Drug-Induced Fatty Liver Disease (DIFLD): A Comprehensive Analysis of Clinical, Biochemical, and Histopathological Data for Mechanisms Identification and Consistency with Current Adverse Outcome Pathways.

Drug induced fatty liver disease (DIFLD) is a form of drug-induced liver injury (DILI), which can also be included in the more general metabolic dysfunction-associated steatotic liver disease (MASLD), which specifically refers to the accumulation of fat in the liver unrelated to alcohol intake. A bi-directional relationship between DILI and MASLD is likely to exist: while certain drugs can cause MASLD by acting as pro-steatogenic factors, MASLD may make hepatocytes more vulnerable to drugs. Having a pre-existing MASLD significantly heightens the likelihood of experiencing DILI from certain medications. Thus, the prevalence of steatosis within DILI may be biased by pre-existing MASLD, and it can be concluded that the genuine true incidence of DIFLD in the general population remains unknown. In certain individuals, drug-induced steatosis is often accompanied by concomitant injury mechanisms such as oxidative stress, cell death, and inflammation, which leads to the development of drug-induced steatohepatitis (DISH). DISH is much more severe from the clinical point of view, has worse prognosis and outcome, and resembles MASH (metabolic-associated steatohepatitis), as it is associated with inflammation and sometimes with fibrosis. A literature review of clinical case reports allowed us to examine and evaluate the clinical features of DIFLD and their association with specific drugs, enabling us to propose a classification of DIFLD drugs based on clinical outcomes and pathological severity: Group 1, drugs with low intrinsic toxicity (e.g., ibuprofen, naproxen, acetaminophen, irinotecan, methotrexate, and tamoxifen), but expected to promote/aggravate steatosis in patients with pre-existing MASLD; Group 2, drugs associated with steatosis and only occasionally with steatohepatitis (e.g., amiodarone, valproic acid, and tetracycline); and Group 3, drugs with a great tendency to transit to steatohepatitis and further to fibrosis. Different mechanisms may be in play when identifying drug mode of action: (1) inhibition of mitochondrial fatty acid ß-oxidation; (2) inhibition of fatty acid transport across mitochondrial membranes; (3) increased de novo lipid synthesis; (4) reduction in lipid export by the inhibition of microsomal triglyceride transfer protein; (5) induction of mitochondrial permeability transition pore opening; (6) dissipation of the mitochondrial transmembrane potential; (7) impairment of the mitochondrial respiratory chain/oxidative phosphorylation; (8) mitochondrial DNA damage, degradation and depletion; and (9) nuclear receptors (NRs)/transcriptomic alterations. Currently, the majority of, if not all, adverse outcome pathways (AOPs) for steatosis in AOP-Wiki highlight the interaction with NRs or transcription factors as the key molecular initiating event (MIE). This perspective suggests that chemical-induced steatosis typically results from the interplay between a chemical and a NR or transcription factors, implying that this interaction represents the primary and pivotal MIE. However, upon conducting this exhaustive literature review, it became evident that the current AOPs tend to overly emphasize this interaction as the sole MIE. Some studies indeed support the involvement of NRs in steatosis, but others demonstrate that such NR interactions alone do not necessarily lead to steatosis. This view, ignoring other mitochondrial-related injury mechanisms, falls short in encapsulating the intricate biological mechanisms involved in chemically induced liver steatosis, necessitating their consideration as part of the AOP's map road as well.

Efficient hepatocyte differentiation of primary human hepatocyte-derived organoids using three dimensional nanofibers (HYDROX) and their possible application in hepatotoxicity research.

Human liver organoids are in vitro three dimensionally (3D) cultured cells that have a bipotent stem cell phenotype. Translational research of human liver organoids for drug discovery has been limited by the challenge of their low hepatic function compared to primary human hepatocytes (PHHs). Various attempts have been made to develop functional hepatocyte-like cells from human liver organoids. However, none have achieved the same level of hepatic functions as PHHs. We here attempted to culture human liver organoids established from cryopreserved PHHs (PHH-derived organoids), using HYDROX, a chemically defined 3D nanofiber. While the proliferative capacity of PHH-derived organoids was lost by HYDROX-culture, the gene expression levels of drug-metabolizing enzymes were significantly improved. Enzymatic activities of cytochrome P450 3A4 (CYP3A4), CYP2C19, and CYP1A2 in HYDROX-cultured PHH-derived organoids (Org-HYDROX) were comparable to those in PHHs. When treated with hepatotoxic drugs such as troglitazone, amiodarone and acetaminophen, Org-HYDROX showed similar cell viability to PHHs, suggesting that Org-HYDROX could be applied to drug-induced hepatotoxicity tests. Furthermore, Org-HYDROX maintained its functions for up to 35 days and could be applied to chronic drug-induced hepatotoxicity tests using fialuridine. Our findings demonstrated that HYDROX could possibly be a novel biomaterial for differentiating human liver organoids towards hepatocytes applicable to pharmaceutical research.

Hepatic recruitment of myeloid-derived suppressor cells upon liver injury promotes both liver regeneration and fibrosis.

BACKGROUND: The liver regeneration is a highly complicated process depending on the close cooperations between the hepatocytes and non-parenchymal cells involving various inflammatory cells. Here, we explored the role of myeloid-derived suppressor cells (MDSCs) in the processes of liver regeneration and liver fibrosis after liver injury. METHODS: We established four liver injury models of mice including CCl4-induced liver injury model, bile duct ligation (BDL) model, concanavalin A (Con A)-induced hepatitis model, and lipopolysaccharide (LPS)-induced hepatitis model. The intrahepatic levels of MDSCs (CD11b+Gr-1+) after the liver injury were detected by flow cytometry. The effects of MDSCs on liver tissues were analyzed in the transwell co-culture system, in which the MDSCs cytokines including IL-10, VEGF, and TGF-ß were measured by ELISA assay and followed by being blocked with specific antibodies. RESULTS: The intrahepatic infiltrations of MDSCs with surface marker of CD11b+Gr-1+ remarkably increased after the establishment of four liver injury models. The blood served as the primary reservoir for hepatic recruitment of MDSCs during the liver injury, while the bone marrow appeared play a compensated role in increasing the number of MDSCs at the late stage of the inflammation. The recruited MDSCs in injured liver were mainly the M-MDSCs (CD11b+Ly6G-Ly6Chigh) featured by high expression levels of cytokines including IL-10, VEGF, and TGF-ß. Co-culture of the liver tissues with MDSCs significantly promoted the proliferation of both hepatocytes and hepatic stellate cells (HSCs). CONCLUSIONS: The dramatically and quickly infiltrated CD11b+Gr-1+ MDSCs in injured liver not only exerted pro-proliferative effects on hepatocytes, but also accounted for the activation of profibrotic HSCs.

Peroxynitrite imaging in ferroptosis-mediated drug-induced liver injury with a near-infrared fluorescence probe.

BACKGROUND: Over-consumption of drugs can result in drug-induced liver damage (DILI), which can worsen liver failure. Numerous studies have shown the significant role ferroptosis plays in the pathophysiology of DILI, which is typified by a marked imbalance between the generation and breakdown of lipid reactive oxygen species (ROS). The content of peroxynitrite (ONOO-) rapidly increased during this process and was thought to be a significant marker of early liver injury. Therefore, the construction of fluorescence probe for the detection and imaging of ONOO- holds immense importance in the early diagnosis and treatment of ferroptosis-mediated DILI. RESULTS: We designed a probe DILI-ONOO based on the ICT mechanism for the purpose of measuring and visualizing ONOO- in ferroptosis-mediated DILI processes and associated studies. This probe exhibited significant fluorescence changes with good sensitivity, selectivity, and can image exogenous and endogenous ONOO- in cells with low cytotoxicity. Using this probe, we were able to show changes in ONOO- content in ferroptosis-mediated DILI cells and mice models induced by the intervention of acetaminophen (APAP) and isoniazid (INH). By measuring the concentration of ferroptosis-related indicators in mice liver tissue, we were able to validate the role of ferroptosis in DILI. It is worth mentioning that compared to existing alanine transaminase (ALT) and aspartate aminotransferase (AST) detection methods, this probe can achieve early identification of DILI prior to serious liver injury. SIGNIFICANCE: This work has significant reference value in researching the relationship between ferroptosis and DILI and visualizing research. The results indicate a strong correlation between the progression of DILI and ferroptosis. Additionally, the use of DILI-ONOO shows promise in investigating the DILI process and assessing the effectiveness of medications in treating DILI.

Exposure to polystyrene nanoplastics induces hepatotoxicity involving NRF2-NLRP3 signaling pathway in mice.

Nanoplastic contamination has been of intense concern by virtue of the potential threat to human and ecosystem health. Animal experiments have indicated that exposure to nanoplastics (NPs) can deposit in the liver and contribute to hepatic injury. To explore the mechanisms of hepatotoxicity induced by polystyrene-NPs (PS-NPs), mice and AML-12 hepatocytes were exposed to different dosages of 20 nm PS-NPs in this study. The results illustrated that in vitro and in vivo exposure to PS-NPs triggered excessive production of reactive oxygen species and repressed nuclear factor erythroid-derived 2-like 2 (NRF2) antioxidant pathway and its downstream antioxidase expression, thus leading to hepatic oxidative stress. Moreover, PS-NPs elevated the levels of NLRP3, IL-1ß and caspase-1 expression, along with an activation of NF-κB, suggesting that PS-NPs induced hepatocellular inflammatory injury. Nevertheless, the activaton of NRF2 signaling by tert-butylhydroquinone mitigated PS-NPs-caused oxidative stress and inflammation, and inbihited NLRP3 and caspase-1 expression. Conversely, the rescuing effect of NRF2 signal activation was dramatically supressed by treatment with NRF2 inhibitor brusatol. In summary, our results demonstrated that NRF2-NLRP3 pathway is involved in PS-NPs-aroused hepatotoxicity, and the activation of NRF2 signaling can protect against PS-NPs-evoked liver injury. These results provide novel insights into the hepatotoxicity elicited by NPs exposure.

Acute liver failure histopathology: A 5-year retrospective study.

BACKGROUND: Acute liver failure (ALF) is a rare, life-threatening disease of diverse etiology. It is defined as severe acute liver injury for fewer than 26 weeks' duration with encephalopathy and impaired synthetic function (international normalized ratio [INR] of 1.5 or higher) in a patient without cirrhosis or pre-existing liver disease. The diagnosis rests mainly on the clinical ground with wide range of pathological features. The present study seeks to explore the diverse histological patterns observed in cases for ALF and assess their usefulness in determining the underlying causes for the condition. METHODOLOGY: A retrospective cross-sectional study was conducted among patients of ALF who underwent liver transplant and transjugular liver biopsy over a five-year period. From 1082 explant liver and 2446 liver biopsies, 22 cases of ALF (10 explants and 12 liver biopsies) were included in the study. Clinical and laboratory details were retrieved and histological findings were reviewed. RESULT: Age ranged from 10 to 72 years (mean age, 40 years). There was a female predominance with a male:female ratio of 1:1.7. The commonest cause for ALF was virus-induced hepatocellular damage in 36.3% (eight patients), followed by autoimmune hepatitis in 22.7% (five patients), drug-induced liver injury (DILI) in 18.1% (four patients), cryptogenic in 13.6% (three patients) and ischemic injury secondary to large vein thrombosis in 9.0% (two) patients. The histological patterns identified were categorized into six categories. A more comprehensive morphological evaluation was conducted specifically for cases of ALF associated with autoimmune hepatitis (AIH) and compared with other cases of ALF. CONCLUSION: In summary, our present study illustrates a morphological overlap in various patterns for the purpose of etiological assessment. In cases of AIH ALF, the presence of portal plasma cell infiltrate and central perivenulitis were identified as significant histological features to guide diagnosis.

Enhanced hepatotoxicity in zebrafish due to co-exposure of microplastics and sulfamethoxazole: Insights into ROS-mediated MAPK signaling pathway regulation.

The combined pollution of microplastics (MPs) and sulfamethoxazole (SMZ) often occurs in aquatic ecosystems, posing a serious threat to animal and human health. However, little is known about the liver damage caused by the single or co-exposure of MPs and SMZ, and its specific mechanisms are still poorly understood. In this study, we investigated the effects of co-exposure to 20 µm or 80 nm MPs and SMZ in both larval and adult zebrafish models. Firstly, we observed a significant decrease in the number of hepatocytes and the liver damage in larval zebrafish worsened following co-exposure to SMZ and MPs. Additionally, the number of macrophages and neutrophils decreased, while the expression of inflammatory cytokines and antioxidant enzyme activities increased after co-exposure in larval zebrafish. Transcriptome analysis revealed significant changes in gene expression in the co-exposed groups, particularly in processes related to oxidation-reduction, inflammatory response, and the MAPK signaling pathway in the liver of adult zebrafish. Co-exposure of SMZ and MPs also promoted hepatocyte apoptosis and inhibited proliferation levels, which was associated with the translocation of Nrf2 from the cytoplasm to the nucleus and an increase in protein levels of Nrf2 and NF-kB p65 in the adult zebrafish. Furthermore, our pharmacological experiments demonstrated that inhibiting ROS and blocking the MAPK signaling pathway partially rescued the liver injury induced by co-exposure both in larval and adult zebrafish. In conclusion, our findings suggest that co-exposure to SMZ and MPs induces hepatic dysfunction through the ROS-mediated MAPK signaling pathway in zebrafish. This information provides novel insights into the potential environmental risk of MPs and hazardous pollutants co-existence in aquatic ecosystems.

Bifidobacterium adolescentis-derived hypaphorine alleviates acetaminophen hepatotoxicity by promoting hepatic Cry1 expression.

Acetaminophen (APAP)-induced liver injury (AILI) is a pressing public health concern. Although evidence suggests that Bifidobacterium adolescentis (B. adolescentis) can be used to treat liver disease, it is unclear if it can prevent AILI. In this report, we prove that B. adolescentis significantly attenuated AILI in mice, as demonstrated through biochemical analysis, histopathology, and enzyme-linked immunosorbent assays. Based on untargeted metabolomics and in vitro cultures, we found that B. adolescentis generates microbial metabolite hypaphorine. Functionally, hypaphorine inhibits the inflammatory response and hepatic oxidative stress to alleviate AILI in mice. Transcriptomic analysis indicates that Cry1 expression is increased in APAP-treated mice after hypaphorine treatment. Overexpression of Cry1 by its stabilizer KL001 effectively mitigates liver damage arising from oxidative stress in APAP-treated mice. Using the gene expression omnibus (GEO) database, we verified that Cry1 gene expression was also decreased in patients with APAP-induced acute liver failure. In conclusion, this study demonstrates that B. adolescentis inhibits APAP-induced liver injury by generating hypaphorine, which subsequently upregulates Cry1 to decrease inflammation and oxidative stress.

In Situ Self-Assembling Liver Spheroids with Synthetic Nanoscaffolds for Preclinical Drug Screening Applications.

Drug-induced liver injury (DILI) is one of the most common reasons for acute liver failure and a major reason for the withdrawal of medications from the market. There is a growing need for advanced in vitro liver models that can effectively recapitulate hepatic function, offering a robust platform for preclinical drug screening applications. Here, we explore the potential of self-assembling liver spheroids in the presence of electrospun and cryomilled poly(caprolactone) (PCL) nanoscaffolds for use as a new preclinical drug screening tool. This study investigated the extent to which nanoscaffold concentration may have on spheroid size and viability and liver-specific biofunctionality. The efficacy of our model was further validated using a comprehensive dose-dependent acetaminophen toxicity protocol. Our findings show the strong potential of PCL-based nanoscaffolds to facilitate in situ self-assembly of liver spheroids with sizes under 350 µm. The presence of the PCL-based nanoscaffolds (0.005 and 0.01% w/v) improved spheroid viability and the secretion of critical liver-specific biomarkers, namely, albumin and urea. Liver spheroids with nanoscaffolds showed improved drug-metabolizing enzyme activity and greater sensitivity to acetaminophen compared to two-dimensional monolayer cultures and scaffold-free liver spheroids. These promising findings highlight the potential of our nanoscaffold-based liver spheroids as an in vitro liver model for drug-induced hepatotoxicity and drug screening.

Cu exposure induces liver inflammation via regulating gut microbiota/LPS/liver TLR4 signaling axis.

Copper (Cu) serves as an essential cofactor in all organisms, yet excessive Cu exposure is widely recognized for its role in inducing liver inflammation. However, the precise mechanism by which Cu triggers liver inflammation in ducks, particularly in relation to the interplay in gut microbiota regulation, has remained elusive. In this investigation, we sought to elucidate the impact of Cu exposure on liver inflammation through gut-liver axis in ducks. Our findings revealed that Cu exposure markedly elevated liver AST and ALT levels and induced liver inflammation through upregulating pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) and triggering the LPS/TLR4/NF-κB signaling pathway. Simultaneously, Cu exposure induced alterations in the composition of intestinal flora communities, notably increasing the relative abundance of Sphingobacterium, Campylobacter, Acinetobacter and reducing the relative abundance of Lactobacillus. Cu exposure significantly decreased the protein expression related to intestinal barrier (Occludin, Claudin-1 and ZO-1) and promoted the secretion of intestinal pro-inflammatory cytokines. Furthermore, correlation analysis was observed that intestinal microbiome and gut barrier induced by Cu were closely related to liver inflammation. Fecal microbiota transplantation (FMT) experiments further demonstrated the microbiota-depleted ducks transplanting fecal samples from Cu-exposed ducks disturbed the intestinal dysfunction, which lead to impaire liver function and activate the liver inflammation. Our study provided insights into the mechanism by which Cu exposure induced liver inflammation in ducks through the regulation of gut-liver axis. These results enhanced our comprehension of the potential mechanisms driving Cu-induced hepatotoxicity in avian species.

Multi-omics and chemical profiling approaches to understand the material foundation and pharmacological mechanism of sophorae tonkinensis radix et rhizome-induced liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Sophorae tonkinensis Radix et Rhizoma (STR) is an extensively applied traditional Chinese medicine (TCM) in southwest China. However, its clinical application is relatively limited due to its hepatotoxicity effects. AIM OF THE STUDY: To understand the material foundation and liver injury mechanism of STR. MATERIALS AND METHODS: Chemical compositions in STR and its prototypes in mice were profiled by ultra-performance liquid chromatography coupled quadrupole-time of flight mass spectrometry (UPLC-Q/TOF MS). STR-induced liver injury (SILI) was comprehensively evaluated by STR-treated mice mode. The histopathologic and biochemical analyses were performed to evaluate liver injury levels. Subsequently, network pharmacology and multi-omics were used to analyze the potential mechanism of SILI in vivo. And the target genes were further verified by Western blot. RESULTS: A total of 152 compounds were identified or tentatively characterized in STR, including 29 alkaloids, 21 organic acids, 75 flavonoids, 1 quinone, and 26 other types. Among them, 19 components were presented in STR-medicated serum. The histopathologic and biochemical analysis revealed that hepatic injury occurred after 4 weeks of intragastric administration of STR. Network pharmacology analysis revealed that IL6, TNF, STAT3, etc. were the main core targets, and the bile secretion might play a key role in SILI. The metabolic pathways such as taurine and hypotaurine metabolism, purine metabolism, and vitamin B6 metabolism were identified in the STR exposed groups. Among them, taurine, hypotaurine, hypoxanthine, pyridoxal, and 4-pyridoxate were selected based on their high impact value and potential biological function in the process of liver injury post STR treatment. CONCLUSIONS: The mechanism and material foundation of SILI were revealed and profiled by a multi-omics strategy combined with network pharmacology and chemical profiling. Meanwhile, new insights were taken into understand the pathological mechanism of SILI.

Inhibition of mitochondrial ROS-mediated necroptosis by Dendrobium nobile Lindl. alkaloids in carbon tetrachloride induced acute liver injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Dendrobium nobile Lindl. (DNL) is a well-known traditional Chinese medicine that has been recorded in the Chinese Pharmacopoeia (2020 edition). The previous data showed that Dendrobium nobile Lindl. alkaloids (DNLA) protect against CCl4-induced liver damage via oxidative stress reduction and mitochondrial function improvement, yet the exact regulatory signaling pathways remain undefined. AIM OF THE STUDY: The aim of the present study was to investigate the role of necroptosis in the mode of CCl4-induced liver injury and determine whether DNLA protects against CCl4-induced acute liver injury (ALI) by inhibiting mitochondrial ROS (mtROS)-mediated necroptosis. MATERIALS AND METHODS: DNLA was extracted from DNL, and the content was determined using liquid chromatograph mass spectrometer (LC-MS). In vivo experiments were conducted in C57BL/6J mice. Animals were administrated with DNLA (20 mg/kg/day, ig) for 7 days, and then challenged with CCl4 (20 µL/kg, ip). CCl4-induced liver injury in mice was evaluated through the assessment of biochemical indicators in mouse serum and histopathological examination of hepatic tissue using hematoxylin and eosin (H&E) staining. The protein and gene expressions were determined with western blotting and quantitative real-time PCR (RT-qPCR). Reactive oxygen species (ROS) production was detected using the fluorescent probe DCFH-DA, and mitochondrial membrane potential was evaluated using a fluorescent probe JC-1. The mtROS level was assessed using a fluorescence probe MitoSOX. RESULTS: DNLA lessened CCl4-induced liver injury, evident by reduced AST and ALT levels and improved liver pathology. DNLA suppressed necroptosis by decreasing RIPK1, RIPK3, and MLKL phosphorylation, concurrently enhancing mitochondrial function. It also broke the positive feedback loop between mtROS and RIPK1/RIPK3/MLKL activation. Similar findings were observed with resveratrol and mitochondrial SOD2 overexpression, both mitigating mtROS and necroptosis. Further mechanistic studies found that DNLA inhibited the oxidation of RIPK1 and reduced its phosphorylation level, whereby lowering the phosphorylation of RIPK3 and MLKL, blocking necroptosis, and alleviating liver injury. CONCLUSIONS: This study demonstrates that DNLA inhibits the necroptosis signaling pathway by reducing mtROS mediated oxidation of RIPK1, thereby reducing the phosphorylation of RIPK1, RIPK3, and MLKL, and protecting against liver injury.

Bifenox induces hepatotoxicity and vascular toxicity in zebrafish embryos via ROS production and alterations in signaling pathways.

Existing evidence shows that currently used pesticides pose toxicological risks to exposed wildlife. Chemically, bifenox belongs to diphenyl ethers, a well-known group of herbicides. Its mechanism of action primarily involves inducing lipid peroxidation and blocking protoporphyrinogen oxidases. Toxicity of diphenyl ether herbicides has been elucidated in animal cells; however, in vivo toxicological evaluations of bifenox are required to determine its unexpected effects. This study aimed to determine the negative effects of bifenox, and its effects on higher eukaryotes. We found that early stages of zebrafish embryo exposed to bifenox demonstrated increased mortality and physiological defects, based on the LC50 value. Bifenox severely inhibited blood vessel growth by reducing key elements of complex connectivity; fluorescently tagged transgenic lines (fli1a:EGFP) showed morphological changes. Additionally, transgenic lines that selectively identified hepatocytes (fabp10a:DsRed) showed reduced fluorescence, indicating that bifenox may inhibit liver development. To evaluate the level of oxidative stress, we used 2',7'-dichlorofluorescein diacetate (DCFH-DA) probes in zebrafish embryos to identify the underlying mechanisms causing developmental damage. Our findings demonstrate that exposure to bifenox causes abnormalities in the hepatic and cardiovascular systems during zebrafish embryogenesis. Therefore, this study provides new information for the evaluation of toxicological risks of bifenox in vertebrates.

Salvia miltiorrhiza polysaccharide mitigates AFB1-induced liver injury in rabbits.

Aflatoxin B1 (AFB1) is one of the common dietary contaminants worldwide, which can harm the liver of humans and animals. Salvia miltiorrhiza polysaccharide (SMP) is a natural plant-derived polysaccharide with numerous pharmacological activities, including hepatoprotective properties. The purpose of this study is to explore the intervention effect of SMP on AFB1-induced liver injury and its underlying mechanisms in rabbits. The rabbits were administered AFB1 (25 µg/kg/feed) and or treatment with SMP (300, 600, 900 mg/kg/feed) for 42 days. The results showed that SMP effectively alleviated the negative impact of AFB1 on rabbits' productivity by increasing average daily weight gain (ADG) and feed conversion rate (FCR). SMP reduced aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels in serum, ameliorating AFB1-induced hepatic pathological changes. Additionally, SMP enhanced superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) activity, and inhibited reactive oxygen species (ROS), malondialdehyde (MDA), 4-Hydroxynonenal (4-HNE), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression, thus mitigating AFB1-induced oxidative stress and inflammatory responses. Moreover, SMP upregulated the expression of nuclear factor E2 related factor 2 (Nrf2), heme oxygenase 1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1) and B-cell lymphoma 2 (Bcl2) while downregulating kelch like ECH associated protein 1 (Keap1), cytochrome c (cyt.c), caspase9, caspase3, and Bcl-2-associated X protein (Bax) expression, thereby inhibiting AFB1-induced hepatocyte apoptosis. Consequently, our findings conclude that SMP can mitigate AFB1-induced liver damage by activating the Nrf2/HO-1 pathway and inhibiting mitochondria-dependent apoptotic pathway in rabbits.

Curcumin alleviates zearalenone-induced liver injury in mice by scavenging reactive oxygen species and inhibiting mitochondrial apoptosis pathway.

Curcumin (CUR) is a compound extracted from turmeric that has a variety of functions including antioxidant and anti-inflammatory. As an estrogen-like mycotoxin, zearalenone (ZEN) not only attacks the reproductive system, but also has toxic effects on the liver. However, whether CUR can alleviate ZEN-induced liver injury remains unclear. This paper aims to investigate the protective effect of CUR against ZEN-induced liver injury in mice and explore the molecular mechanism involved. BALB/c mice were randomly divided into control (CON) group, CUR group (200 mg/kg b. w. CUR), ZEN group (40 mg/kg b. w. ZEN) and CUR+ZEN group (200 mg/kg b. w. CUR+40 mg/kg b. w. ZEN). 28 d after ZEN exposure and CUR treatment, blood and liver samples were collected for subsequent testing. The results showed that CUR reversed ZEN-induced hepatocyte swelling and necrosis in mice. It significantly reduced the serum alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice (p < 0.05). In addition, CUR significantly reduced hepatic ROS, malondialdehyde, hydrogen peroxide and apoptosis levels in mice (p < 0.05). Quantitative RT-PCR and Western blot results showed that CUR significantly reduced the expression of Bax and Caspase3, and reversed the increase of Nrf2, HO-1 and NQO1 expression in the liver of mice induced by ZEN (p < 0.05). In conclusion, CUR alleviated ZEN-induced liver injury in mice by scavenging ROS and inhibiting the mitochondrial apoptotic pathway.

Aqueous extract of Epimedium sagittatum (Sieb. et Zucc.) Maxim. induces liver injury in mice via pyroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Epimedium sagittatum (Sieb. et Zucc.) Maxim. has been used traditionally in Asia. It can dispel wind and cold, tonify the kidney, and strengthen bones and tendons. However, adverse effects of E. sagittatum have been reported, and the underlying mechanisms remain unclear. AIM OF THE STUDY: This study aimed to investigate liver injury caused by an aqueous extract of E. sagittatum in Institute of Cancer Research (ICR) mice and explore its potential mechanisms. MATERIALS AND METHODS: Dried E. sagittatum leaves were decocted in water to prepare aqueous extracts for ultra-high performance liquid chromatography analysis. Mice were administered an aqueous extract of E. sagittatum equivalent to either 3 g raw E. sagittatum/kg or 10 g raw E. sagittatum/kg once daily via intragastric injection for three months. The liver weights and levels of the serum biochemical parameters including alanine transaminase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), total bilirubin (TBIL), and alkaline phosphatase were measured. Hematoxylin-eosin staining was performed for histopathology. Apoptosis was detected using the TUNEL apoptosis assay kit. IL-1ß was detected using ELISA kits. Proteomics was used to identify the differentially expressed proteins. Western blot analysis was performed to determine the levels of proteins significantly affected by the aqueous extract of E. sagittatum. RESULTS: E. sagittatum treatment increased the liver weights and liver coefficients, and ALT and AST levels significantly increased (p < 0.05). A high dose of E. sagittatum significantly increased LDH and TBIL levels (p < 0.05). Ruptured cell membranes and multiple sites of inflammatory cell infiltration were also observed. No evidence of apoptosis was observed. IL-1ß levels were significantly increased (p < 0.05). The expressions of PIK3R1, p-MAP2K4, p-Jun N-terminal kinase (JNK)/JNK, p-c-Jun, VDAC2, Bax, and CYC were upregulated, whereas that of Bcl-2 was inhibited by E. sagittatum. The expression of cleaved caspase-1 was significantly increased; however, its effects on GSDMD and GSDMD-N were significantly decreased. The expression levels of cleaved caspase-3 and its effector proteins GSDME and GSDME-N significantly increased. CONCLUSIONS: Our results suggest that the aqueous extract of E. sagittatum induces liver injury in ICR mice after three months of intragastric injection via inflammatory pyroptosis.

Paeonia lactiflora Pall. ameliorates acetaminophen-induced oxidative stress and apoptosis via inhibiting the PKC-ERK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeonia lactiflora Pall. (PLP), a traditional Chinese medicine, is recognized for its antioxidative and anti-apoptotic properties. Despite its potential medicinal value, the mechanisms underlying its efficacy have been less explored, particularly in alleviating acute liver injury (ALI) caused by excessive intake of acetaminophen (APAP). AIM OF THE STUDY: This study aims to elucidate the role and mechanisms of PLP in mitigating oxidative stress and apoptosis induced by APAP. MATERIALS AND METHODS: C57BL/6 male mice were pre-treated with PLP for seven consecutive days, followed by the induction of ALI using APAP. Liver pathology was assessed using HE staining. Serum indicators, immunofluorescence (IF), immunohistochemical (IHC), and transmission electron microscopy were employed to evaluate levels of oxidative stress, ferroptosis and apoptosis. Differential expression proteins (DEPs) in the APAP-treated and PLP pre-treated groups were analyzed using quantitative proteomics. Subsequently, the potential mechanisms of PLP pre-treatment in treating ALI were validated using western blotting, molecular docking, molecular dynamics simulations, and surface plasmon resonance (SPR) analysis. RESULTS: The UHPLC assay confirmed the presence of three compounds, i.e., albiflorin, paeoniflorin, and oxypaeoniflorin. Pre-treatment with PLP was observed to ameliorate liver tissue pathological damage through HE staining. Further confirmation of efficacy of PLP in alleviating APAP-induced liver injury and oxidative stress was established through liver function serum biochemical indicators, IF of reactive oxygen species (ROS) and IHC of glutathione peroxidase 4 (GPX4) detection. However, PLP did not demonstrate a significant effect in alleviating APAP-induced ferroptosis. Additionally, transmission electron microscopy and TUNEL staining indicated that PLP can mitigate hepatocyte apoptosis. PKC-ERK pathway was identified by proteomics, and subsequent molecular docking, molecular dynamics simulations, and SPR verified binding of the major components of PLP to ERK protein. Western blotting demonstrated that PLP suppressed protein kinase C (PKC) phosphorylation, blocking extracellular signal-regulated kinase (ERK) phosphorylation and inhibiting oxidative stress and cell apoptosis. CONCLUSION: This study demonstrates that PLP possesses hepatoprotective abilities against APAP-induced ALI, primarily by inhibiting the PKC-ERK cascade to suppress oxidative stress and cell apoptosis.

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

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

Acrylamide-Aggravated Liver Injury by Activating Endoplasmic Reticulum Stress in Female Mice with Diabetes.

Acrylamide (ACR) is a common industrial contaminant with endocrine-disrupting toxicity. Numerous studies have indicated that females and diabetics are more sensitive to environmental contaminants. However, it remains unknown whether female diabetics are susceptible to ACR-induced toxicity and its potential mechanisms. Thus, the female ACR-exposure diabetic Balb/c mice model was established to address these issues. Results showed that ACR could induce liver injury in normal mice and cause more serious inflammatory cell infiltration, hepatocyte volume increase, and fusion in diabetic mice liver. Meanwhile, ACR could lead to exacerbation of diabetic symptoms in diabetic mice by disturbing the glucose and lipid metabolism in the liver, which mainly manifests as the accumulation of liver glycogen and liver lipids, the reduction of the activity/content of glycolytic and metabolizing enzyme as well as pentose phosphatase, upregulation of the gene expression in fatty acid transporter and gluconeogenesis, and downregulation of the gene expression in fatty acid synthesis and metabolism. Moreover, ACR exposure could induce oxidative stress, inflammation, and endoplasmic reticulum stress in the liver by a decrease in hepatic antioxidant enzyme activity and antioxidant content, an increase in inflammatory factor levels, and a change in the related protein expression of endoplasmic reticulum stress (ERS) and apoptosis-related pathways in diabetic mice. Statistical analysis results revealed that ACR-induced liver injury was highly correlated with inflammation and oxidative stress, and ERS and diabetic mice had a higher risk of liver injury than normal mice. Overall results suggested that female diabetic mice easily suffer from ACR-induced toxicity, and the reason was that ACR could induce further damage to the liver by worsening the condition of inflammation, oxidative stress, and ERS in the liver.

Rosmarinic acid alleviates toosendanin-induced liver injury through restoration of autophagic flux and lysosomal function by activating JAK2/STAT3/CTSC pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rosmarinic acid (RA), a natural polyphenol abundant in numerous herbal remedies, has been attracting growing interest owing to its exceptional ability to protect the liver. Toosendanin (TSN), a prominent bioactive compound derived from Melia toosendan Siebold & Zucc., boasts diverse pharmacological properties. Nevertheless, TSN possesses remarkable hepatotoxicity. Intriguingly, the potential of RA to counteract TSN-induced liver damage and its probable mechanisms remain unexplored. AIM OF THE STUDY: This study is aimed at exploring whether RA can alleviate TSN-induced liver injury and the potential mechanisms involved autophagy. MATERIALS AND METHODS: CCK-8 and LDH leakage rate assay were used to evaluate cytotoxicity. Balb/c mice were intraperitoneally administered TSN (20 mg/kg) for 24 h after pretreatment with RA (0, 40, 80 mg/kg) by gavage for 5 days. The autophagic proteins P62 and LC3B expressions were detected using western blot and immunohistochemistry. RFP-GFP-LC3B and transmission electron microscopy were applied to observe the accumulation levels of autophagosomes and autolysosomes. LysoTracker Red and DQ-BSA staining were used to evaluate the lysosomal acidity and degradation ability respectively. Western blot, immunohistochemistry and immunofluorescence staining were employed to measure the expressions of JAK2/STAT3/CTSC pathway proteins. Dual-luciferase reporter gene was used to measure the transcriptional activity of CTSC and RT-PCR was used to detect its mRNA level. H&E staining and serum biochemical assay were employed to determine the degree of damage to the liver. RESULTS: TSN-induced damage to hepatocytes and livers was significantly alleviated by RA. RA markedly diminished the autophagic flux blockade and lysosomal dysfunction caused by TSN. Mechanically, RA alleviated TSN-induced down-regulation of CTSC by activating JAK2/STAT3 signaling pathway. CONCLUSION: RA could protect against TSN-induced liver injury by activating the JAK2/STAT3/CTSC pathway-mediated autophagy and lysosomal function.