Acetaminophen-induced Liver Injury In Mice Research Articles

Wogonin mitigates acetaminophen-induced liver injury in mice through inhibition of the PI3K/AKT signaling pathway

Ethnopharmacological relevanceScutellaria baicalensis Georgi (SBG), a widely used traditional Chinese medicine, exhibits anti-inflammatory and antioxidant properties. Wogonin is one of the primary bioactive components of SBG. Acetaminophen (APAP)-induced liver injury (AILI) represents a prevalent form of drug-induced liver damage and is primarily driven by inflammatory responses and oxidative stress. Aim of studyTo investigate the therapeutic effects of Wogonin on AILI and the underlying mechanisms. Materials and methodsC57BL/6 J mice were pre-treated with Wogonin (1, 2.5, and 5 mg/kg bodyweight) for 3 days, followed by treatment with APAP (300 mg/kg bodyweight). The serum and liver tissue samples were collected at 24 h post-APAP treatment. Bone marrow-derived macrophages and RAW264.7 cells were cultured and pre-treated with Wogonin (5, 10, and 20 μM) for 30 min, followed by stimulation with lipopolysaccharide (LPS; 100 ng/mL) for 3 h. To examine the role of the PI3K/AKT signaling pathway in the therapeutic effect of Wogonin on AILI, mice and cells were treated with LY294002 (a PI3K inhibitor) and MK2206 (an AKT inhibitor). ResultsWogonin pre-treatment dose-dependently alleviated AILI in mice. Additionally, Wogonin suppressed oxidative stress and inflammatory responses. Liver transcriptome analysis indicated that Wogonin primarily regulates immune function and cytokines in AILI. Wogonin suppressed inflammatory responses of macrophages by inhibiting the PI3K/AKT signaling pathway. Consistently, Wogonin exerted therapeutic effects on AILI in mice through the PI3K/AKT signaling pathway. ConclusionsWogonin alleviated AILI and APAP-induced hepatotoxicity in mice through the PI3K/AKT signaling pathway.

The thrombopoietin mimetic JNJ-26366821 reduces the late injury and accelerates the onset of liver recovery after acetaminophen-induced liver injury in mice.

Acetaminophen (APAP)-induced hepatotoxicity is comprised of an injury and recovery phase. While pharmacological interventions, such as N-acetylcysteine (NAC) and 4-methylpyrazole (4-MP), prevent injury there are no therapeutics that promote recovery. JNJ-26366821 (TPOm) is a novel thrombopoietin mimetic peptide with no sequence homology to endogenous thrombopoietin (TPO). Endogenous thrombopoietin is produced by hepatocytes and the TPO receptor is present on liver sinusoidal endothelial cells in addition to megakaryocytes and platelets, and we hypothesize that TPOm activity at the TPO receptor in the liver provides a beneficial effect following liver injury. Therefore, we evaluated the extent to which TPOm, NAC or 4-MP can provide a protective and regenerative effect in the liver when administered 2h after an APAP overdose of 300mg/kg in fasted male C57BL/6J mice. TPOm did not affect protein adducts, oxidant stress, DNA fragmentation and hepatic necrosis up to 12h after APAP. In contrast, TPOm treatment was beneficial at 24h, i.e., all injury parameters were reduced by 42-48%. Importantly, TPOm enhanced proliferation by 100% as indicated by PCNA-positive hepatocytes around the area of necrosis. When TPOm treatment was delayed by 6h, there was no effect on the injury, but a proliferative effect was still evident. In contrast, 4MP and NAC treated at 2h after APAP significantly attenuated all injury parameters at 24h but failed to enhance hepatocyte proliferation. Thus, TPOm arrests the progression of liver injury by 24h after APAP and accelerates the onset of the proliferative response which is essential for liver recovery.

Preventive and Therapeutic Effects of Epicatechin on Acetaminophen-Induced Liver Injury in Mice

Background: Nowadays, the use of over-the-counter drugs such as acetaminophen (APAP) may cause severe liver injury, which can occur not only in high doses but also in therapeutic doses due to nutritional deficiency, alcoholism, or using cytochrome p450 inducers. Objectives: In this study, the protective effect of epicatechin (EPC) was evaluated against APAP-induced hepatotoxicity to find an effective and inexpensive therapy. Methods: The animals were divided into preventive and therapeutic groups. In the preventive study, the animals received EPC (25, 50, and 100 mg/kg/day) for five days, and the last dose was administered 1 hour before APAP (400 mg/kg). In the therapeutic groups, the animals received EPC just before and 2 hours after the APAP injection. All the animals were killed, and blood and liver samples were taken for further analysis. The liver pathology, enzymes, and oxidant, antioxidant, inflammatory, and anti-inflammatory factors were evaluated. Results: EPC significantly decreased the serum activity level of the liver biomarkers ALT and AST in the APAP-treated mice. Furthermore, the hepatic levels of thiobarbituric acid-reactive substances were noticeably lowered, and the levels of total thiol and catalase activity increased significantly with EPC. Histopathological results were strongly consistent with those of the biochemical estimations. The most effective dose was observed at EPC 100 mg/kg, and the therapeutic groups showed better results than the preventive groups. Conclusions: EPC attenuated the liver toxicity in the mice by suppressing oxidative stress and can be considered a preventive and therapeutic agent for inhibiting and resolving the liver damage induced by APAP.

High-throughput screening of novel TFEB agonists in protecting against acetaminophen-induced liver injury in mice

Macroautophagy (referred to as autophagy hereafter) is a major intracellular lysosomal degradation pathway that is responsible for the degradation of misfolded/damaged proteins and organelles. Previous studies showed that autophagy protects against acetaminophen (APAP)-induced injury (AILI) via selective removal of damaged mitochondria and APAP protein adducts. The lysosome is a critical organelle sitting at the end stage of autophagy for autophagic degradation via fusion with autophagosomes. In the present study, we showed that transcription factor EB (TFEB), a master transcription factor for lysosomal biogenesis, was impaired by APAP resulting in decreased lysosomal biogenesis in mouse livers. Genetic loss-of and gain-of function of hepatic TFEB exacerbated or protected against AILI, respectively. Mechanistically, overexpression of TFEB increased clearance of APAP protein adducts and mitochondria biogenesis as well as SQSTM1/p62-dependent non-canonical nuclear factor erythroid 2-related factor 2 (NRF2) activation to protect against AILI. We also performed an unbiased cell-based imaging high-throughput chemical screening on TFEB and identified a group of TFEB agonists. Among these agonists, salinomycin, an anticoccidial and antibacterial agent, activated TFEB and protected against AILI in mice. In conclusion, genetic and pharmacological activating TFEB may be a promising approach for protecting against AILI.

The Protective Effect and Mechanism of a Phytochemical Extract from the Wild Vegetable Shutou (Crateva unilocularis Buch.) against Acetaminophen-Induced Liver Injury in Mice.

Acetaminophen (APAP) abuse is a common public health problem which can cause severe liver damage. However, strategies for dealing with this situation safely and effectively are very limited. The goal of the current work was to evaluate the protection and potential molecular mechanisms of an ethanol extract from shoots of the wild vegetable shutou (Crateva unilocularis Buch.) (ECS) against APAP-induced liver damage in mice. Mice orally received ECS for seven days (300 or 600 mg/kg b.w. per day) before being intraperitoneally injected with APAP (250 mg/kg). Results exhibited that ECS obviously decreased the content of alkaline phosphatase, alanine aminotransferase, aspartate transaminase, and malondialdehyde (p < 0.05). Catalase and superoxide dismutase were notably restored (p < 0.05), and the content of reduced glutathione was obviously increased (p < 0.05). Moreover, ECS significantly inhibited the secretion of interleukin-1β and tumor necrosis factor-α (p < 0.05). Further analyses of the mechanisms showed that ECS may alleviate oxidative stress in the liver by increasing the expression of the nuclear factor erythroid-2-related factor 2 and NADH quinone oxidoreductase 1 proteins, and may suppress liver inflammation by inhibiting the expression of the phosphorylated-inhibitor kappa B alpha/inhibitor kappa B alpha, phosphorylated-nuclear factor κB/nuclear factor κB, and cyclooxygenase-2 proteins. Meanwhile, ECS inhibited hepatocyte apoptosis by enhancing B-cell lymphoma gene 2 and suppressing Bcl-2-associated X protein. In summary, ECS may be used as a dietary supplement to prevent the liver damage caused by APAP abuse.

Efficacy of oltipraz in preventing acetaminophen-induced liver injury in mice.

Oltipraz (OPZ) is a synthetic dithiolethione with potential as a cancer chemopreventive agent, which can work by inducing detoxification enzymes. OPZ is an activator of nuclear factor erythroid 2-related factor 2 (Nrf2), suggesting its involvement in enzyme induction and possible protection against drug-induced liver injury. In this study, we present OPZ-mediated protection of mice against acetaminophen (APAP)-induced liver injury and discuss its possible contributing factors. Overnight-fasted male CD-1 mice were administered APAP intraperitoneally, and some mice were administered OPZ 16h before APAP. Hepatotoxicity was assessed by measuring serum alanine aminotransferase leakage and histopathological evaluation. The hepatic mRNA expressions of CYP2E1, glutamate cysteine ligase (GCL), and NAD(P)H:quinone oxidoreductase (NQO1) were measured by real-time reverse-transcription polymerase chain reaction. OPZ protected mice from APAP-induced liver injury in a dose-dependent manner, but did not alter hepatic glutathione (GSH) content or GCL expression in control mice, indicating that its hepatoprotective effect is not due to changes in basal GSH levels. OPZ did not affect CYP2E1 expression or APAP-induced early GSH depletion, suggesting it does not inhibit the metabolic activation of APAP to produce N-acetyl-p-benzoquinone imine. In contrast, after GSH depletion, OPZ accelerated hepatic GSH recovery. APAP significantly increased GCL expression during liver injury, but OPZ treatment only led to additional NQO1 expression. This suggests that NQO1 is responsible for the enhanced GSH recovery and protection against APAP-induced liver injury seen in OPZ-treated mice. In summary, OPZ protects against APAP-induced liver injury by inducing NQO1 expression and resulting in improved GSH recovery.

Ameliorative Potentials of Vernonia amygdalina on Acetaminophen-induced Acute Liver Injury in Mice

Aims: To investigate the ameliorative potentials of Vernonia amygdalina on acetaminophen-induced liver injury in mice.&#x0D; Methodology: Acetaminophen-induced acute liver injury was conducted on Swiss-albino mice weighing 25 - 30 g. Group A was maintained as a negative control (healthy liver) while group B was assigned as a positive control (damaged liver). Group C, D and E were orally administered 100 mg/kg, 200 mg/kg and 400 mg/kg of dried Vernonia amygdalina leaf extract, respectively. Meanwhile, group F, G and H were orally administered 100 mg/kg, 200 mg/kg and 400 mg/kg of fresh Vernonia amygdalina leaf extract, respectively, for 21 days. All mice were subjected to the assessment of symptoms including the severity of liver injury, biochemical assay and histological features. Phytochemical screening was also performed as per standard test methods.&#x0D; Results: Phytochemical screening revealed the presence of biological active compounds including alkaloids, flavonoids, saponins and terpenoids. Steroids were found in dried Vernonia amygdalina leaf extract, but not in the fresh Vernonia amygdalina leaf extract. Compared to the pathological changes observed in the liver parenchyma, sinusoids and hepatocytes in the positive control group, lesions were determined to be less severe particularly in the group D receiving 200 mg/kg of dried Vernonia amygdalina leaf extract and group H receiving of 400 mg/kg of fresh Vernonia amygdalina leaf extract. This was in agreement with the biochemical assessment whose ALT and AST levels were significantly decreased into the normal range as compared to the negative and positive control groups. However, dose of 400 mg/kg dried Vernonia amygdalina leaf extract significantly elevated the serum ALT and AST.&#x0D; Conclusion: Vernonia amygdalina has proven its ameliorative potentials on acetaminophen-induced liver injury in mice as determined by marked indicators on liver tissue and biochemical parameters including ALT and AST.

The hepatoprotective effect of Sophora viciifolia fruit extract against acetaminophen-induced liver injury in mice.

This research demonstrated the protective effect and possible mechanism of the Sophora viciifolia extract (SVE) against acetaminophen-induced liver injury in mice. The levels of ALT and AST in the serum and antioxidant enzyme activity in the liver were measured. We used immunohistochemistry to detect CYP2E1, Nrf2, and Keap1 protein expression in the liver. The mRNA expression in the liver of TNF-α, NF-κB, and IL-6, Nrf2 and its downstream genes HO-1 and GCLC were measured by qRT-PCR. We found that SVE could decrease the ALT and AST levels, promote the activities of SOD, CAT, GSH-Px, and GSH, and ameliorate pathological liver lesions. SVE could down-regulate the mRNA expression of inflammatory factors and up-regulate Nrf2, HO-1 and GCLC. SVE reduced the protein expression of the CYP2E1 and increased the Nrf2 and Keap1. SVE has been shown to have a protective effect against APAP-induced liver injury, possibly through activation of the Keap1-Nrf2 pathway.

Single-cell RNA sequencing to reveal non-parenchymal cell heterogeneity and immune network of acetaminophen-induced liver injury in mice.

The role of non-parenchymal cells (NPCs) in the early phase of acetaminophen (APAP)-induced liver injury (AILI) remains unclear. Therefore, single-cell sequencing (scRNA-seq) was performed to explore the heterogeneity and immune network of NPCs in the livers of mice with AILI. Mice were challenged with saline, 300mg/kg APAP, or 750mg/kg APAP (n = 3 for each group). After 3h, the liver samples were collected, digested, and subjected to scRNA-seq. Immunohistochemistry and immunofluorescence were performed to confirm the expression of Makorin ring finger protein 1 (Mkrn1). We identified 14 distinct cell subtypes among the 120,599 cells. A variety of NPCs were involved, even in the early stages of AILI, indicating highly heterogeneous transcriptome dynamics. Cholangiocyte cluster 3, which had high deleted in malignant brain tumors 1 (Dmbt1) expression, was found to perform drug metabolism and detoxification functions. Liver sinusoidal endothelial cells exhibited fenestrae loss and angiogenesis. Macrophage cluster 1 displayed a M1 polarization phenotype, whereas cluster 3 tended to exhibit M2 polarization. Kupffer cells (KCs) exhibited pro-inflammatory effects due to the high expression of Cxcl2. qRT-PCR and western blotting verified that the LIFR-OSM axis might promote the activation of MAPK signaling pathway in RAW264.7 macrophages. Mkrn1 was highly expressed in the liver macrophages of AILI mice and AILI patients. Interaction patterns between macrophages/KCs and other NPCs were complex and diverse. NPCs were highly heterogeneous and were involved in the immune network during the early phase of AILI. In addition, we propose that Mkrn1 may serve as a potential biomarker of AILI.

High Fischer ratio oligopeptides from hard-shelled mussel: Preparation and hepatoprotective effect against acetaminophen-induced liver injury in mice

High Fischer ratio oligopeptides (HFOPs) have a variety of physiological activities, but there are few literatures reporting the hepatoprotective effects of HFOPs against acetaminophen (APAP)-induced liver injury. The objectives of this study were to establish the preparation process of HFOPs from hard-shelled mussel (Mytilus coruscus) (HFOPs-HM) and investigate the hepatoprotective effects and potential mechanisms of HFOPs-HM against APAP-induced liver injury in mice. Therefore, trypsin and papain were selected as the endo and exogenous proteases for preparing HFOPs-HM according to the degree of hydrolysis (DH) of hard-shelled mussel hydrolysates, and activated carbon of XHJ-200 displayed the best effect to remove aromatic amino acids according to the Fischer ratio (OD220/OD280) and its parameters were optimized as adsorption time of 3.0 h and solid-liquid ratio of 1:10. The HFOPs-HM's Fischer ratio and average molecular weight were 26.03 (>20) and 878.54 Da, respectively. Furthermore, a mice model of liver injury induced by APAP was established. HFOPs-HM could significantly improve liver index and histopathological features, decrease the serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities, increase the glutathione content and superoxide dismutase (SOD) and catalase (CAT) activities. Further research demonstrated that HFOPs-HM could alleviate liver oxidative stress in liver tissue via activating the Nrf2 pathway to initiate the intracellular antioxidant enzyme system. In addition, HFOPs-HM also suppressed liver inflammation by downregulating the secretion of TNF-α, IL-1β, and IL-6. Thus, HFOPs-HM could serve as an auxiliary functional dietary molecule applied in function products to alleviate drug-induced liver injury.

Role of NADPH Oxidase-Derived ROS-Mediated IL-6/STAT3 and MAPK/NF-κB Signaling Pathways in Protective Effect of Corilagin against Acetaminophen-Induced Liver Injury in Mice

Acetaminophen (APAP) overdose causes acute liver injury via oxidative stress, uncontrolled inflammatory response, and subsequent hepatocyte death. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a potent source of cellular reactive oxygen species (ROS) and may contribute to oxidative stress in many inflammatory processes. Corilagin, a component of Phyllanthus urinaria, possesses antioxidant, anti-inflammatory, and hepatoprotective effects. We evaluated the mechanisms underlying the protective effect of corilagin against acetaminophen-induced liver injury. Mice were intraperitoneally administrated 300 mg/kg APAP or equal volume of saline (control), with or without various concentrations of corilagin (0, 1, 5, or 10 mg/kg) administered after 30 min. All animals were sacrificed 16 h after APAP administration, and serum and liver tissue assays including histology, immunohistochemistry, and Western blot assay were performed. Corilagin post-treatment significantly attenuated APAP-induced liver injury (p < 0.005), inflammatory cell infiltration, hepatic proinflammatory cytokine levels, and hepatic oxidative stress. Furthermore, corilagin attenuated the protein levels of NOX1, NOX2, signal transducer and activator of transcription 3 (STAT3), and nuclear factor kappa B (NF-κB) in APAP-induced liver injury. These results indicated that the antioxidant, anti-inflammatory, and protective effects of corilagin in APAP-induced liver injury might involve the regulation of interleukin (IL)-6/STAT3 and mitogen-activated protein kinase (MAPK)/NF-κB signaling pathways through NOX-derived ROS.

Cathelicidin promotes liver repair after acetaminophen-induced liver injury in mice

Acetaminophen (APAP)-induced acute liver injury (AILI) is a leading cause of acute liver failure (ALF). N-acetylcysteine (NAC) is only effective within 24h after APAP intoxication, raising an urgent need for alternative approaches to treat this disease. This study aimed to test whether cathelicidin (Camp), which is a protective factor in chronic liver diseases, protects mice against APAP-induced liver injury and ALF. A clinically relevant AILI model and an APAP-induced ALF model were generated in mice. Genetic and pharmacological approaches were used to interfere with the levels of cathelicidin invivo. An increase in hepatic pro-CRAMP/CRAMP (the precursor and mature forms of mouse cathelicidin) was observed in APAP-intoxicated mice. Upregulated cathelicidin was derived from liver-infiltrating neutrophils. Compared with wild-type littermates, Camp knockout had no effect on hepatic injury but dampened hepatic repair in AILI and reduced survival in APAP-induced ALF. CRAMP administration reversed impaired liver recovery observed in APAP-challenged Camp knockout mice. Delayed CRAMP, CRAMP(1-39) (the extended form of CRAMP), or LL-37 (the mature form of human cathelicidin) treatment exhibited a therapeutic benefit for AILI. Co-treatment of cathelicidin and NAC in AILI displayed a stronger hepatoprotective effect than NAC alone. A similar additive effect of CRAMP(1-39)/LL-37 and NAC was observed in APAP-induced ALF. The pro-reparative role of cathelicidin in the APAP-damaged liver was attributed to an accelerated resolution of inflammation at the onset of liver repair, possibly through enhanced neutrophil phagocytosis of necrotic cell debris in an autocrine manner. Cathelicidin reduces APAP-induced liver injury and ALF in mice by promoting liver recovery via facilitating inflammation resolution, suggesting a therapeutic potential for late-presenting patients with AILI with or without ALF. Acetaminophen-induced acute liver injury is a leading cause of acute liver failure. The efficacy of N-acetylcysteine, the only clinically approved drug against acetaminophen-induced acute liver injury, is significantly reduced for late-presenting patients. We found that cathelicidin exhibits a great therapeutic potential in mice with acetaminophen-induced liver injury or acute liver failure, which makes up for the limitation of N-acetylcysteine therapy by specifically promoting liver repair after acetaminophen intoxication. The pro-reparative role of cathelicidin, as a key effector molecule of neutrophils, in the APAP-injured liver is attributed to an accelerated resolution of inflammation at the onset of liver repair, possibly through enhanced phagocytic function of neutrophils in an autocrine manner.

Potential Curative Effects of Aqueous Extracts of Cissus quadrangularis (Vitaceae) and Jatropha gossypiifolia (Euphorbiaceae) on Acetaminophen-Induced Liver Injury in Mice

Acetaminophen-induced liver injury remains a significant public health problem because available treatments are limited due to their adverse effects. Medicinal plants, which are an important source of bioactive molecules, could be an alternative treatment for liver disease. This study was designed to investigate the curative effect of aqueous extracts of Cissus quadrangularis (Vitaceae) and Jatropha gossypiifolia (Euphorbiaceae) on acetaminophen-induced liver injury in mice. Mice were divided into groups and treated with distilled water, silymarin (50 mg/kg), a reference hepatoprotective agent, and aqueous extracts of C quadrangularis and J gossypiifolia (50 and 100 mg/kg, PO, respectively). These substances were given as a single daily dose 4 hours after acetaminophen administration (300 mg/kg, PO) for 2 days. Mice were humanely put to death 24 hours after the last dose and serum alanine aminotransferase and aspartate aminotransferase activities, total bilirubin and protein levels, reduced glutathione, superoxide dismutase, malondialdehyde, catalase, and nitrite tissue levels were assessed. Histology of the livers of the mice was performed by hematoxylin and eosin staining. Acetaminophen administration induced a significant (P < 0.05) mean (SEM) body weight loss (-14.45% [5.92%]), a significant elevation of alanine aminotransferase activity (15.08%), total protein and bilirubin levels (25.80%), and a significant (P < 0.05) increase in liver superoxide dismutase (67.71%), catalase (63.00%), glutathione (40.29%), malondialdehyde (30.67%), and nitrite levels compared with the control group. In curative treatment, C quadrangularis and J gossypiifolia (50 and 100 mg/kg) significantly (P < 0.05) reduced mean (SEM) body weight loss (16.67% [7.16%] and 1.25% [0.51%], respectively), serum alanine aminotransferase activity (17.62% and 11.14%, respectively), bilirubin level (29.62% and 49.14%, respectively) compared with acetaminophen group, and J gossypiifolia normalized serum total protein level. Both extracts significantly (P < 0.05) reduced the levels of glutathione and malondialdehyde and normalized that of nitrite, superoxide dismutase, and catalase compared with the acetaminophen group. Hepatocyte necrosis and inflammatory cell infiltration were remarkably reduced by the plant extracts. The results obtained are evidence in favor of the development of a formulation based on the extracts of these plants against liver diseases.

Paeoniflorin Protects against Acetaminophen-Induced Liver Injury in Mice via JNK Signaling Pathway

Drug-induced liver injury (DILI), represented by acetaminophen (APAP), is a common cause of acute liver failure in clinics. Paeoniflorin (PF) has been proven to demonstrate a significant hepatoprotective effect. However, it is still unclear whether it can be a potential agent against hepatotoxicity induced by APAP. This study aimed to explore the preventive and therapeutic effects and mechanisms of PF on APAP-induced liver injury. Different doses of PF (50, 100, and 200 mg/kg) were given to C57BL/6 male mice for five consecutive days. After 12 h of APAP (250 mg/kg i.p.) treatment, blood and liver tissues were collected and isolated for detection. The results showed that the therapeutic effects of PF on APAP mice were presented in the downregulation of the content of serum indices and significantly improved hepatic tissue edema and inflammatory infiltration. Meanwhile, PF reduces the level of the mitochondrial metabolic enzyme. Ulteriorly, it was found that PF has a downregulating effect on the apoptotic reaction and could inhibit the protein expression of CYP2E1/JNK signaling, which in turn reduces the damage of APAP. Our findings showed that PF acted as a protective agent against APAP-induced hepatotoxicity by inhibiting JNK-related signals, suggesting a novel insight into treating APAP-induced liver injury.

A novel humanized Chi3l1 blocking antibody attenuates acetaminophen-induced liver injury in mice.

Acetaminophen (APAP) overdose is a leading cause of acute liver injury in the USA. The chitinase 3-like-1 (Chi3l1) protein contributes to APAP-induced liver injury (AILI) by promoting hepatic platelet recruitment. Here, we report the development of a Chi3l1-targeting antibody as a potential therapy for AILI. By immunizing a rabbit successively with the human and mouse Chi3l1 proteins, we isolated cross-reactive monoclonal antibodies (mAbs) from single memory B cells. One of the human and mouse Chi3l1 cross-reactive mAbs was humanized and characterized in both in vitro and in vivo biophysical and biological assays. X-ray crystallographic analysis of the lead antibody C59 in complex with the human Chi3l1 protein revealed that the kappa light contributes to majority of the antibody-antigen interaction; and that C59 binds to the 4α-5β loop and 4α-helix of Chi3l1, which is a functional epitope and hotspot for the development of Chi3l1 blocking antibodies. We humanized the C59 antibody by complementarity-determining region grafting and kappa chain framework region reverse mutations. The humanized C59 antibody exhibited similar efficacy as the parental rabbit antibody C59 in attenuating AILI in vivo. Our findings validate Chi3l1 as a potential drug target for AILI and provide proof of concept of developing Chi3l1 blocking antibody as a therapy for the treatment of AILI.

Schisandrin C improves acetaminophen-induced liver injury in mice by regulating Nrf2 signaling pathway

Excess acetaminophen(APAP) can be converted by the cytochrome P450 system to the toxic metabolite N-acetyl-p-benzoquinoneimine(NAPQI), which consumes glutathione(GSH). When GSH is depleted, NAPQI covalently binds with proteins, inducing mitochondrial dysfunction and oxidative stress and thereby leading to hepatotoxicity. Schisandrin C(SinC) is a dibenzocyclooctadiene derivative isolated from Schisandra chinensis. Although there is some evidence showing that SinC has hepatoprotective activity, its protective effect and mechanism on APAP-induced liver injury remain unclear. In this paper, an acute liver injury mouse model was established by intraperitoneal injection of APAP at a dose of 400 mg·kg~(-1) to evaluate the effect of SinC administration on the APAP-induced liver injury and its mechanism through an animal experiment. At the same time, a potential candidate drug was provi-ded for traditional Chinese medicine(TCM) prevention and treatment of overdose APAP-induced liver injury. In the APAP-induced liver injury mouse model, we found that SinC can relieve hepatic histopathological lesions and significantly reduce the activities of alanine aminotransferase(ALT), aspartate aminotransferase(AST) and alkaline phosphatase(ALP). It was also capable of increasing the content of GSH and superoxide dismutase(SOD) and decreasing the levels of total bilirubin(TBIL), direct bilirubin(DBIL), malondialdehyde(MDA), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α). Further analysis showed that SinC decreased the content of CYP2 E1 in liver tissues at protein and mRNA levels and increased nuclear factor erythroid 2-related factor 2(Nrf2) and the expression of its downstream targets(including HO-1, NQO1 and GCLC). Taken together, the above results indicate that SinC can alleviate APAP-induced liver injury by reducing the expression of CYP2 E1, suppressing apoptosis, improving inflammatory response and activating the Nrf2 signaling pathway to inhibit oxidative stress.

73 Therapeutic Effects of Melittin Against Acetaminophen-Induced Liver Injury in Mice

Study Objective: An overdose of acetaminophen (APAP) frequently causes severe liver injury, but the treatment options remain limited. Melittin is a bioactive peptide derived from bee venom and possesses anti-oxidative, anti- apoptotic, and anti-inflammatory properties. Emerging evidence suggests that this peptide exerts beneficial effects on several inflammatory disorders. However, whether melittin has a protective effect on APAP toxicity remains undetermined. The purpose of the current study was to investigate the effect of melittin on APAP-induced hepatotoxicity.Methods: Melittin was intraperitoneally administered at a dose of 0.01 mg/kg to mice 1 hour after APAP injection (400 mg/kg).Results: Administration of melittin after APAP injection reduced serum aspartate aminotransferase and alanine aminotransferase levels and alleviated histological alterations in APAP-injected mice. Melittin inhibited lipid peroxidation and DNA oxidation with restoration of glutathione content and upregulation of antioxidant enzymes. Melittin also attenuated hepatocyte apoptosis with inhibition of caspase-3 activation. In addition, serum and hepatic levels of cytokines were reduced by melittin. This effect was associated with inhibition of nuclear factor-κB activation. Furthermore, melittin attenuated infiltration of neutrophils and macrophages into the liver.Conclusion: Altogether, these data suggest that melittin might be served as a treatment agent for APAP-induced hepatotoxicity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)No, authors do not have interests to disclose Study Objective: An overdose of acetaminophen (APAP) frequently causes severe liver injury, but the treatment options remain limited. Melittin is a bioactive peptide derived from bee venom and possesses anti-oxidative, anti- apoptotic, and anti-inflammatory properties. Emerging evidence suggests that this peptide exerts beneficial effects on several inflammatory disorders. However, whether melittin has a protective effect on APAP toxicity remains undetermined. The purpose of the current study was to investigate the effect of melittin on APAP-induced hepatotoxicity. Methods: Melittin was intraperitoneally administered at a dose of 0.01 mg/kg to mice 1 hour after APAP injection (400 mg/kg). Results: Administration of melittin after APAP injection reduced serum aspartate aminotransferase and alanine aminotransferase levels and alleviated histological alterations in APAP-injected mice. Melittin inhibited lipid peroxidation and DNA oxidation with restoration of glutathione content and upregulation of antioxidant enzymes. Melittin also attenuated hepatocyte apoptosis with inhibition of caspase-3 activation. In addition, serum and hepatic levels of cytokines were reduced by melittin. This effect was associated with inhibition of nuclear factor-κB activation. Furthermore, melittin attenuated infiltration of neutrophils and macrophages into the liver. Conclusion: Altogether, these data suggest that melittin might be served as a treatment agent for APAP-induced hepatotoxicity. No, authors do not have interests to disclose

Novel carvedilol-loaded pro-phytomicelles: formulation, characterization and enhanced protective efficacy against acetaminophen-induced liver injury in mice

The work describes a novel, small-molecule phytochemicals as nanomaterials based pro-micelles (pro-phytomicelles) drug delivery system, for oral delivery of carvedilol (CAR). This novel nanoformulation of CAR, named CAR pro-phytomicelles, was prepared with rebaudioside A (RA) and dipotassium glycyrrhizinate (DG) as mixed nanomaterials. The formulation was optimized, leading to a 502-fold increase in solubility of CAR in water as a result of encapsulation within mixed phytomicelles based on DG and RA. CAR pro-phytomicelles samples could be instantly dissolved into aqueous media to formulate clear phytomicelle solutions with CAR encapsulation efficiency of 99.67 ± 0.02 %, and small micelle size of 15.62 ± 0.27 nm. CAR pro-phytomicelles exhibited good storage stability, rapid in vitro release in simulated intestinal fluid, and improved in vitro antioxidant activity. CAR pro-phytomicelles had good biocompatibility. Protective efficacy evaluation revealed that acetaminophen overdose could induce high mortality and severe liver injury in mice, while CAR pro-phytomicelle treatment exhibited significant protective effect against acetaminophen overdose. This protective efficacy was due to a mechanism that involved the regulation of high-mobility group box 1 and its signaling-related proinflammatory cytokines. These results show that pro-phytomicelles could provide a new concept and promising therapeutics as nanomedicines for improving the activities of CAR against acetaminophen-induced liver injury.

Recovered Hepatocytes Promote Macrophage Apoptosis Through CXCR4 After Acetaminophen-Induced Liver Injury in Mice.

Acetaminophen (APAP) overdose is the main cause of acute liver failure in Western countries. The mechanism of APAP hepatotoxicity is associated with centrilobular necrosis which initiates infiltration of neutrophils, monocytes, and other leukocytes to the area of necrosis. Although it has been recognized that this infiltration of immune cells plays a critical role in promoting liver repair, mechanism of immune cell clearance that is important for resolution of inflammation and the return to normal homeostasis are not well characterized. CXCR4 is a chemokine receptor expressed on hepatocytes as well as neutrophils, monocytes, and hematopoietic stem cells. CXCR4 function is dependent on its selective expression on different cell types and thus can vary depending on the pathophysiology. This study aimed to investigate the crosstalk between hepatocytes and macrophages through CXCR4 to promote macrophage apoptosis after APAP overdose. C57BL/6J mice were subjected to APAP overdose (300 mg/kg). Flow cytometry and immunohistochemistry were used to determine the mode of cell death of macrophages and expression pattern of CXCR4 during the resolution phase of APAP hepatotoxicity. The impact of CXCR4 in regulation of macrophage apoptosis and liver recovery was assessed after administration of a monoclonal antibody against CXCR4. RNA sequencing analysis was performed on flow cytometry sorted CXCR4+ macrophages at 72 h to confirm the apoptotic cell death of macrophages. Our data indicate that the inflammatory response is resolved by recovering hepatocytes through induction of CXCR4 on macrophages, which triggers their cell death by apoptosis at the end of the recovery phase.

Alterations in circadian rhythms aggravate Acetaminophen-induced liver injury in mice by influencing Acetaminophen metabolization and increasing intestinal permeability

ABSTRACT Acetaminophen (APAP) is the most common antipyretic and analgesic drug causing drug-induced liver injury (DILI). Alterations in circadian rhythms can adversely affect liver health, especially metabolic and detoxification functions. However, the effect of circadian rhythm alterations induced by environmental factors on APAP-induced liver injury and the underlying mechanisms are not well known. In this study, a mouse model of circadian rhythm alterations was established by light/dark cycle shift and then treated with excessive APAP. The liver injury indexes, APAP-related metabolic enzymes, and intestinal permeability in mice were evaluated by biochemical analysis, quantitative real-time PCR, enzyme-linked immunosorbent assays, and histopathology. Results showed that circadian rhythm alterations resulted in increased reactive oxygen species (ROS) and malondialdehyde (MDA) and decreased liver superoxide dismutase (SOD), glutathione, and CYP1A2 and CYP3A11 mRNA expression, and increased serum diamine oxidase, lipopolysaccharide, and D-lactate in the mice. Compared with control mice, APAP induced higher serum alanine aminotransferase and aspartate aminotransferase, liver interleukin-1β and tumor necrosis factor-α mRNA, ROS and MDA, lower SOD, glutathione, and UDP-glucuronosyltransferases /sulfotransferases mRNA and more severe liver necrosis and intestinal damage in mice with alterations in circadian rhythms. In conclusion, circadian rhythm alterations by light/dark cycle shift resulted in increased oxidative stress and intestinal permeability in the mice and exacerbated APAP-induced liver injury by influencing APAP metabolization and increasing intestinal permeability.