Inhibition of NAD kinase elevates the hepatic NAD+ pool and alleviates acetaminophen-induced acute liver injury in mice

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

Acetaminophen (APAP) in high doses causes acute liver injury and acute liver failure. Ethyl gallate (EG) is a natural polyphenol, possessing antioxidant, anti-inflammatory, and anti-microbial properties. Therefore, in this study, we evaluated the protective role of EG against APAP-induced acute liver injury in mice. Acute liver injury was induced by a single dose of APAP (400 mg/kg., i.p.). In separate groups, EG (10 mg/kg), EG (20 mg/kg), and N-acetylcysteine (NAC; 1200 mg/kg., i.p.) were administered concurrently with APAP. The mice were sacrificed after 24 h of treatment. Liver marker enzymes of hepatotoxicity, antioxidant markers, inflammatory markers, and histopathological studies were done. APAP administration caused a significant elevation of marker enzymes of hepatotoxicity and lipid peroxidation. APAP administration also decreased enzymic and nonenzymic antioxidants. Acute APAP intoxication induced nuclear factor κ B, tumor necrosis factor-α, interleukin-1, p65, and p52 and downregulated IκB gene expressions. Our histopathological studies have confirmed the presence of centrilobular necrosis, 24 h after APAP intoxication. All the above abnormalities were significantly inhibited in groups of mice that were concurrently administered with APAP + EG and APAP + NAC. Our in silico analysis further confirms that hydroxyl groups of EG interact with the above inflammatory proteins at the 3,4,5-trihydroxybenzoic acid region. These effects of EG against APAP-induced acute liver injury could be attributed to its antioxidative, free radical scavenging, and anti-inflammatory potentials. Therefore, this study suggests that EG can be an efficient therapeutic approach to protect the liver from APAP intoxication.

This study explored the protective effect of atractylenolide Ⅰ(AO-Ⅰ) against acetaminophen(APAP)-induced acute liver injury(ALI) in mice and its underlying mechanism. C57 BL/6 J mice were randomly divided into a control group, an APAP group(500 mg·kg~(-1)), a low-dose combination group(500 mg·kg~(-1) APAP + 60 mg·kg~(-1) AO-Ⅰ), and a high-dose combination group(500 mg·kg~(-1) APAP + 120 mg·kg~(-1) AO-Ⅰ). ALI was induced by intraperitoneal injection of APAP(500 mg·kg~(-1)). AO-Ⅰ by intragastric administration was performed 2 hours before APAP treatment, and the control group received the same dose of solvent by intragastric administration or intraperitoneal injection. The protective effect of AO-Ⅰ against APAP-induced ALI was evaluated by detecting alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels in the plasma and H&E staining in liver tissues of mice. The malondialdehyde(MDA) and glutathione(GSH) content and catalase(CAT) activity in mouse liver tissues were detected to evaluate the effect of AO-Ⅰ on APAP-induced oxidative stress in the liver. The proteins in the liver p38 mitogen-activated protein kinase(p38 MAPK), c-jun N-terminal kinase(JNK), and nuclear factor kappa-B p65(NF-κB p65) signaling pathways were measured by Western blot, and the liver inflammatory cytokines interleukin-1β(IL-1β) and interleukin-6(IL-6) were detected by real-time PCR. Compared with the APAP group, the combination groups showed reduced APAP-induced ALT level and liver MDA content, potentiated liver CAT activity, and elevated GSH content. Mechanistically, AO-Ⅰ treatment significantly inhibited APAP-up-regulated MAPK phosphorylation and NF-κB p65, and significantly reduced the transcriptional activities of IL-1β and IL-6, downstream targets of NF-κB p65. AO-Ⅰ can improve APAP-induced ALI and the underlying mechanism is related to the inhibition of the MAPK/NF-κB p65 signaling pathway in APAP-challenged mice.

Acetaminophen (APAP), a common antipyretic and analgesic drug, is considered the most common cause of drug-induced liver injury (DILI). The mechanism of liver injury induced by APAP is mainly related to oxidative stress and inflammatory reaction. Herein, we report a simple and efficient one-step synthesis of Prussian blue (PB) nanozymes with multiple antioxidant enzymatic activities that effectively treat APAP-induced DILI. At the cellular level, reaching 10 μg/mL PB nanozymes can effectively scavenge intracellular reactive oxygen species (ROS), reduce mitochondrial membrane potential drop, and inhibit hepatocyte apoptosis. According to in vivo experimental studies, the levels of serum biochemical indicators and histopathological examination of DILI mice livers showed that 12.5 mg/kg PB nanozymes could effectively inhibit liver necrosis and 25 mg/kg PB nanozymes achieved the same therapeutic effect as 300 mg/kg NAC. More importantly, compared with NAC, PB nanozymes can still attenuate APAP-induced acute liver injury in mice after APAP-induced acute liver injury in mice for 3 h. Therefore, PB nanozymes can effectively prolong the therapeutic time window, revealing the potential of PB nanozymes in clinical applications for advanced DILI treatment. Furthermore, the therapeutic mechanism studies have shown that PB nanozymes with abundant and variable valence states could not only directly scavenge ROS but also through the Keap1-Nrf2/HO-1 pathway to reduce oxidative stress. Moreover, the decreased expression levels of myeloperoxidase and F4/80 in the liver, which are markers of neutrophil and macrophage infiltration, indicated that the Prussian blue nanozymes modulates inflammation to protect against APAP-induced acute liver injury. Consequently, our findings suggested that PB nanozymes have excellent clinical application prospects for acetaminophen-induced acute liver injury.

Pro-Regenerative Signaling after Acetaminophen-Induced Acute Liver Injury in Mice Identified Using a Novel Incremental Dose Model

Mitophagy protects against acetaminophen-induced acute liver injury in mice through inhibiting NLRP3 inflammasome activation

Excessive acetaminophen (APAP) can induce neutrophil activation and hepatocyte death. Along with hepatocyte dysfunction and death, NETosis (a form of neutrophil-associated inflammation) plays a vital role in the progression of acute liver injury (ALI) induced by APAP overdose. It has been shown that activated neutrophils tend to migrate towards the site of injury and participate in inflammatory processes via formation of neutrophil extracellular traps (NETs). In this study we investigated whether NETs were involved in hepatocyte injury and contributed to APAP-induced ALI progression. ALI mouse model was established by injecting overdose (350 mg/kg) of APAP. After 24 h, blood and livers were harvested for analyses. We showed that excessive APAP induced multiple programmed cell deaths of hepatocytes including pyroptosis, apoptosis and necroptosis, accompanied by significantly increased NETs markers (MPO, citH3) in the liver tissue and serum. Preinjection of DNase1 (10 U, i.p.) for two consecutive days significantly inhibited NETs formation, reduced PANoptosis and consequently alleviated excessive APAP-induced ALI. In order to clarify the communication between hepatocytes and neutrophils, we induced NETs formation in isolated neutrophils, and treated HepaRG cells with NETs. We found that NETs treatment markedly increased the activation of GSDMD, caspase-3 and MLKL, while pre-treatment with DNase1 down-regulated the expression of these proteins. Knockdown of AIM2 (a cytosolic innate immune receptor) abolished NETs-induced PANoptosis in HepaRG cells. Furthermore, excessive APAP-associated ALI was significantly attenuated in AIM2KO mice, and PANoptosis occurred less frequently. Upon restoring AIM2 expression in AIM2KO mice using AAV9 virus, both hepatic injury and PANoptosis was aggravated. In addition, we demonstrated that excessive APAP stimulated mtROS production and mitochondrial DNA (mtDNA) leakage, and mtDNA activated the TLR9 pathway to promote NETs formation. Our results uncover a novel mechanism of NETs and PANoptosis in APAP-associated ALI, which might serve as a therapeutic target.

Acetaminophen (APAP) overdose-induced acute liver injury (ALI) is characterized by extensive oxidative stress, and the clinical interventions for this adverse effect remain limited. Astilbin is an active compound found in the rhizome of Smilax glabra Roxb. with anti-inflammatory and antioxidant activities. Due to its low oral bioavailability, astilbin can accumulate in the intestine, which provides a basis for the interaction between astilbin and gut microbiota (GM). In the present study we investigated the protective effects of astilbin against APAP-induced ALI by focusing on the interaction between astilbin and GM. Mice were treated with astilbin (50 mg·kg-1·d-1, i.g.) for 7 days. After the last administration of astilbin for 2 h, the mice received APAP (300 mg/kg, i.g.) to induce ALI. We showed that oral administration of astilbin significantly alleviated APAP-induced ALI by altering the composition of GM and enriching beneficial metabolites including hydroxytyrosol (HT). GM depletion using an "antibiotics cocktail" or paraoral administration of astilbin abolished the hepatoprotective effects of astilbin. On the other hand, administration of HT (10 mg/kg, i.g.) caused similar protective effects in APAP-induced ALI mice. Transcriptomic analysis of the liver tissue revealed that HT inhibited reactive oxygen species and inflammation-related signaling in APAP-induced ALI; HT promoted activation of the Nrf2 signaling pathway to combat oxidative stress following APAP challenge in a sirtuin-6-dependent manner. These results highlight that oral astilbin ameliorates APAP-induced ALI by manipulating the GM and metabolites towards a more favorable profile, and provide an alternative therapeutic strategy for alleviating APAP-induced ALI.

Nicotinamide improves NAD+ levels to protect against acetaminophen-induced acute liver injury in mice.

Correction

Intrabody against prolyl hydroxylase 2 ameliorates acetaminophen-induced acute liver injury in mice via concomitant promotion of angiogenesis and redox homeostasis

Most cases of acute liver failure are caused by acetaminophen (APAP) overdose. Oxidative stress is a key factor in APAP toxicity. Although augmenter of liver regeneration (ALR) has both antioxidative and antiapoptotic effects, its therapeutic potential in APAP hepatotoxicity remains unknown. The current study assessed the protective effects and associated mechanisms of ALR against APAP-induced acute liver injury in female BALB/c mice. We found that serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, intrahepatic hemorrhage and necrosis were increased at 3, 6, 12, 24, 48, and 72 h after 600 mg/kg APAP i.p. injection. During the early stages (before 12 h) of acute liver injury, ALR levels increased significantly, followed by a decrease to control level at 24 h after APAP administration. ALR treatment increased autophagosomes, promoted the conversion of LC3 I to LC3 II, and the degradation of p62. ALR attenuated APAP-stimulated increases in ALT, AST, myeloperoxidase (MPO), malondialdehyde (MDA), and reactive oxidative species (ROS) levels; intrahepatic hemorrhage; and necrosis as well as superoxide dismutase (SOD) and Glutathione (GSH) depletion. We found that APAP caused release of the mitochondrial intermembrane proteins apoptosis-inducing factor (AIF) and cytochrome c and that ALR inhibited this change. Meanwhile, ALR decreased expression of cleaved-caspase 3 and apoptotic cells. Subsequently, we investigated the significance of autophagy in APAP-induced acute liver injury by treatment with 3-methyladenine (3-MA), which were classical pharmaceuticals for suppressing autophagy. ALR directly induced autophagy flux; and the inhibition of autophagy reversed the beneficial effects of ALR on hepatotoxicity. Our findings suggest that ALR protects against APAP hepatotoxicity by activating the autophagy pathway.

ADAMTS13 Plays a Critical Role in Acetaminophen-Induced Acute Liver Injury in Mice, and Can be a Novel Therapeutic Option

BackgroundAcetaminophen (APAP) overdose causes hepatotoxicity and even acute liver failure. Recent studies indicate that sterile inflammation and innate immune cells may play important roles in damage-induced hepatocytes regeneration and liver repair. The scavenger receptor CD36 has its crucial functions in sterile inflammation. However, the roles of CD36 in APAP induced acute liver injury remain unclear and warrant further investigation.MethodsWT C57BL/6 J and CD36−/− mice were intraperitoneally injected with APAP (300 mg/kg) after fasting for 16 h. Liver injury was evaluated by serum alanine aminotransferase (ALT) level and liver tissue hematoxylin and eosin (H&E) staining. Liver inflammatory factor expression was determined by real-time polymerase chain reaction (PCR). The protein adducts forming from the metabolite of APAP and the metabolism enzyme cytochrome P450 2E1 (CYP2E1) levels were measured by Western blot. Liver infiltrating macrophages and neutrophils were characterized by flow cytometry. RNA sequencing and Western blot were used to evaluate the effect of damage-associated molecular patterns (DAMP) molecule high mobility group B1 (HMGB1) on WT and CD36−/− macrophages. Moreover, PP2, a Src kinase inhibitor, blocking CD36 signaling, was applied in APAP model.ResultsThe expression of CD36 was increased in the liver of mice after APAP treatment. Compared with WT mice, APAP treated CD36−/− mice show less liver injury. There was no significant difference in APAP protein adducts and CYP2E1 expression between these two strains. However, reduced pro-inflammatory factor mRNA expression and serum IL-1β level were observed in APAP treated CD36−/− mice as well as infiltrating macrophages and neutrophils. Moreover, CD36 deficiency impaired the activation of c-Jun N-terminal kinase (JNK) caused by APAP. Interestingly, the lack of CD36 reduced the activation of extracellular regulated protein kinases (Erk) and v-akt murine thymoma viral oncogene homolog (Akt) induced by HMGB1. RNA transcription sequencing data indicated that HMGB1 has a different effect on WT and CD36−/− macrophages. Furthermore, treatment with PP2 attenuated APAP induced mouse liver injury.ConclusionOur data demonstrated that CD36 deficiency ameliorated APAP-induced acute liver injury and inflammatory responses by decreasing JNK activation. CD36 might serve as a new target to reduce acute liver injury.

The aim of the current study is to investigate the effects of growth hormone releasing hormone (GHRH) antagonist on acetaminophen (APAP)-induced acute liver injury in mice. Healthy C57/B6L mice were orally treated with 200 mg/kg APAP with or without a 30-min pre-treatment with 300 µg/kg GHRH antagonist MZ-5-156. After 12 hours, serum, plasma, and liver samples from each mouse were collected for analyses. Our results showed that twelve-hour treatment with APAP caused obvious liver injury, elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, increased oxidative stress, reduced expressions of antioxidant enzymes, accumulated expression of pro-inflammatory cytokines, and increased circulating levels of growth hormone (GH) and insulin-like growth factor-I (IGF-I). Pre-treatment with MZ-5-156 aggravated liver injury, further increased serum ALT and AST levels, exacerbated oxidative stress and inflammation induced by APAP. Treatment of MZ-5-156 also blocked the phosphorylation form and total form of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). Treatment of GHRH super-agonist JI-38 immediately after MZ-5-156 treatment partly reversed the liver injury caused by APAP and MZ-5-156. In conclusion, GHRH plays essential protective role in APAP-induced acute liver injury in vivo. The protective properties of GHRH are partially through GH/IGF-I axis and JAK/STAT pathway.