FAK inhibition delays liver repair after acetaminophen-induced acute liver injury by suppressing hepatocyte proliferation and macrophage recruitment.

Overdose of acetaminophen (APAP), a commonly used antipyretic analgesic, can lead to severe liver injury and failure. Current treatments are only effective in the early stages of APAP-induced acute liver injury (ALI). Therefore, a detailed examination of the mechanisms involved in liver repair following APAP-induced ALI could provide valuable insights for clinical interventions. 4D-label-free proteomics analysis was used to identify dysregulated proteins in the liver of APAP-treated mice. RNA-Seq, hematoxylin-eosin staining, immunohistochemical staining, immunofluorescence staining, quantitative PCR, western blotting, transwell were used to explore the underlying mechanisms. Utilizing high throughput 4D-label-free proteomics analysis, we observed a notable increase in proteins related to the "focal adhesion" pathway in the livers of APAP-treated mice. Inhibiting focal adhesion kinase (FAK) activation with a specific inhibitor, 1,2,4,5-Benzenetetraamine tetrahydrochloride (also called Y15), resulted in reduced macrophage numbers, delayed necrotic cell clearance, and inhibited liver cell proliferation in the necrotic regions of APAP-treated mice. RNA-Seq analysis demonstrated that Y15 downregulated genes associated with "cell cycle" and "phagosome" pathways in the livers of APAP-treated mice. Furthermore, blocking extracellular matrix (ECM)-integrin activation with a competitive peptide inhibitor, Gly-Arg-Gly-Asp-Ser (GRGDS), suppressed FAK activation and liver cell proliferation without affecting macrophage recruitment to necrotic areas. Mechanistically, ECM-induced FAK activation upregulated growth-promoting cell cycle genes, leading to hepatocyte proliferation, while CCL2 enhanced FAK activation and subsequent macrophage recruitment via F-actin rearrangement. Overall, these findings underscore the pivotal role of FAK activation in liver repair post-APAP overdose by promoting liver cell proliferation and macrophage recruitment.

Li Q ，Xu Q ，Shi J ，Dong W ，Jin J ，Zhang C ... -  《Hepatology Communications》

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 2 h after an APAP overdose of 300 mg/kg in fasted male C57BL/6J mice. TPOm did not affect protein adducts, oxidant stress, DNA fragmentation and hepatic necrosis up to 12 h after APAP. In contrast, TPOm treatment was beneficial at 24 h, 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 6 h, there was no effect on the injury, but a proliferative effect was still evident. In contrast, 4MP and NAC treated at 2 h after APAP significantly attenuated all injury parameters at 24 h but failed to enhance hepatocyte proliferation. Thus, TPOm arrests the progression of liver injury by 24 h after APAP and accelerates the onset of the proliferative response which is essential for liver recovery.

Adelusi OB ，Akakpo JY ，Eichenbaum G ，Sadaff E ，Ramachandran A ，Jaeschke H ... -  《-》

Artemisia Argyi essential oil ameliorates acetaminophen-induced hepatotoxicity via CYP2E1 and γ-glutamyl cycle reprogramming.

The hepatotoxicity induced by acetaminophen (APAP), a commonly used antipyretic, analgesic and anti-inflammatory drug in clinical practice, has received accumulated attention. Artemisia argyi essential oil (AAEO), a volatile oil component extracted from traditional Chinese medicine Artemisia argyi H.Lév. & Vaniot, has great hepatoprotective effects. However, the potential role of AAEO in APAP-induced hepatotoxicity has not been characterized. The present study aimed to investigate the effects of AAEO on hepatic metabolic changes in mice exposed to APAP. In this study, 300.00 mg/kg acetaminophen was used to establish liver injury model in C57BL/6 J mice. Hepatoprotective effect of AAEO on APAP-induced hepatotoxicity in mice was investigated by detecting liver function enzymes and histopathological examination. Secondly, UPLC-MS/MS was used to analyze the to analyze the small molecule metabolites and metabolic pathways induced by AAEO treatment; In addition, the effect of AAEO on APAP-induced oxidative stress and inflammation were evaluated by detecting the levels of glutathione peroxidase 4, malondialdehyde, reactive oxygen species and inflammatory factors. Finally, the active components of AAEO were preliminarily screened by cellular assays. The hepatoprotective effect of AAEO against APAP-induced hepatotoxicity was examined through the Western blotting, after the CYP2E1 gene was knocked down in AML12 cells by siRNA transfection. Compared with the APAP group, AAEO could reduce the abnormal increase in the levels of liver function enzymes caused by APAP. AAEO could enhance the antioxidant capacity by down-regulating the biosynthesis pathway of unsaturated fatty acids and promoting the activity of antioxidant enzymes SOD and CAT in liver tissue induced by APAP. Our study revealed that AAEO promoted GSH synthesis and covalently combined to form APAP-GSH conjugates to reduce the accumulation of APAP in liver tissue. In addition, the chemical constituents in AAEO were analyzed by GC-MS/MS, and it was determined to identify that dihydro-beta-ionone and (-)-verbenone in AAEO might have a significant protective effect on hepatocyte survival after APAP exposure. Further studies on the hepatoprotective mechanism of AAEO indicated that it might reduce the production of toxic metabolites by regulating CYP2E1 levels. AAEO exerted hepatoprotective effects on acetaminophen-induced hepatotoxicity in mice via regulating the activity of CYP2E1 and regulating the γ-glutamyl cycle pathway.

Cui W ，Cao Q ，Liu L ，Yin X ，Wang X ，Zhao Y ，Wang Y ，Wei B ，Xu X ，Tang Y ... -  《-》

Shenqi Pill alleviates acetaminophen-induced liver injury: a comprehensive strategy of network pharmacology and spectrum-effect relationship reveals mechanisms and active components.

Acetaminophen (APAP), commonly used for its antipyretic and analgesic properties, can cause severe liver injury or even acute liver failure when overdosed. However, the options for treating APAP-induced liver toxicity are limited. Shenqi Pill (SQP), a traditional Chinese herbal formula, has shown effectiveness in treating various liver ailments. SQP consists of cinnamon, aconite, rehmannia, cornus, peony bark, Chinese yam, poria, and alisma in a ratio of 1:1:8:4:3:4:3:3. However, the mechanisms and active components of SQP that counteract drug-induced liver injury (DILI) are not well understood. This study aimed to explore the protective effects of SQP against APAP-induced liver injury in both laboratory and animal settings. It seeks to identify the active components and potential mechanisms by which SQP targets mitochondria to alleviate liver damage. A mouse model with APAP-induced liver injury was established to assess SQP's therapeutic impact. This study then analyzed the components of SQP using UPLC-Q-TOF-MS in both in vivo and in vitro environments. Network pharmacology and the GEO database helped predict potential pathways and targets. Potential active components were identified through spectrum-effect relationship analysis and validated their efficacy using Seahorse assays and molecular docking. Treatment with SQP significantly reduced liver dysfunction, tissue damage, lipid metabolic disruptions, and inflammation caused by APAP in mice. In cellular tests, SQP-treated serum notably enhanced mitochondrial function, maintained membrane potential, decreased ROS levels, and prevented mitochondrial permeability transition pore opening. Biochemically, SQP reversed the suppression of p-AMPK, p-ACC, CPT1, and ACADM expression caused by APAP overdose. This study identified 97 in vitro and 24 in vivo components of SQP, with eight showing significant mitochondrial benefits. Molecular docking studies suggest that fuziline and paeoniflorin could activate AMPK. SQP effectively mitigates APAP-induced liver injury by enhancing mitochondrial function via the AMPK-ACC-CPT1-ACADM pathway. Moreover, this study introduces a novel strategy for analyzing the relationship between the chemical and pharmacological properties of drug-containing serum, successfully identifying compounds with mitochondrial activity. Fuziline and paeoniflorin, in particular, emerge as promising mitochondrial protectants and warrant further investigation. This research underpins the development of innovative treatments for DILI using SQP and its components.

Luo Q ，Li X ，Huang J ，Zhao L ，Liu L ，Huang S ，Xu Y ，Qiu P ，Li C ... -  《-》

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.

Zhang C ，Liu X ，Liu X ，Hua R ，Liu H ，Ma J ，Zou D ，Wang G ，Yuan Q ，Wang B ，Wei S ，Chen Y ... -  《-》