRP3, Gasdermin D, Caspase-1, ASC, IL-1ß). Additionally, administration of P2X4R antagonist (5-BDBD) or agonist (cytidine 5'-triphosphate) either improved or worsened ConA-induced autoimmune hepatitis, respectively. This study is the first to reveal that the absence of the P2X4 receptor may mitigate immune-mediated liver damage, potentially by restraining inflammation, oxidation, and programmed cell death mechanisms. And highlight P2X4 receptor is essential for ConA-induced acute hepatitis.

NEDD4 lactylation promotes APAP induced liver injury through Caspase11 dependent non-canonical pyroptosis.

Caspase-11 detection of intracellular lipopolysaccharide mediates non-canonical pyroptosis, which could result in inflammatory damage and organ lesions in various diseases such as sepsis. Our research found that lactate from the microenvironment of acetaminophen-induced acute liver injury increased Caspase-11 levels, enhanced gasdermin D activation and accelerated macrophage pyroptosis, which lead to exacerbation of liver injury. Further experiments unveiled that lactate inhibits Caspase-11 ubiquitination by reducing its binding to NEDD4, a negative regulator of Caspase-11. We also identified that lactates regulated NEDD4 K33 lactylation, which inhibits protein interactions between Caspase-11 and NEDD4. Moreover, restraining lactylation reduces non-canonical pyroptosis in macrophages and ameliorates liver injury. Our work links lactate to the exquisite regulation of the non-canonical inflammasome, and provides a basis for targeting lactylation signaling to combat Caspase-11-mediated non-canonical pyroptosis and acetaminophen-induced liver injury.

Therapeutic potentials of N-acetylcysteine immobilized polyrhodanine nanoparticles toward acetaminophen-induced acute hepatotoxicity in rat.

N-acetylcysteine (NAC) is a recommended drug for treating acetaminophen (APAP) intoxication. Due to NAC's low bioavailability, this study aimed to use polyrhodanine (PR) nanoparticles (NPs) as a drug carrier to improve the effectiveness of NAC. After preparation and characterization of NAC loaded on PR, 30 rats were randomly divided into five groups of six. The first group (control) received normal saline. Groups 2-5 were treated with normal saline, PR, NAC, and NAC loaded on PR, respectively. The treatments were started 4 h after oral administration of APAP (2000 mg kg-1 ). After 48 h, the animals were anesthetized, and liver function indices and oxidative stress were measured in tissue and serum samples. The APAP administration can increase aminotransferases and alkaline phosphatase enzymes in serum, decreasing the total antioxidant capacity and thiol groups and increasing lipid peroxidation in liver tissue. Administration of PR-NAC could effectively improve the level of serum-hepatic enzymes, total antioxidant capacity and thiol groups, lipid peroxidation, and pathological changes in liver tissue in animals poisoned with APAP. PR-NAC has a significant therapeutic effect on preventing acute hepatotoxicity caused by APAP, and its effectiveness can be associated with an improvement in the oxidant/antioxidant balance of liver tissue.

Serine synthesis via reversed SHMT2 activity drives glycine depletion and acetaminophen hepatotoxicity in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects one-third of the global population. Understanding the metabolic pathways involved can provide insights into disease progression and treatment. Untargeted metabolomics of livers from mice with early-stage steatosis uncovered decreased methylated metabolites, suggesting altered one-carbon metabolism. The levels of glycine, a central component of one-carbon metabolism, were lower in mice with hepatic steatosis, consistent with clinical evidence. Stable-isotope tracing demonstrated that increased serine synthesis from glycine via reverse serine hydroxymethyltransferase (SHMT) is the underlying cause for decreased glycine in steatotic livers. Consequently, limited glycine availability in steatotic livers impaired glutathione synthesis under acetaminophen-induced oxidative stress, enhancing acute hepatotoxicity. Glycine supplementation or hepatocyte-specific ablation of the mitochondrial SHMT2 isoform in mice with hepatic steatosis mitigated acetaminophen-induced hepatotoxicity by supporting de novo glutathione synthesis. Thus, early metabolic changes in MASLD that limit glycine availability sensitize mice to xenobiotics even at the reversible stage of this disease.

ARRB1 downregulates acetaminophen-induced hepatoxicity through binding to p-eIF2α to inhibit ER stress signaling.

Acetaminophen (APAP) stands as the predominant contributor to drug-induced liver injury (DILI), and limited options are available. ß-Arrestin1 (ARRB1) is involved in numerous liver diseases. However, the role of ARRB1 in APAP-induced liver injury remained uncertain. Wild-type (WT) and ARRB1 knockout (KO) mice were injected with APAP and sacrificed at the indicated times. The histological changes, inflammation, endoplasmic reticulum (ER) stress, and apoptosis were then evaluated. Hepatic cell lines AML-12 and primary hepatocytes were used for in vitro analyses. Systemic ARRB1-KO mice were susceptible to APAP-induced hepatotoxicity, as indicated by larger areas of centrilobular necrosis area and higher levels of ALT, AST, and inflammation level. Moreover, ARRB1-KO mice exhibited increased ER stress (indicated by phosphorylated α subunit of eukaryotic initiation factor 2 (p-eIF2α)-activating transcription factor 4 (ATF4)-CCAAT-enhancer-binding protein homologous protein (CHOP)) and apoptosis (indicated by cleaved caspase 3). Further rescue experiments demonstrated that the induction of apoptosis was partially mediated by ER stress. Overexpression of ARRB1 alleviated APAP-induced ER stress and apoptosis. Moreover, co-IP analysis revealed that ARRB1 directly bound to p-eIF2α and eIF2α. ARRB1 protected against APAP-induced hepatoxicity through targeting ER stress and apoptosis. ARRB1 is a prospective target for treating APAP-induced DILI.

Spatial analysis of renal acetaminophen metabolism and its modulation by 4-methylpyrazole with DESI mass spectrometry imaging.

Acute kidney injury (AKI) is a common complication in acetaminophen (APAP) overdose patients and can negatively impact prognosis. Unfortunately, N-acetylcysteine, which is the standard of care for the treatment of APAP hepatotoxicity does not prevent APAP-induced AKI. We have previously demonstrated the renal metabolism of APAP and identified fomepizole (4-methylpyrazole, 4MP) as a therapeutic option to prevent APAP-induced nephrotoxicity. However, the kidney has several functionally distinct regions, and the dose-dependent effects of APAP on renal response and regional specificity of APAP metabolism are unknown. These aspects were examined in this study using C57BL/6J mice treated with 300-1200 mg/kg APAP and mass spectrometry imaging (MSI) to provide spatial cues relevant to APAP metabolism and the effects of 4MP. We find that renal APAP metabolism and generation of the nonoxidative (APAP-GLUC and APAP-SULF) and oxidative metabolites (APAP-GSH, APAP-CYS, and APAP-NAC) were dose-dependently increased in the kidney. This was recapitulated on MSI which revealed that APAP overdose causes an accumulation of APAP and APAP GLUC in the inner medulla and APAP-CYS in the outer medulla of the kidney. APAP-GSH, APAP-NAC, and APAP-SULF were localized mainly to the outer medulla and the cortex where CYP2E1 expression was evident. Interestingly, APAP also induced a redistribution of reduced GSH, with an increase in oxidized GSH within the kidney cortex. 4MP ameliorated these region-specific variations in the formation of APAP metabolites in renal tissue sections. In conclusion, APAP metabolism has a distinct regional distribution within the kidney, the understanding of which provides insight into downstream mechanisms of APAP-induced nephrotoxicity.

Network pharmacology and experimental validation of effects of total saponins extracted from Abrus cantoniensis Hance on acetaminophen-induced liver injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Abrus cantoniensis Hance (AC), an abrus cantoniensis herb, is a Chinese medicinal herb used for the treatment of hepatitis. Total saponins extracted from AC (ACS) are a compound of triterpenoid saponins, which have protective properties against both chemical and immunological liver injuries. Nevertheless, ACS has not been proven to have an influence on drug-induced liver injury (DILI). AIM OF THE STUDY: This study used network pharmacology and experiments to investigate the effects of ACS on acetaminophen (APAP)-induced liver injury. MATERIALS AND METHODS: The targets associated with ACS and DILI were obtained from online databases. Cytoscape software was utilized to construct a "compound-target" network. In addition, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to analyze the related signaling pathways impacted by ACS. AutoDock Vina was utilized to evaluate the binding affinity between bioactive compounds and the key targets. To validate the findings of network pharmacology, in vitro and in vivo experiments were conducted. Cell viability assay, transaminase activity detection, immunofluorescence assay, immunohistochemistry staining, RT-qPCR, and western blotting were utilized to explore the effects of ACS. RESULTS: 25 active compounds and 217 targets of ACS were screened, of which 94 common targets were considered as potential targets for ACS treating APAP-induced liver injury. GO and KEGG analyses showed that the effects of ACS exert their effects on liver injury through suppressing inflammatory response, oxidative stress, and apoptosis. Molecular docking results demonstrated that core active compounds of ACS were successfully docked to core targets such as CASP3, BCL2L1, MAPK8, MAPK14, PTGS2, and NOS2. In vitro experiments showed that ACS effectively attenuated APAP-induced damage through suppressing transaminase activity and attenuating apoptosis. Furthermore, in vivo studies demonstrated that ACS alleviated pathological changes in APAP-treated mice and attenuated inflammatory response. Additionally, ACS downregulated the expression of iNOS, COX2, and Caspase-3, and upregulated the expression of Bcl-2. ACS also suppressed the MAPK signaling pathway. CONCLUSIONS: This study demonstrated that ACS is a hepatoprotective drug through the combination of network pharmacology and in vitro and in vivo experiments. The findings reveal that ACS effectively attenuate APAP-induced oxidative stress, apoptosis, and inflammation through inhibiting the MAPK signaling pathway. Consequently, this research offers novel evidence supporting the potential preventive efficacy of ACS.

Screening of Multitarget Compounds against Acetaminophen Hepatic Toxicity Using In Silico, In Vitro, and In Vivo Approaches.

Combination therapy and multitarget drugs have recently attracted much attention as promising tools to fight against many challenging diseases and, thus, represent a new research focus area. The aim of the current project was to screen multitarget compounds and to study their individual and combined effects on acetaminophen-induced liver injury. In this study, 2 of the best hepatoprotective multitargeting compounds were selected from a pool of 40 major compounds present in Curcuma longa and Cinnamomum zeylanicum by using molecular docking, ADMET profiling, and Pfizer's rule of five. The two selected compounds, quercetin and curcumin, showed a high binding affinity for the CYP2E1 enzyme, MAPK, and TLR4 receptors that contribute to liver injury. The candidates caused the decreased viability of cancer cell lines (HepG2 and Huh7) but showed no effect on a normal cell line (Vero). Examination of biochemical parameters (ALT, AST, ALP, and bilirubin) showed the hepatoprotective effect of the candidate drugs in comparison with the control group, which was confirmed by histological findings. Taken together, quercetin and curcumin not only satisfied the drug-like assessment criterion and proved to be multitargeting by preventing liver damage but also showed anticancer activities.

Non-canonical BIM-regulated energy metabolism determines drug-induced liver necrosis.

Paracetamol (acetaminophen, APAP) overdose severely damages mitochondria and triggers several apoptotic processes in hepatocytes, but the final outcome is fulminant necrotic cell death, resulting in acute liver failure and mortality. Here, we studied this switch of cell death modes and demonstrate a non-canonical role of the apoptosis-regulating BCL-2 homolog BIM/Bcl2l11 in promoting necrosis by regulating cellular bioenergetics. BIM deficiency enhanced total ATP production and shifted the bioenergetic profile towards glycolysis, resulting in persistent protection from APAP-induced liver injury. Modulation of glucose levels and deletion of Mitofusins confirmed that severe APAP toxicity occurs only in cells dependent on oxidative phosphorylation. Glycolytic hepatocytes maintained elevated ATP levels and reduced ROS, which enabled lysosomal recycling of damaged mitochondria by mitophagy. The present study highlights how metabolism and bioenergetics affect drug-induced liver toxicity, and identifies BIM as important regulator of glycolysis, mitochondrial respiration, and oxidative stress signaling.

Coculture model of a liver sinusoidal endothelial cell barrier and HepG2/C3a spheroids-on-chip in an advanced fluidic platform.

The liver is one of the main organs involved in the metabolism of xenobiotics and a key organ in toxicity studies. Prior to accessing the hepatocytes, xenobiotics pass through the hepatic sinusoid formed by liver sinusoidal endothelial cells (LSECs). The LSECs barrier regulates the kinetics and concentrations of the xenobiotics before their metabolic processing by the hepatocytes. To mimic this physiological situation, we developed an in vitro model reproducing an LSECs barrier in coculture with a hepatocyte biochip, using a fluidic platform. This technology made dynamic coculture and tissue crosstalk possible. SK-HEP-1 and HepG2/C3a cells were used as LSECs and as hepatocyte models, respectively. We confirmed the LSECs phenotype by measuring PECAM-1 and stabilin-2 expression levels and the barrier's permeability/transport properties with various molecules. The tightness of the SK-HEP-1 barrier was enhanced in the dynamic coculture. The morphology, albumin secretion, and gene expression levels of markers of HepG2/C3a were not modified by coculture with the LSECs barrier. Using acetaminophen, a well-known hepatotoxic drug, to study tissue crosstalk, there was a reduction in the expression levels of the LSECs markers stabilin-2 and PECAM-1, and a modification of those of CLEC4M and KDR. No HepG2/C3a toxicity was observed. The metabolisation of acetaminophen by HepG2/C3a monocultures and cocultures was confirmed. Although primary cells are required to propose a fully relevant model, the present approach highlights the potential of our system for investigating xenobiotic metabolism and toxicity.