small molecules

2026-09-27 | An in Vivo Factor Connecting Sustained Reactive Oxygen Species Capture with Rapid Disposition: Quercetin Distribution into Hemoglobin.

The in vivo antioxidant effects of freely available quercetin (Qc) on human health are poorly defined by Qc pharmacokinetics, with high binding capacity to plasma albumin, wide distribution, and a short half-life. To pharmacokinetically investigate reactive oxygen species (ROS)-scavenging pathways of Qc, we focused on in vivo upstream factors (splenic macrophage-generated ROS and deformed erythrocytes) of mouse acetaminophen (APAP)-induced liver damage. Qc co-treatment rescued the hepatic accumulation of blood cells and deformed blood cells without affecting APAP metabolite-blood cell adduct levels. It clearly reduced lipid hydroperoxidation of blood cells after APAP overdose, suggesting in vivo protective effects against splenic ROS attack. Our in vitro data with Qc derivatives showed that the 3-, 3'-, and 4'-OH groups in the Qc molecule participated in formation of the Qc-blood cell complex. 5-Hydroxyl-lacking fisetin competitively bound to Qc-binding site(s) in the mouse hemoglobin (Hb) protein molecule. When Qc was injected into mice, crude Hb strongly interacted with Qc even after 3 h, differing from rapid Qc disposition within 30 min from plasma. The Qc-Hb interaction suggested a source of the short half-life and wide distribution of Qc. Therefore, we suggest that rapid formation of the Qc-Hb protein complex is essential in determining an in vivo upstream factor connecting in vivo ROS-scavenging effects with Qc disposition. When Qc is intravenously and orally administered, the Qc-Hb protein complex rather than Qc in plasma is an important factor in analyzing Qc pharmacokinetics, leading to determination of intake safely available for human healthcare and re-evaluation of the poor Qc bioavailability reported.

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2026-09-23 | Breaking the ROS inflammation loop: a hierarchical nanozyme natural enzyme cascade for acetaminophen induced acute liver injury.

Acetaminophen (APAP) induced acute liver injury (ALI) is driven by a self amplifying cascade of reactive oxygen species (ROS) and lipid peroxidation, yet current therapies fail to interrupt this pathological loop. Here we engineer a liver targeted hierarchical nanozyme, SOD@ZIF-8/Mn3O4, which integrates natural superoxide dismutase (SOD) and Mn3O4 nanozymes within a ZIF‑8 nanoplatform. Unlike conventional single step scavengers, this system implements a catalytic hierarchy matching strategy. SOD rapidly converts O2·- to H2O2, while Mn3O4 simultaneously decomposes H2O2 and intercepts ·OH, thereby converting the entire ROS cascade into benign water. Density functional theory calculations reveal that the SOD-Mn3O4 interface lowers the energy barriers for catalase like and glutathione peroxidase like steps, establishing a synergistic channeling effect. In APAP injured AML-12 cells and a murine ALI model, the nanozyme not only alleviates oxidative stress and lipid peroxidation but also breaks the ROS inflammation feed forward loop by restoring the Keap1/Nrf2/HO‑1 redox switch and suppressing NF‑κB activation. Transcriptomic and functional analyses confirm that treatment rewires hepatic transcriptional programs associated with lipid metabolism and inflammatory signaling. This work establishes a modular nanozyme natural enzyme hybrid platform where the MOF acts as a spatiotemporal organizer, transforming the pathological ROS chain into a controlled detoxification pathway. The concept is readily extensible to other diseases driven by sequential ROS cascades, offering a paradigm shift from single enzyme mimicry to pathway reprogramming nanobiocatalysis.

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2026-09-14 | Integrated Microneedle Patches Combining PROTAC-Derived Phytochemicals, Ceria Nanozymes, and Puerarin Hydrogels for Targeted Treatment of Acetaminophen-Induced Acute Liver Injury.

Acute liver failure (ALF) is a critical clinical condition associated with rapid deterioration and substantial mortality, in which dysregulated crosstalk between oxidative stress and inflammatory cascades plays a pivotal pathogenic role. However, current therapeutic strategies remain inadequate due to the pharmacokinetic barrier of achieving efficient hepatic drug accumulation. Herein, we report a multifunctional microneedle-based therapeutic platform (MN(PUE)-Hed#3/CeO2) for precise hepatic delivery and spatiotemporal modulation of the oxidative stress-inflammation axis in ALF management. Through rational PROTAC-mediated structural optimization of hederagenin, a novel proteolysis-targeting chimera (Hed#3) was developed, which retains the intrinsic antioxidant activity of the parent compound while exhibiting enhanced protein degradation efficiency, thereby amplifying cytoprotective effects. Integration with cerium oxide nanozymes yielded a Hed#3/CeO2@PLGA nanocomposite that synergistically couples molecular antioxidation with enzymatic reactive oxygen species (ROS) scavenging. Leveraging the thermoresponsive sol-gel transition and inherent anti-inflammatory activity of puerarin (PUE), this hybrid system was incorporated into a dissolvable microneedle array for localized and controlled hepatic drug release. Comprehensive pharmacodynamic studies demonstrated that MN(PUE)-Hed#3/CeO2 markedly attenuated acetaminophen-induced hepatotoxicity via concurrent activation of the Nrf2/HO-1 antioxidant defense pathway and inhibition of NF-κB-mediated proinflammatory signaling. This work establishes a therapeutically viable and translatable strategy for ALF treatment and presents a conceptual paradigm for targeted intervention of oxidative stress-associated inflammatory diseases.

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2026-08-25 | The Bacterial Metabolite Lndole-3-Propionic Acid Alleviates Acetaminophen-Induced Acute Liver Injury Partly through the Aryl Hydrocarbon Receptor Pathway.

Acetaminophen (APAP)-induced liver injury is a major cause of acute liver injury (ALI). Indole-3-propionic acid (IPA), a gut microbiota-derived tryptophan metabolite with potent anti-inflammatory and antioxidant properties, has not been fully explored for its therapeutic potential in APAP-induced ALI. APAP-induced liver injury is often accompanied by dysregulation of both the gut microbiota and the intrahepatic immune system. In our study, IPA levels were markedly reduced in the ALI model, reflecting impaired gut tryptophan metabolism. In vitro and in vivo experiments demonstrated that IPA mitigates cytotoxicity, reduces hepatocyte apoptosis, and alleviates liver inflammation, thereby conferring significant hepatoprotective effects. The aryl hydrocarbon receptor (AhR), a key mediator of IPA's effects, was substantially downregulated in APAP-induced ALI and negatively correlated with serum AST and ALT levels, suggesting that AhR may serve as a potential early biomarker for APAP-induced hepatotoxicity. Notably, IPA treatment restored hepatic and serum AhR expression and attenuated liver injury. Collectively, these findings underscore the critical role of IPA in modulating AhR signaling and highlight its therapeutic potential in APAP-induced ALI.

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2026-08-21 | Differentiating hepatic and renal toxicity reveals CYP-independent mechanisms of acetaminophen-induced acute kidney injury.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF), with acute kidney injury (AKI) contributing substantially to morbidity and mortality in those patients. To determine whether APAP-induced AKI depends on hepatic CYP2E1-mediated bioactivation, we used CYP2E1^flox/flox^ mice treated with AAV8-TBG-Cre to selectively delete hepatic CYP2E1 while preserving renal metabolism. Male and female mice received APAP (600 mg/kg) and were evaluated up to 48 h for liver and kidney injury. Liver-specific CYP2E1 deletion reduced APAP hepatotoxicity, confirming the reduction of hepatic NAPQI formation. Despite this protection, both male and female mice treated with AAV8-TBG-Cre and APAP developed progressive renal injury, with marked increases in blood urea nitrogen (BUN) and creatinine, tubular vacuolation, and strong induction of KIM-1 and osteopontin, along with cell death at 48 h. Notably, female mice lacking renal CYP2E1 and showing no detectable renal protein adducts still progressed to AKI, demonstrating that kidney injury can occur through CYP-independent mechanisms. Given that APAP-induced AKI is a delayed injury, we further considered p-aminophenol (PAP), a deacetylation product of APAP, as a potential CYP-independent contributor. These findings support the concept that non-CYP pathways, including PAP formation, may contribute to kidney injury during the later phase of toxicity, although this pathway likely represents only one component of a multifactorial injury process. Together, these results demonstrate that APAP-induced AKI is a kidney-intrinsic process that can develop independently of both hepatic and renal CYP2E1 activity, emphasizing the need for kidney-specific therapeutic strategies for preventing APAP-induced renal injury.

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cell therapies

2026-09-03 | From pathogenesis to therapy: Extracellular vesicles orchestrate cellular crosstalk in acetaminophen hepatotoxicity.

Drug-induced liver injury (DILI) is a major cause of acute liver failure, with acetaminophen (APAP) overdose representing the leading cause of intrinsic hepatotoxicity worldwide. Although the mechanisms of APAP-induced liver injury have been extensively characterized, recent evidence highlights a critical role for extracellular vesicles (EVs) in mediating intercellular communication during hepatic stress and injury. EVs are lipid bilayer-enclosed vesicles released by hepatocytes and non-parenchymal liver cells under physiological and pathological conditions, carrying diverse bioactive cargo, including microRNAs, mRNAs, proteins, and mitochondrial components. Following APAP exposure, EV release is rapidly increased, often preceding overt hepatocellular necrosis and elevations in conventional biomarkers such as alanine aminotransferase (ALT). EVs actively contribute to the progression of liver injury by transferring stress signals that promote oxidative stress, activate c-Jun N-terminal kinase (JNK) signaling, and stimulate innate immune responses, including neutrophil recruitment and cGAS-STING-mediated inflammation. EV-associated cargo, particularly liver-specific microRNAs such as miR-122, demonstrates greater stability and diagnostic sensitivity than traditional compared serum biomarkers, supporting its utility as an early and reliable biomarker of hepatocellular injury. Beyond their diagnostic potential, EVs also exhibit therapeutic potential. Mesenchymal stromal cell-derived EVs have been shown to attenuate APAP-induced liver injury by delivering regulatory microRNAs, such as miR-186-5p, which suppresses chemokine signaling and reduces inflammatory cell infiltration. Collectively, EVs function as dynamic mediators of liver injury, serving as biomarkers and intercellular communicators, while also showing potential as therapeutic agents. A better understanding of EV biology may facilitate the development of novel diagnostic and therapeutic strategies for APAP-induced liver injury.

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2026-09-01 | MSCs overexpressing FGF21 alleviate acetaminophen-induced acute liver injury by eliciting macrophage-mediated phagocytosis.

Acetaminophen (APAP) is a widely used analgesic and antipyretic agent that is safe at therapeutic doses. However, due to its extensive misuse, APAP-induced liver injury has become a major public health concern. Although mesenchymal stem/stromal cells (MSCs) represent a promising emerging therapy for APAP-induced liver injury, considerable research has focused on enhancing their efficacy, notably through genetic modification. Fibroblast growth factor 21 (FGF21), an endocrine hormone activated by metabolic stress, is known to regulate energy homeostasis, glucose and lipid metabolism, and to promote the homing of MSCs to sites of injury. Consequently, this study was designed to determine whether genetically engineering MSCs to overexpress FGF21 (FGF21-MSCs) augments their therapeutic potential against APAP-induced acute liver injury (ALI). In this investigation, MSCs were employed as a platform for FGF21 gene delivery. The MSCs were transduced with lentiviral vectors encoding the FGF21 gene to facilitate sustained FGF21 overexpression. We subsequently assessed the therapeutic potential of these FGF21-MSCs in a murine model of APAP-induced ALI. The extent of liver injury was comprehensively evaluated. Furthermore, the underlying mechanisms were elucidated using techniques including immunohistochemistry, immunofluorescence, and flow cytometry. Our results demonstrated that FGF21-MSCs significantly enhanced the therapeutic efficacy of conventional MSCs against APAP-induced ALI via a biphasic mechanism: attenuating oxidative stress and inflammation during the acute injury phase, while actively fostering tissue repair during the subsequent regenerative phase. This protective effect is primarily mediated through the enhancement of macrophage phagocytic capacity, thereby accelerating tissue repair and regeneration. Our findings demonstrate that FGF21-MSCs significantly augment therapeutic efficacy against APAP-induced ALI in mice, thereby yielding critical insights and revealing novel therapeutic targets for ALI prevention and treatment.

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2026-07-21 | Adoptive transfer of VSIG4+ macrophages interrupts the CCL2-CCR2 inflammatory amplification loop to mitigate acetaminophen-induced acute liver injury in murine and human organoid models.

Acute liver injury (ALI) can rapidly progress to life-threatening acute liver failure. Liver transplantation remains the only definitive treatment, despite critical donor shortages. Macrophage-based therapies have shown promise in ALI but face challenges related to subset imprecision and limited humanized validation. VSIG4 is generally considered a marker of Kupffer cells; however, it is also expressed in monocyte-derived macrophages (MoMFs), and its role remains unclear during ALI. By integrating single-cell RNA sequencing (scRNA-seq) data from human and mouse liver tissues with clinical ALI tissue samples, we elucidated the dynamic changes in VSIG4+ macrophages (VSIG4+ Mφ) within the liver. We developed a reversible immunomagnetic nanoparticle system for the non-destructive isolation of viable VSIG4+ Mφ. The therapeutic efficacy and potential mechanisms of VSIG4+ Mφ were evaluated through tissue and molecular-level analyses in an acetaminophen (APAP)-induced ALI mouse model, as well as in a newly established vascularized human liver organoid ALI and monocyte chemotaxis model. scRNA-seq revealed a previously underrecognized subpopulation of VSIG4+ MoMFs, which increases following ALI, while the number of resident VSIG4+ Kupffer cells decreases significantly. Clinical ALI tissue samples also confirmed the presence of CCR2+VSIG4+ cells in the livers of patients with ALI. In the APAP-induced ALI animal model, adoptive transfer of isolated VSIG4+ MoMFs dramatically decreased serum alanine transaminase, hepatic necrosis, and apoptosis while increasing anti-inflammatory cytokines. In contrast, adoptive transfer of unselected BMDM failed to improve liver injury and instead exacerbated certain pro-inflammatory responses, including elevated TNF-α and reduced CD206 expression. Mechanistically, VSIG4+ MoMFs prevented inflammatory amplification by suppressing NF-κB-dependent CCL2 transcription, thereby disrupting the CCL2-CCR2 chemotactic axis and reducing pro-inflammatory CCR2+ monocyte and macrophage recruitment. We further developed vascularized human liver organoids and APAP-induced hepatocyte injury and monocyte chemotaxis, finding that the chemotaxis-interrupting mechanism was fully recapitulated in the human liver organoid ALI model. This study identifies VSIG4+ MoMFs as a therapeutically viable subset for ALI, with clear superiority over unselected BMDM. By blocking the CCL2-CCR2 inflammatory amplification loop, these cells attenuate liver injury in both mouse and humanized models. These consistent findings provide robust preclinical evidence to support the advancement of VSIG4+ Mφ-based immunotherapies into clinical practice.

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2026-06-18 | Vitamin A-coupled MSCs- derived extracellular vesicles relieve acute liver injury by targeting hepatic stellate cells to deliver REDD1.

Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) offer great potential for treating liver injury. However, owing to their intrinsic surface characteristics, bare EVs are non-specifically distributed in the liver tissue after systemic administration, leading to limited therapeutic efficacy. Acute liver injury, often induced by acetaminophen overdose, can progress to life-threatening fibrosis, with hepatic stellate cells (HSCs) recognized as central drivers of this pathological process. While regulated in development and DNA damage response 1 (REDD1) has demonstrated antifibrotic effects by inhibiting HSCs activation, its clinical application has been hindered by challenges in targeted delivery. Utilizing the natural affinity of vitamin A (VA) for retinol binding protein receptors on HSCs, this study addresses the critical need for targeted therapies in acute liver injury by developing a novel delivery system based on VA-conjugated EVs (V-EVs) to transport the therapeutic gene REDD1 specifically to activated HSCs (aHSCs). After loading with REDD1 (V-EVREDD1), the system showed enhanced cellular uptake in activated HSCs in vitro and effective hepatic accumulation in vivo. Treatment with V-EVREDD1 significantly suppressed HSCs activation, reduced inflammation and hepatocyte apoptosis, improved liver function, and alleviated liver fibrosis in experimental models. This work highlights a promising strategy that combines targeted vesicle delivery with gene therapy, offering a potential avenue for improving the precision and efficacy of treatments for liver injury and fibrosis.

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2026-03-18 | Paracetamol Poisoning and Liver Transplantation: An Updated Review

Introduction:: Regarding clinical recovery and survival of patients with severe paracetamol- induced acute liver failure and hepatic encephalopathy, liver transplantation is recommended as a good approach to management. This review summarizes the findings of previous studies associated with paracetamol-induced hepatotoxicity and liver transplantation. Methods:: All relevant articles (n=113) were searched systematically between the relevant record initiation and 14 October 2025 (the date of our final search), across PubMed and Web of Science. The search terms included the main concepts and their synonyms, including the problem: “paracetamol- over-dose”, “paracetamol poisoning”, the intervention “assessment of hepatotoxicity”, and the outcome “liver transplantation”, combined with the AND, OR operators. Synonyms for this concept were extracted from thesauri such as Medical Subject Headings (MeSH). Results:: An arterial pH lower than 7.3 or all three of the following: 1) International Normalized ratio of Prothrombin Time > 6.5, 2) Creatinine > 3.39 mg/dl, and a grade 3 or 4 hepatic encephalopathy should be considered as poor prognosis criteria in the patient. To avoid the need for liver transplantation associated with end-stage liver disease as the most available treatment nowadays, attention to the other new therapeutics approach such as: procalcitonin serum levels, vitamin K1, sustained c-Jun N-terminal kinase (JNK) activation, targeted metabolomics method based on the metabolic profiling of fatty acids, initial verification of keratin-18 and high-mobility group box-1 protein molecular, combination of adult stem cell transplantation with conservative medication recommended. Discussion:: The creation of N-acetyl-p-benzoquinone imine by cytochrome P450 and the generation of reactive oxygen species by paracetamol overdose could cause severe hepatic failure. The escalation in cellular and mitochondrial glutathione disulfide levels after paracetamol overdose suggests the participation of oxidative stress in the pathophysiology Conclusion:: With the advancements in technology and immunotherapies, the cost of liver transplantation continues to increase. Therefore, consideration should be given to further restrictions on paracetamol due to its availability as an over-the-counter drug.

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antibodies

2026-09-15 | The Role of Systemic Inflammation in Paracetamol Poisoning-An Experimental Model.

Accumulating evidence indicates that acute, life-threatening acetaminophen (APAP) poisoning is associated with systemic inflammation (SI). Investigating SI in experimental models holds promise for the development of pathogenetic therapies. This study aimed to evaluate SI manifestations in a murine model of APAP-induced toxic liver injury. Ten-week-old male C57Bl/6 mice received a single intraperitoneal dose of 600 mg/kg APAP, corresponding to the median lethal dose (LD50). To assess signs of multiorgan damage and SI, we performed histological examinations of internal organs as well as immunoenzymatic, hematological, and biochemical analyses of blood samples. Histological analysis revealed tissue alterations in the liver, kidneys, spleen, lungs, and heart, accompanied by functional impairment. Statistically significant changes were observed in plasma levels of tumor necrosis factor-α (TNF-α), IL-1α, IL-6, IL-10, C-reactive protein (CRP), platelet count, total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and troponin I. Severe liver damage induced by APAP was associated with a cytokine storm, coagulopathy (thrombocytopenia), systemic alterations, pulmonary edema, and multiorgan dysfunction. Collectively, these findings indicate that severe APAP-induced liver damage triggers SI as a characteristic pathological process.

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2025-11-25 | An intrabody targeting PHD2 protects mice against acetaminophen-induced liver injury by attenuating ferroptosis.

Acetaminophen (APAP) overdose has become the most common cause of acute liver failure. Ferroptosis plays a critical role in APAP-induced liver injury (AILI). Although inhibiting prolyl hydroxylase 2 (PHD2)-mediated degradation of hypoxia-inducible factor-1α (HIF-1α) can alleviate APAP hepatotoxicity, the function and underlying mechanism of PHD2/HIF-1 axis in APAP-induced ferroptosis are not fully understood. Here, we developed a novel anti-PHD2 cytoplasmic intrabody (INP2) and elucidated the anti-ferroptosis mechanism by which INP2 inhibits APAP hepatotoxicity. Our findings demonstrated that INP2 specifically recognized PHD2 and disrupted its interaction with HIF-1α, inhibiting HIF-1α hydroxylation and increasing HIF-1α stability. In the murine AILI model, INP2 pretreatment significantly protects against AILI by attenuating ferroptosis via restoring redox homeostasis, attenuating malondialdehyde and ROS accumulation, and decreasing ferrous iron content. Importantly, INP2 enhanced glutathione biosynthesis and eliminated oxidative stress by upregulating SLC7A11 and glutathione peroxidase 1 (GPX1), thereby decreasing cytotoxicity in APAP/H2O2-exposed hepatocytes. GPX1 knockdown markedly impaired INP2-mediated hepatoprotection and anti-ferroptosis by suppressing glutathione-dependent reduction of cytotoxic lipid hydroperoxides including 12-HPETE. Collectively, a PHD2-targeted intrabody protects acetaminophen-induced liver injury by attenuating ferroptosis via regulating PHD2/HIF-1α/GPX1 axis, which enhances glutathione biosynthesis, suppresses oxidative stress and ferroptosis, offering a potential strategy for the prevention and treatment of AILI.

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2025-08-17 | Platelets mediate neutrophil infiltration and exacerbate liver injury and sinusoidal endothelial cell damage after a severe acetaminophen overdose in mice.

Acetaminophen (APAP) overdose, the leading cause of acute liver failure in the US, is accompanied by hepatocyte necrosis and liver sinusoidal endothelial cell (LSEC) damage, with hepatic neutrophil infiltration and platelet deposition. However, the exact role played by platelets in the pathophysiology is not fully understood. To investigate this, we depleted platelets in C57Bl/6 J mice by injecting 2 mg/kg anti-CD41 antibody or IgG control 12 and 2 h before a moderate (300 mg/kg) or severe (600 mg/kg) APAP overdose. Platelet depletion did not affect liver injury as measured by plasma ALT levels and hepatic areas of necrosis 24 h after a moderate APAP overdose, but reduced hepatic neutrophil infiltration. After a severe overdose, platelet depletion caused a significant reduction in hepatic neutrophil infiltration, but contrary to the moderate overdose, it also significantly reduced liver injury. These findings were confirmed with a second platelet-depleting antibody (CD42b antibody). These results corroborate our previous findings, which showed that neutrophils exacerbate liver injury only after a severe APAP overdose but not a moderate one. Furthermore, severe APAP overdose induced LSEC injury 24 h after APAP, which was significantly reduced in platelet-depleted mice as measured by intrahepatic hemorrhage, increased hepatic von Willebrand factor deposition, and elevated plasma levels of hyaluronan. Thus, platelets contribute to endothelial cell injury and mediate neutrophil recruitment, which aggravates liver injury after a severe APAP overdose, underscoring the role of platelets in the pathophysiology.

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2025-04-12 | Modular Nanotransporters Containing Keap1 Monobodies Are Capable of Reducing the Toxic Effect of Acetaminophen on the Liver of Mice.

Previously, we created a modular nanotransporter (MNT) containing a monobody to Keap1, an intracellular protein inhibitor of the Nrf2 transcription factor that controls cellular protection from oxidative stress and is capable of interacting with Keap1 in hepatocytes and protect these cells from the effects of hydrogen peroxide. Oxidative liver damage by acetaminophen was used as a model to study the antitoxic effect of this MNT. Intraperitoneal injection of acetaminophen to mice resulted in an increase in the level of alanine aminotransferase and aspartate aminotransferase in the blood, as well as in liver edema. A significant decrease in the level of these enzymes in the blood, along with a decrease in liver edema, was observed after preliminary intravenous administration of MNT 2 h before the acetaminophen injection. The results obtained can be used as a basis for developing drugs to treat diseases associated with oxidative stress.

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2025-01-20 | Dynamic analysis of intrahepatic T cells reveals a unique group of restorative Cxcr3+ tissue-resident CD4 T cells in acute liver injury.

Acetaminophen (APAP) stands as one of the most prevalent triggers of drug-induced acute liver injury (ALI). The intricate modulation of immune system activation and inflammatory cascades by hepatic immune cells is paramount in managing liver injury and subsequent restoration. In this study, we employed an integrative approach that fused our proprietary flow cytometry analyses across various time points post-APAP injury with publicly available single-cell RNA sequencing (scRNA-seq) datasets, encompassing time-series data from liver tissue of mice subjected to APAP intoxication. This allowed us to delve into the dynamics of T cell profiles during APAP-induced ALI. Our comprehensive analyses unveiled the intricate temporal shifts in intrahepatic T cell populations across different phases following APAP-induced ALI. Notably, we observed a persistent augmentation of intrahepatic CD4+ T cells post-APAP injury. Amongst these, a distinct population of restorative Cxcr3+ tissue-resident CD4+ T cells emerged. Inhibition of CXCR3 using a neutralizing antibody exacerbated APAP-induced liver function impairment and hepatocyte death. Furthermore, we identified that the Cxcr3+ tissue-resident CD4+ T cells were tightly regulated by intrahepatic ''Lgals9-Cd45'' and 'CXCL13-Cxcr3' signaling pathways. These discoveries underscore the novel protective function of CXCR3, a vital biological macromolecule, in mitigating APAP-induced ALI, and may shed lights on new therapeutic strategies targeting this condition.

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proteins

2026-09-15 | Annexin A5 Maintains Mitochondrial Integrity by Inhibiting mPTP Opening to Protect Against Acetaminophen-Induced Acute Liver Injury.

Acetaminophen (APAP) represents a major cause of drug-induced liver injury (DILI), and effective pharmacological interventions remain limited. Annexin A5 (AnxA5), a Ca2+-dependent phospholipid-binding protein, participates in diverse biological processes related to tissue repair. In this study, we evaluated AnxA5 expression in APAP-challenged mouse livers and clinical samples from patients with liver injury. Using hepatic cell lines AML12 and HepG2, we performed overexpression-based functional assays to assess the cytoprotective effects of AnxA5 against APAP toxicity. Co-immunoprecipitation assays were applied to characterize protein interactions, and mitochondrial functional parameters were measured to dissect the underlying molecular mechanism. We found that AnxA5 was significantly upregulated in both APAP exposed mice and APAP DILI patients. Cellular functional assays showed that AnxA5 overexpression mitigated APAP triggered cytotoxicity in AML12 and HepG2 cells. Mechanistically, AnxA5 bound to voltage dependent anion channel 1 (VDAC1), restrained VDAC1 mediated mitochondrial Ca2+ influx, and suppressed VDAC1 oligomerization, which further inhibited mitochondrial permeability transition pore (mPTP) opening. In an APAP-induced liver injury mouse model, exogenous recombinant AnxA5 treatment maintained mitochondrial integrity and ameliorated hepatic inflammation and liver damage. Collectively, our data reveal AnxA5 as an endogenous mitochondrial protective factor and support its therapeutic potential against APAP-induced liver injury.

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2026-05-01 | DNAJB4/HLJ1 protects against acetaminophen-induced liver injury by attenuating ER stress via HSP70.

Drug-induced liver injury (DILI) remains a leading cause of acute liver failure; however, current clinical strategies lack reliable biomarkers, predictors of susceptibility, and effective therapeutic interventions. Among these etiologies, acetaminophen (APAP) overdose is the most common cause of DILI worldwide. Heat shock proteins (HSPs), particularly members of the HSP40 family, are central regulators of cellular stress responses, yet the specific role of DNAJB4/HLJ1 in APAP-induced hepatotoxicity remains poorly defined. To address this gap, we employed DNAJB4/HLJ1-deficient mice (Dnajb4-/-) to investigate the function of DNAJB4/HLJ1 in APAP-induced liver injury. Following APAP administration (> 400 mg/kg), Dnajb4-/- exhibited exacerbated hepatic necrosis, elevated liver enzymes, and enhanced c-jun/JNK activation compared with Dnajb4+/+ controls. Metabolic profiling revealed altered APAP metabolism, with reduced detoxification products and excessive oxidative metabolites, and pronounced glutathione (GSH) depletion. Transcriptomic analysis implicated DNAJB4/HLJ1 in metabolism, protein folding, and endoplasmic reticulum (ER) stress via interaction with HSP70. Consistently, ATF6, XBP1, and CHOP expression confirmed aggravated ER stress in Dnajb4-/- livers. AlphaFold-Multimer modeling and co-immunoprecipitation validated physical interaction between DNAJB4/HLJ1 and HSP70. Restoration of DNAJB4/HLJ1 expression attenuated ER stress, c-jun/JNK activation, and liver injury, while pharmacological inhibition of ER stress confirmed its mechanistic involvement. Collectively, these findings identify DNAJB4/HLJ1 as a previously unrecognized regulator of stress signaling in APAP-induced hepatotoxicity. By modulating ER stress within the integrated cellular stress network, DNAJB4/HLJ1 limits injury progression and promotes hepatocellular resilience, highlighting its potential as a novel therapeutic target for preventing or mitigating DILI.

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2026-04-22 | Protective effect of latex proteins from Plumeria pudica against acetaminophen-induced acute liver injury.

The protein fraction from Plumeria pudica latex (LPPp) was evaluated against APAP-induced liver injury in mice. Key measurements included total leukocyte count, liver weight, AST, ALT, liver levels of MDA, GSH, SOD, MPO, NO3/NO2 concentration, cytokines TNF-α, IL-β, IL-6, IL-10, IFN- γ, and CXCL1. Histopathological and immunohistochemical analysis were conducted, and proteins in LPPp were identified by mass spectrometry. Molecular docking analyses were performed with LPPp proteins and APAP, NAPQI, TNF-α, and IFN-α. LPPp reduced liver weight, ALT, and AST levels, and prevented the decrease in total leukocyte count, indicating protection against liver injury. It also reduced MDA levels and preserved GSH and SOD levels, showing its antioxidant properties. LPPp significantly inhibited MPO and NO3/NO2 levels, reducing inflammation. LPPp decreased levels of TNF-α, IL-β, IL-6, IFN-γ, and CXCL1, while IL-10 levels remained elevated, suggesting an anti-inflammatory response. Histological analysis showed preserved liver tissue, and anti-Ly6C immunostaining indicated reduced neutrophil accumulation. Molecular docking revealed that latex cysteine proteases had a higher affinity for APAP and NAPQI, while a proteinase inhibitor showed affinity for CYP2EI, TNF-α, and IFN-α. These findings suggest that LPPp protects against APAP hepatotoxicity by interfering with APAP and NAPQI metabolism, reducing oxidative stress and inflammation, thus preventing hepatocyte damage.

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2026-03-16 | PLGA nanoparticles encapsulating fibroblast growth factor 21 alleviate acetaminophen-induced acute liver injury.

The short half-life of fibroblast growth factor 21 (FGF21) in vivo limits its clinical application in acetaminophen (APAP)-induced acute liver injury (ALI). This study aimed to construct a novel PLGA-based nanoparticle delivery system for FGF21 and to investigate its protective effects and underlying mechanisms in APAP-induced ALI. PLGA-FGF21 nanoparticles were prepared using a double-emulsion solvent evaporation method and characterized by transmission electron microscopy and Zetasizer Nano for morphology, size, and stability. Their release profile and cytotoxicity were evaluated in vitro using AML12 cells, while hepatoprotective effects were assessed in an APAP-induced ALI mouse model. Histopathology, biochemical assays, immunofluorescence, and Western blot were employed to examine liver function, oxidative stress, and molecular mechanisms. The nanoparticles displayed uniform spherical morphology with an average size of 222.04 ± 1.08 nm and remained stable for 7 days in aqueous suspension. Encapsulation conferred sustained-release properties, and the particles showed no cytotoxicity while maintaining biological activity for at least 28 days. In vivo, PLGA-FGF21 significantly alleviated APAP-induced liver damage, reduced serum ALT and AST, and exhibited superior hepatoprotective efficacy compared with free FGF21. Furthermore, it suppressed inflammatory responses by decreasing TNF-α and MDA levels, while enhancing antioxidant defenses through increased SOD activity. Mechanistically, PLGA-FGF21 activated the PGC-1α/Nrf2/HO-1 signaling pathway, contributing to enhanced anti-inflammatory and antioxidant effects. PLGA-FGF21 nanoparticles provide potent protection against APAP-induced ALI by reducing inflammation and oxidative stress, which may be associated with activation of the PGC-1α/Nrf2 signaling pathway, offering a promising strategy for FGF21-based therapy in liver injury.

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2026-03-01 | MOTS-c Protects Against Acetaminophen-induced Liver Injury through the MAPK Signaling Pathway.

Acetaminophen (APAP)-induced liver injury (AILI) is a leading cause of acute liver failure worldwide, but effective therapeutic strategies are still lacking. MOTS-c, a mitochondrial-derived peptide, has demonstrated hepatoprotective properties in models of nonalcoholic steatohepatitis (NASH) and hepatitis B virus (HBV) infection. This study aims to explore the role and underlying mechanisms of MOTS-c in AILI. An AILI model was established in male C57BL/6 mice via intraperitoneal (i.p.) injection of APAP (300 mg/kg). The therapeutic potential of MOTS-c and its mechanisms were assessed using behavioral tests, qPCR, western blotting, ELISA, immunohistochemistry, immunofluorescence, and TUNEL staining. MOTS-c levels in both plasma and liver tissues were significantly reduced in APAPinduced AILI mice compared with controls. Administration of MOTS-c via i.p. injection markedly attenuated APAP-induced increases in AST and ALT levels, histopathological liver damage, and other liver injury markers. MOTS-c treatment suppressed the release of pro-inflammatory factors (TNF-α, IL-1β, IL-6, and COX-2) and macrophage infiltration induced by APAP. Furthermore, MOTS-c treatment significantly restored GSH content, diminished reactive oxygen species (ROS) production, and oxidative stress. TUNEL staining confirmed that increased apoptosis in APAPtreated livers was significantly attenuated by MOTS-c, which are key contributors to hepatocyte death and liver injury. Mechanistic studies revealed that MOTS-c inhibited APAP-induced phosphorylation of MAPK pathway components, including ERK, JNK, and p38. The protective effects of MOTS-c on serum ALT and AST levels were abolished by co-treatment with inhibitors of ERK, JNK, and p38. This study reveals that the mitochondrial peptide MOTS-c can alleviate drug-induced liver injury by suppressing oxidative stress and inflammation via the MAPK pathway. This positions MOTS-c as a promising therapeutic candidate for treating APAP-induced liver injury. This study demonstrates that administering MOTS-c effectively protects against APAP-induced liver injury in mice. The protective mechanism involves suppressing the damaging MAPK signaling pathway (ERK, JNK, p38), which in turn reduces oxidative stress, inflammation, and cell death.

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other

2025-08-27 | CpG-oligodeoxynucleotides challenged macrophages ameliorate acetaminophen induced liver injury by activating TLR9/IRG1/itaconate metabolic pathway.

Acetaminophen, or N-acetyl-para-aminophenol (APAP), causes severe liver damage and acute liver failure when overdosed. Oligodeoxynucleotides containing CpG motifs (CpG ODN) can regulate the function of macrophages, which play an important role in drug-induced liver injury. It is unclear whether CpG ODN-treated macrophages play an immune regulation role in APAP-induced liver injury. In the present study, we aim to explore the role of CpG ODN-activated macrophages in APAP-induced liver injury and the underlying mechanism in protecting against the cytotoxicity of APAP. In vivo, C57BL/6 mice were treated with APAP (300 mg/Kg) or/and CpG ODN (ODN 1826, 1.65 mg/Kg) by intraperitoneal injection, then survival rate, histopathological evaluation, and inflammatory factors were observed to ascertain the protective effect of CpG ODN. Then, CpG ODN-treated macrophages were reinfused into the animal model to determine the effector cells. In vitro, RNA sequencing and untargeted metabolomics detection were performed to illustrate the underlying mechanism. Last, Acod1 siRNA interference was used to clarify the role of IRG1 in resistance to APAP cytotoxicity by ROS and apoptosis indicator detections. We found that CpG ODN showed a protective effect against APAP cytotoxicity by stimulating macrophages rather than hepatic parenchymal cells. In particular, reinfusion of CpG ODN-treated macrophages to mice can alleviate APAP-induced liver injury. Transcriptome and metabolome analysis revealed that the expression of aconitate decarboxylase 1 (Acod1; also known as immune responsive gene 1, IRG1) and the metabolite itaconate generated by IRG1 catalysis increased after CpG ODN stimulation. In addition, we found that the mechanism of this protective effect is ascribed to the increased expression of Acod1 and the antioxidative function of itaconate by the activation of the TLR9/NF-κB signaling pathway. CpG ODN alleviated liver injury induced by APAP through the activation of the TLR9/NF-κB signaling pathway in macrophages, upregulating the expression of IRG1 protein, promoting the production of endogenous metabolite itaconate, and inhibiting macrophage apoptosis which was regulated by upregulating the expression of Nrf2 to inhibit ROS production. This study sheds new light on CpG ODN as a therapeutic strategy in resistance to APAP-induced liver injury.

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2025-07-02 | miR-375 protects against acetaminophen-induced acute liver failure by orchestrating pharmacogene expression.

Acetaminophen (APAP) overdose is a leading cause of acute liver failure (ALF), primarily through the excessive production of N-acetyl-p-benzoquinone imine (NAPQI). N-acetylcysteine (NAC) is the Food and Drug Administration-approved treatment for APAP overdose, but there is a growing interest in microRNAs as potential therapeutic agents. We delivered miR-375 ectopically via a liver-tropic adeno-associated virus serotype 8 (AAV8) and demonstrated its potent protection in a murine model of APAP overdose-induced ALF. Slc16a2, Cyb5b, and Acsl5 were identified as critical targets acting synergistically to mitigate toxicity. Liver transcriptome revealed that miR-375 overexpression or silencing of the targets of miR-375 increased Gstm3 expression in mice. AAV8-mediated Gstm3 expression protects against APAP-ALF, and the protection was further enhanced by disrupting the expression of Cyp2e1. Additionally, CYP2E1 and GSS, which contribute to APAP detoxification, were down- and upregulated by miR-375, respectively. These findings suggest that miR-375 prevents APAP-ALF by orchestrating the expression of pharmacogenes and enhancing glutathione synthesis. We conclude that miR-375 and its targets are promising therapeutic targets for APAP-ALF.

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2025-02-28 | Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure, with oxidative stress being a critical factor in this process. Glutathione (GSH) plays a vital defensive role. Activation of nuclear factor erythroid 2 like 2 (Nrf2) pathway mitigates APAP-induced liver damage by promoting GSH biosynthesis and enhancing drug detoxification. Although the role of B cell leukemia/lymphoma 3 (Bcl3) in regulating inflammatory responses, cellular oncogenesis, and immune balance is well-documented, its function in APAP-induced liver injury remains unclear. We employed liver-specific Bcl3 knockout (Bcl3hep-/-) mice and adeno-associated virus (AAV)-8-mediated Bcl3 overexpression (AAV-Bcl3) mice to model APAP-induced liver injury. Liver damage was assessed through hematoxylin and eosin staining and serum alanine aminotransferase and aspartate aminotransferase measurements. The interaction between Bcl3 and Nrf2 was examined using immunofluorescence and co-immunoprecipitation assays. Our study reveals a significant upregulation of Bcl3 expression in the livers of male mice following APAP administration, suggesting Bcl3's potential involvement in this pathological process. In Bcl3hep-/- mice, a reduced severity of liver damage was observed at both 6 and 24 hours post-APAP treatment compared with controls. Notably, Bcl3-deficient mice exhibited accelerated GSH replenishment due to the rapid induction of Gclc and Gclm genes following 6 hours of APAP exposure. Through immunofluorescence and co-immunoprecipitation analyses, we identified an interaction between Bcl3 and Nrf2. The loss of Bcl3 enhanced Nrf2 translocation upon APAP challenge, leading to the upregulation of antioxidant gene expression. These findings suggest that Bcl3 knockout alleviates oxidative stress resulting from APAP overdose. We uncovered a previously uncharacterized role of Bcl3 in APAP-induced liver injury, emphasizing the role of the Bcl3-Nrf2 axis in oxidative stress-related liver damage.

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2024-07-28 | Adenosine kinase protects against acetaminophen-induced acute liver injury by activating autophagy in hepatocytes.

Acute liver injury (ALI) is a common life-threatening condition with a high mortality rate due to liver disease-related death. However, current therapeutic interventions for ALI remain ineffective, and the development of effective novel therapies is urgently needed. Liver samples from patients with drug-induced ALI were collected to detect adenosine kinase (ADK) expression. Male C57BL/6 J mice, hepatocyte-specific ADK knockout (ADKHKO) mice, and their controls (ADKf/f) were exposed to acetaminophen (APAP) and other treatments to investigate the mechanisms of APAP-related ALI. ADK expression was significantly decreased in APAP-injured livers. Hepatocyte-specific ADK deficiency exacerbated APAP-induced ALI, while a gain-of-function approach delivering AAV-ADK, markedly alleviated APAP-induced ALI, as indicated by changes in alanine aminotransferases (ALT) levels, aspartate aminotransferase (AST) levels, neutrophil infiltration and hepatocyte death. This study showed that ADK played a critical role in ALI by activating autophagy through two signaling pathways, the adenosine monophosphate-activated protein kinase (AMPK)-mTOR pathway and the adenosine receptor A1 (ADORA1)-Akt-mTOR pathway. Furthermore, we found that metformin upregulated ADK expression in hepatocytes and protected against APAP-induced ALI. These results demonstrate that ADK is critical in protecting against APAP-induced ALI and that developing therapeutics targeting ADK-adenosine-ADORA1 is a new approach for ALI treatment. Metformin is a potential candidate for preventing ALI by upregulating ADK.

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2024-03-26 | Deletion of hepatocyte cysteine dioxygenase type 1, a bile acid repressed gene, enhances glutathione synthesis and ameliorates acetaminophen hepatotoxicity.

Liver is a major organ that metabolizes sulfur amino acids cysteine, which is the substrate for the synthesis of many essential cellular molecules including GSH, taurine, and coenzyme A. Bile acid-activated farnesoid x receptor (FXR) inhibits cysteine dioxygenase type 1 (CDO1), which mediates hepatic cysteine catabolism and taurine synthesis. To define the impact of bile acid inhibition of CDO1 on hepatic sulfur amino acid metabolism and antioxidant capacity, we developed hepatocyte-specific CDO1 knockout mice (Hep-CDO1 KO) and hepatocyte specific CDO1 transgenic mice (Hep-CDO1 Tg). Liver metabolomics revealed that genetic deletion of hepatic CDO1 reduced de novo taurine synthesis but had no impact on hepatic taurine abundance or bile acid conjugation. Consistent with reduced cysteine catabolism, Hep-CDO1 KO mice showed increased hepatic cysteine abundance but unaltered methionine cycle intermediates and coenzyme A synthesis. Upon acetaminophen overdose, Hep-CDO1 KO mice showed increased GSH synthesis capacity and alleviated liver injury. In contrast, hepatic CDO1 overexpression in Hep-CDO1 Tg mice stimulated hepatic cysteine to taurine conversion, resulting in reduced hepatic cysteine abundance. However, Hep-CDO1 Tg mice and WT showed similar susceptibility to acetaminophen-induced liver injury. Hep-CDO1 Tg mice showed similar hepatic taurine and coenzyme A compared to WT mice. In summary, these findings suggest that bile acid and FXR signaling inhibition of CDO1-mediated hepatic cysteine catabolism preferentially modulates hepatic GSH synthesis capacity and antioxidant defense, but has minimal effect on hepatic taurine and coenzyme A abundance. Repression of hepatic CDO1 may contribute to the hepatoprotective effects of FXR activation under certain pathologic conditions.

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