Apoptotic Hepatocytes Research Articles

The effect of chronic predatory stress on the histostructure of the rat liver

One of the topical issues of modern circulatory physiology is the study of the peculiarities of the influence of chronic emotional stress on the histostructure of the liver.Aim: To study changes in the histostructure of the liver in the simulation of chronic predatory stress.Methodology and Research Methods. The study was conducted on 60 male Wistar rats weighing 220–370 g, divided into control and experimental groups of 30 rats each. Modeling of chronic predatory stress was performed according to the method of V.E. Zeilikman et al. (2021). Histological examination of the liver was performed in animals of the control and experimental groups. The preparations were stained with hematoxylin-eosin.Results. Under conditions of chronic predatory stress, stress-induced changes develop in the liver of rats. Transformation of hepatocytes occurs with the development of dystrophic changes, expansion and fullness of sinusoidal capillaries with compression of hepatic beams and destruction of hepatic lobules, a decrease in the parenchymal-stromal ratio. Foci of foamy accumulation of macrophages appear without signs of necrosis, single or group apoptotic hepatocytes. Necrotic foci are formed. The number of Kupffer cells and binucleated hepatocytes increases.Conclusion. With chronic predatory stress, the processes of dystrophy, necrosis and apoptosis of hepatocytes, signs of phagocytosis and regenerative processes, and decreased immune protection develop in the liver of rats.

Integrative bioinformatic and experimental analysis of benzoylbenzodioxol derivatives: hypoglycemic potential in diabetic mice.

We investigated the hypoglycemic activity and pharmacokinetic study of two synthesized benzoyl benzodioxol derivatives, compound I (methyl 2-(6-(2-bromobenzoyl)benzo[d][1,3]dioxol-5-yl)acetate), and compound II, 2-(6-benzoylbenzo[d][1,3]dioxol-5-yl)acetic acid, which showed very strong α-amylase inhibiting activity in our previous study. Then, diabetes was induced by the injection of streptozotocin to mice. The molecular docking simulations and analyses of density functional theory analyses were conducted to study the binding interactions with human pancreatic alpha-amylase, and their pharmacokinetic properties were further evaluated by ADMET profiling. Compound I showed the most important hypoglycemic effect, decreasing the blood glucose by 32.4%, higher than that of compound II by 14.8% and even the positive control acarbose by 22.9%. Histopathological examination revealed that diabetic livers showed portal inflammation with some apoptotic hepatocytes due to streptozotocin treatment, whereas controls without any treatment maintained normal liver architecture. Molecular docking studies gave results for the best binding affinity of the compound I, through its strong water bridges and π-π interactions, and also through analysis with density functional theory, was more stable and reactive when compared to compound II. Further ADMET analysis showed that both compounds shared a promising pharmacokinetic profile, and compound I had the potential for CNS penetration. Thus, compound I was selected as the best candidate for developing new hypoglycemic agents with potent efficacy, good binding interactions, and excellent pharmacokinetic properties.

Putative role of 6-chogaol against tramadol-induced hepatotoxicity in albino rats via anti-inflammatory, antifibrotic, and antiapoptotic effects

Tramadol is a commonly used drug to relieve pain and avoid premature ejaculation in males with hepatotoxic effects, and 6-chogaol has potent anti-inflammatory and hepatoprotective properties.The work impetus is probing the hepatoprotective mechanisms of 6-chogaol against tramadol hepatoxicity. Twenty adult male rats were enrolled to obtain four equal groups [control group (G1), 6-chogaol group (G2), tramadol group (G3), and 6-chogaol+tramadol group (G4)]. Liver specimens were excised and processed to evaluate hepatocyte injury through histopathological (HP), immunohistochemical (IHC), flow cytometry, and biochemical investigations. The HP study exhibited hepatic injury in G3 hepatocytes (inflammatory cell infiltration, hepatic fibrosis, and disturbed liver structure). The IHC study showed a significant rise in caspase-3 and reduced PCNA immuno-expression (IE). Likewise, the flow cytometry and biochemical experiments exhibited a substantial elevation of apoptotic hepatocytes and the serum levels of IL-1β, IL-6, TNF-α, ALP, ALT, and AST in G3. In contrast, G4 rats significantly improved in all HP, IHC, flow cytometry, and biochemical parameters. Collectively, tramadol intake exerted harmful toxic effects on hepatocytes, whereas 6-Shogaol hampered these changes and served as a natural hepatoprotective agent. Therefore, we advise concurrent intake of 6-Shogaol supplement with tramadol to preserve the integrity of hepatic tissues.

Therapeutic Promises of Ferulic Acid and its Derivatives on Hepatic damage Related with Oxidative Stress and Inflammation: A Review with Mechanisms.

Ferulic acid (FA) is a naturally occurring phenolic compound commonly found in the plant Ferula communis. This study aims to investigate the hepatoprotective effect of FA and its derivatives (methyl ferulic acid and trans-ferulic acid) against oxidative stress and inflammation-related hepatotoxicity due to toxicants based on the results of different non-clinical and preclinical tests. For this, data was collected from different reliable electronic databases such as PubMed, Google Scholar, and ScienceDirect, etc. The results of this investigation demonstrated that FA and its derivatives have potent hepatoprotective effects against oxidative stress and inflammation-related damage. The findings also revealed that these protective effects are due to the antioxidant and anti-inflammatory effects of the chemical compound. FA and its analogues significantly inhibit free radical generation and hinder the effects of proinflammatory markers and inflammatory enzymes, resulting in diminished cytotoxic and apoptotic hepatocyte death. The compounds also prevent intracellular lipid accumulation and provide protective effects.

ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner

BackgroundProgrammed cell death is an important mechanism for the development of hepatic ischemia and reperfusion (IR) injury, and multiple novel forms of programmed cell death are involved in the pathological process of hepatic IR. ERRFI1 is involved in the regulation of cell apoptosis in myocardial IR. However, the function of ERRFI1 in hepatic IR injury and its modulation of programmed cell death remain largely unknown.MethodsHere, we performed functional and molecular mechanism studies in hepatocyte-specific knockout mice and ERRFI1-silenced hepatocytes to investigate the significance of ERRFI1 in hepatic IR injury. The histological severity of livers, enzyme activities, hepatocyte apoptosis and ferroptosis were determined.ResultsERRFI1 expression increased in liver tissues from mice with IR injury and hepatocytes under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions. Hepatocyte-specific ERRFI1 knockout alleviated IR-induced liver injury in mice by reducing cell apoptosis and ferroptosis. ERRFI1 knockdown reduced apoptotic and ferroptotic hepatocytes induced by OGD/R. Mechanistically, ERRFI1 interacted with GRB2 to maintain its stability by hindering its proteasomal degradation. Overexpression of GRB2 abrogated the effects of ERRFI1 silencing on hepatocyte apoptosis and ferroptosis.ConclusionsOur results revealed that the ERRFI1-GRB2 interaction and GRB2 stability are essential for ERRFI1-regulated hepatic IR injury, indicating that inhibition of ERRFI1 or blockade of the ERRFI1-GRB2 interaction may be potential therapeutic strategies in response to hepatic IR injury.

TREM2 protects against inflammation by regulating the release of mito-DAMPs from hepatocytes during liver fibrosis

BackgroundLiver fibrosis typically develops as a result of chronic liver injury, which involves inflammatory and regenerative processes. The triggering receptor expressed on myeloid cells 2 (TREM2), predominantly expressing in hepatic non-parenchymal cells, plays a crucial role in regulating the function of macrophages. However, its mechanism in liver fibrosis remains poorly defined. MethodsExperimental liver fibrosis models in wild type and TREM2−/− mice, and in vitro studies with AML-12 cells and Raw264.7 cells were conducted. The expression of TREM2 and related molecular mechanism were evaluated by using samples from patients with liver fibrosis. ResultsWe demonstrated that TREM2 was upregulated in murine model with liver fibrosis. Mice lacking TREM2 exhibited reduced phagocytosis activity in macrophages following carbon tetrachloride (CCl4) intoxication. As a result, there was an increased accumulation of necrotic apoptotic hepatocytes. Additionally, TREM2 knockout aggravated the release of mitochondrial damage-associated molecular patterns (mito-DAMPs) from dead hepatocytes during CCl4 exposure, and further promoted the occurrence of macrophage-mediated M1 polarization. Then, TREM2−/− mice showed more serious fibrosis pathological changes. In vitro, the necrotic apoptosis inhibitor GSK872 effectively alleviated the release of mito-DAMPs in AML-12 cells after CCl4 intoxication, which confirmed that mito-DAMPs originated from dead liver cells. Moreover, direct stimulation of Raw264.7 cells by mito-DAMPs from liver tissue can induce intracellular inflammatory response. More importantly, TREM2 was elevated and inflammatory factors were markedly accumulated surrounding dead cells in the livers of human patients with liver fibrosis. ConclusionOur study highlights that TREM2 serves as a negative regulator of liver fibrosis, suggesting its potential as a novel therapeutic target.

Development of a Large Animal Model of Ischemia-free Liver Transplantation in Pigs.

In organ transplantation, ischemia, and reperfusion injury (IRI) is considered as an inevitable event and the major contributor to graft failure. Ischemia-free liver transplantation (IFLT) is a novel transplant procedure that can prevent IRI and provide better transplant outcomes. However, a large animal model of IFLT has not been reported. Therefore, we develop a new, reproducible, and stable model of IFLT in pigs for investigating mechanisms of IFLT in IRI. Ten pigs were subjected to IFLT or conventional liver transplantation (CLT). Donor livers in IFLT underwent 6-h continuous normothermic machine perfusion (NMP) throughout graft procurement, preservation, and implantation, whereas livers in CLT were subjected to 6-h cold storage before implantation. The early reperfusion injury was compared between the 2 groups. Continuous bile production, low lactate, and liver enzyme levels were observed during NMP in IFLT. All animals survived after liver transplantation. The posttransplant graft function was improved with IFLT when compared with CLT. Minimal histologic changes, fewer apoptotic hepatocytes, less sinusoidal endothelial cell injury, and proinflammatory cytokine (interleukin [IL]-1β, IL-6, and tumor necrosis factor-α) release after graft revascularization were documented in the IFLT group versus the CLT group. We report that the concept of IFLT is achievable in pigs. This innovation provides a potential strategy to investigate the mechanisms of IRI and provide better transplant outcomes for clinical practice.

Apoptotic cell identity induces distinct functional responses to IL-4 in efferocytic macrophages.

Macrophages are functionally heterogeneous cells essential for apoptotic cell clearance. Apoptotic cells are defined by homogeneous characteristics, ignoring their original cell lineage identity. We found that in an interleukin-4 (IL-4)-enriched environment, the sensing of apoptotic neutrophils by macrophages triggered their tissue remodeling signature. Engulfment of apoptotic hepatocytes promoted a tolerogenic phenotype, whereas phagocytosis of T cells had little effect on IL-4-induced gene expression. In a mouse model of parasite-induced pathology, the transfer of macrophages conditioned with IL-4 and apoptotic neutrophils promoted parasitic egg clearance. Knockout of phagocytic receptors required for the uptake of apoptotic neutrophils and partially T cells, but not hepatocytes, exacerbated helminth infection. These findings suggest that the identity of apoptotic cells may contribute to the development of distinct IL-4-driven immune programs in macrophages.

Therapeutic inhibition of monocyte recruitment prevents checkpoint inhibitor-induced hepatitis

BackgroundCheckpoint inhibitor-induced hepatitis (CPI-hepatitis) is an emerging problem with the widening use of CPIs in cancer immunotherapy. Here, we developed a mouse model to characterize the mechanism of CPI-hepatitis and...

Integrated multi-omic analysis identifies fatty acid binding protein 4 as a biomarker and therapeutic target of ischemia–reperfusion injury in steatotic liver transplantation

Background and aimsDue to a lack of donor grafts, steatotic livers are used more often for liver transplantation (LT). However, steatotic donor livers are more sensitive to ischemia–reperfusion (IR) injury and have a worse prognosis after LT. Efforts to optimize steatotic liver grafts by identifying injury targets and interventions have become a hot issue.MethodsMouse LT models were established, and 4D label-free proteome sequencing was performed for four groups: normal control (NC) SHAM, high-fat (HF) SHAM, NC LT, and HF LT to screen molecular targets for aggravating liver injury in steatotic LT. Expression detection of molecular targets was performed based on liver specimens from 110 donors to verify its impact on the overall survival of recipients. Pharmacological intervention using small-molecule inhibitors on an injury-related target was used to evaluate the therapeutic effect. Transcriptomics and metabolomics were performed to explore the regulatory network and further integrated bioinformatics analysis and multiplex immunofluorescence were adopted to assess the regulation of pathways and organelles.ResultsHF LT group represented worse liver function compared with NC LT group, including more apoptotic hepatocytes (P < 0.01) and higher serum transaminase (P < 0.05). Proteomic results revealed that the mitochondrial membrane, endocytosis, and oxidative phosphorylation pathways were upregulated in HF LT group. Fatty acid binding protein 4 (FABP4) was identified as a hypoxia-inducible protein (fold change > 2 and P < 0.05) that sensitized mice to IR injury in steatotic LT. The overall survival of recipients using liver grafts with high expression of FABP4 was significantly worse than low expression of FABP4 (68.5 vs. 87.3%, P < 0.05). Adoption of FABP4 inhibitor could protect the steatotic liver from IR injury during transplantation, including reducing hepatocyte apoptosis, reducing serum transaminase (P < 0.05), and alleviating oxidative stress damage (P < 0.01). According to integrated transcriptomics and metabolomics analysis, cAMP signaling pathway was enriched following FABP4 inhibitor use. The activation of cAMP signaling pathway was validated. Microscopy and immunofluorescence staining results suggested that FABP4 inhibitors could regulate mitochondrial membrane homeostasis in steatotic LT.ConclusionsFABP4 was identified as a hypoxia-inducible protein that sensitized steatotic liver grafts to IR injury. The FABP4 inhibitor, BMS-309403, could activate of cAMP signaling pathway thereby modulating mitochondrial membrane homeostasis, reducing oxidative stress injury in steatotic donors.Graphical

Dandelion Root Extract in Trout Feed and its Effects on the Physiological Performance of Oncorhynchus mykiss and Resistance to Lactococcus garvieae Infection

Abstract In this study, we investigated the dietary effects of dandelion root extract (DRE) on growth performance, body composition, serum biochemical parameters, intestinal and liver histology, and fish resistance against Lactococcus garvieae infection in the rainbow trout. In total, 540 rainbow trout fry (22.05±1.740 g and 13.03±0.470 cm) were used. Six experimental groups (control, 2.5 mL/kg (DRE-1), 5 mL/kg (DRE-2), 10 mL/kg (DRE-3), 20 mL/kg (DRE-4), and 40 mL/kg (DRE-5) dandelion root extract) with three replicates were formed. In each tank, 30 fish were placed and fed for 90 days, and the volume of water was adjusted to 80 L with a flow-through system. The results showed that the final fish weight, weight gain, specific growth rate, protein efficiency ratio, and protein deposition rate were significantly higher in the DRE-4 group, while the feed conversion ratio and fat deposition ratio were significantly lower compared to that in the control group (P&lt;0.05). The viscerosomatic index and the hepatosomatic index decreased in the experimental groups depending on the concentration of the extract used, while the condition factor was higher than that in the control (P&lt;0.05). The DRE significantly decreased glucose, cholesterol, triglyceride, low-density lipoprotein, alanine aminotransferase, and aspartate aminotransferase levels in blood serum in the fish from the experimental groups, but it significantly increased high-density lipoprotein, alkaline phosphatase, and total protein levels (P&lt;0.05). Similarly, the DRE positively affected intestinal histology by increasing the stratum compactum, submucosa, lamina propria, villi length, villi width, absorption area, tunica muscularis thickness, mucosal folds, and goblet cell count. In the liver, depending on the high-fat diet (crude lipid = 20%), vacuolization areas, hepatocellular degeneration areas, apoptotic hepatocyte nucleus, and necrotic areas recorded in the control group decreased or even disappeared completely in the extract groups. The results of the challenge test showed that adding 2.5 mL/kg of the DRE to the feed positively affected the disease resistance against Lactococcus garvieae infection. The results of the quadratic polynomial regression showed that an average level of 23.91±1.048 mL/kg of DRE would be suitable for optimum fish growth.

Electron Microscopic Studying the Impact of Ozone on Chronic Hepatitis C Patients as Antifibrotic

We aimed to evaluate the histopathological and ultrastructural changes detected by light and electron microscopy, induced in patients having chronic hepatitis C receiving ozone/oxygen gas treatment. Twenty six patients with chronic hepatitis C who were at different stages of liver fibrosis, non-responders to interferon plus ribavirin therapy (n = 9), had contraindications (n = 2), or were not compliant (n = 15) were included. At baseline and 12 weeks after administering the ozone/oxygen gas mixture, liver biopsies were carried out utilizing both the major rectal insufflations and auto-hemotherapy along with clinical evaluation, kidney and liver function assessments, liver biopsies and abdominal ultrasonography. Before and 12 weeks after the treatment of ozone, quantitative PCR was performed. Two pathologists evaluated the histological activity index (HAI) using Ishak's score while working in blind settings, taking into account the degree of inflammation and the stage of fibrosis. Electron microscopy was done for all cases before and after treatment. Significant improvements in liver enzymes in hepatic fibrosis and inflammatory activity based on Ishak scoring system were detected. The mean grade of inflammation dropped from 10.3 to 8.4 and the mean stage of fibrosis dropped from 2.3 to 2.0 both with P value &lt; 0.001 and &lt; 0.05 respectively. Mean PCR values showed significant increase after 12 weeks of treatment from 17059 to 218736 with P value &lt;0.05. As regards electron microscopy findings, the ultra-structural manifestations of HCV infection were disclosed in liver specimens exposed or not to ozone treatment. Stellate cells were often encountered in unexposed samples to ozone, meanwhile they disappeared after ozone exposure. Apoptotic hepatocytes which were frequently encountered before treatment are rarely seen in specimens after treatment with ozone. Signs of cellular regeneration in the form of binucleated cells, RER enveloping mitochondria, hepatocyte progenitors insinuating between the cells at the sinusoid pole are seen after treatment. Circulating inflammatory cells in the sinusoids and infilterating the lobule were decreased after treatment. Peroxisomes were increased after exposure to ozone with longitudinal orientation of mitochondrial cristae thus increasing the antioxidative activity of hepatocytes. No single significant complication was recorded in a total of &gt;1000 settings of ozone therapy. In conclusion, ozone oxygen gas mixture is a direct anti-fibrotic and anti-inflammatory agent in treatment of chronic hepatitis C patients without improving viral PCR as evidenced by histopathology, electron microscopy and quantitative PCR.

Improvement of rat hepatocellular carcinoma model induced by diethylnitrosamine

ObjectiveTo construct a new diethylnitrosamine (DEN)-induced rat hepatocellular carcinoma (HCC) model with short induction time, high incidence, and survival rate. Methods60 male Sprague-Dawley rats were randomly divided into 4 groups: the control group, the model A (MA) group, the model B (MB) group, and the model C (MC) group. The control group was intraperitoneally injected with 0.9% saline for 6 weeks. The MA group was injected with the DEN solution at 30 mg/kg three times a week for 6 weeks. The MB group was injected with the DEN solution at 30 mg/kg three times a week for 6 weeks, and discontinued the induction for 2 weeks. The MC group was injected with the DEN solution at 30 mg/kg three times a week for 8 weeks. The levels of albumin (ALB), alanine transaminase (ALT), and aspartate aminotransferase (AST) in serum were assayed. Meanwhile, the pathological conditions, apoptosis of hepatocytes, expression of NF-κBp65, and the reactive oxygen species level were detected. ResultsAll rats in the control group and the MA group survived, and none of the rats occurred HCC. HCC occurred in rats of the MB group and the MC group. The serum ALB level in the MB group was higher than that in the MC group. The serum ALT and AST levels and the number of proliferating and apoptotic hepatocyte cells in the MB group were lower than those in the MC group. The expression of ROS- and NF-κBp6- positive cells in the MA group, MB group, and MC group were significantly higher than that of the control group. ConclusionThis study developed a new DEN-induced rat HCC model with short induction time, high incidence, and survival rate. NF-κB pathway may be one of the main pathways involved in the development of this model.

Deoxyribonuclease I Alleviates Septic Liver Injury in a Rat Model Supported by Venoarterial Extracorporeal Membrane Oxygenation.

Sepsis is an unusual systemic reaction with high mortality and secondary septic liver injury is proposed to be the major cause of mortality. Extracorporeal membrane oxygenation (ECMO) can enhance terminal organ perfusion by elevating circulatory support which is used in severe sepsis patients. However, the interaction of blood components with the biomaterials of the extracorporeal membrane elicits a systemic inflammatory response. Besides, inflammation and apoptosis are the main mediators in the pathophysiology of septic liver injury. Therefore, we investigated the protective effect of Deoxyribonuclease I (DNase I) against septic liver injury supported by ECMO in rats. Sepsis was induced by lipopolysaccharide (LPS) and 24 hours after the administration, the rats were treated with ECMO. Then blood samples and liver tissues were collected. DNase I significantly attenuated the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and significantly decreased hepatic levels of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, myeloperoxidase (MPO), downstream inflammatory factor interleukin-1β (IL-1β) and interleukin-18 (IL-18), and improved neutrophil infiltration. Additionally, DNase I significantly reduced the expression of apoptosis key protein and terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-labeled apoptotic hepatocytes. In summary, our findings demonstrated that DNase I alleviates liver injury in ECMO-supported septic rats by reducing the inflammatory and apoptotic responses.

An unexpected role of neutrophils in clearing apoptotic hepatocytes in vivo.

Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here, we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver, and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.

An unexpected role of neutrophils in clearing apoptotic hepatocytes in vivo

Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here, we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver, and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.

Distinct disease features of acute and persistent genotype 3 hepatitis E virus infection in immunocompetent and immunosuppressed Mongolian gerbils.

Hepatitis E virus (HEV) causes self-limited acute hepatitis in immunocompetent individuals and can establish chronic infection in solid organ transplant recipients taking immunosuppressive drugs. A well characterized small animal model is needed to understand HEV pathogenesis. In this study, we established a robust model to study acute and persistent HEV infection using Mongolian gerbils (Meriones unguiculatus) with or without immunosuppression. Gerbils were implanted subcutaneously with continuous release tacrolimus pellet to induce immunosuppression. Gerbils with or without tacrolimus treatment were inoculated with HEV intraperitoneally. Viremia, fecal virus shedding, serum antibody and ALT levels, liver histopathological lesions, hepatocyte apoptosis, and liver macrophage distribution were assessed. Mild to moderate self-limited hepatitis and IgM and IgG antibody responses against HEV ORF2 were observed in immunocompetent gerbils. Levels of HEV-specific IgM responses were higher and lasted longer in immunocompetent gerbils with higher peak viremia. Persistent viremia and fecal virus shedding with either weak, or absent HEV antibody levels were seen in immunosuppressed gerbils. Following HEV infection, serum ALT levels were increased, with lower and delayed peaks observed in immunosuppressed compared to immunocompetent gerbils. In immunocompetent gerbils, foci of apoptotic hepatocytes were detected that were distributed with inflammatory infiltrates containing CD68+ macrophages. However, these foci were absent in immunosuppressed gerbils. The immunosuppressed gerbils showed no inflammation with no increase in CD68+ macrophages despite high virus replication in liver. Our findings suggest adaptive immune responses are necessary for inducing hepatocyte apoptosis, CD68+ macrophage recruitment, and inflammatory cell infiltration in response to HEV infection. Our studies show that Mongolian gerbils provide a promising model to study pathogenesis during acute and persistent HEV infection.

Hepatotoxic effect of dietary phytoestrogens on juvenile cultured Russian sturgeon (Acipenser gueldenstaedtii)

In the last two decades, much controversy has grown over the use of soybean products in aquafeeds, especially for carnivorous fish like sturgeons. One point of discussion is the effect of soybean phytoestrogens on fish health. There are many aspects of phytoestrogen utilization in aquafeeds, therefore, the aim of this study is to verify if common legume phytoestrogens can affect juvenile cultured sturgeon erythrocyte and hepatocyte genotoxicity and cause liver pathology. Russian sturgeons were fed from 100 till 365 dph11Days post hutching. with daidzein, genistein, and coumestrol supplemented diets in concentrations: 10, 0.05 and 0.001 g kg−1 of feed, respectively. The SCGE22Single Cell Gel Electrophoresis (comet assay). method combined with qPCR of three genes involved in DNA repair and genome maintenance, namely cyp1a1, gaad45a and p53 were analyzed. The results were compared with histopathological evaluation of liver tissue. In fish fed with coumestrol supplemented diet, DNA strand damage was the highest in both erythrocytes and hepatocytes, however, simultaneously the lowest level of oxidative DNA damage was found. Additionally, slightly elevated expression of the p53 gene was observed along with a decreased number of apoptotic hepatocytes, which suggests that low concentration of coumestrol may support DNA repair mechanisms in the liver. Although, daidzein showed a preventive effect only against fibrosis. Isoflavones did not show a significant effect on DNA damage in studied cells. Genistein was found to increase macro- and microvesicular steatosis, portal hepatitis and fibrosis, indicating its negative role in the development of liver injuries. Daidzein alleviated some sturgeon liver damage, especially macrovesicular steatosis and interface hepatitis. However, it increased hepatocyte apoptosis, which may suggest daidzein potentially inducing liver injury, though not manifested by other histopathological lesions. Therefore, it can be concluded that at given concentrations, the tested phytoestrogens did not show clearly hepatoprotective effect in sturgeons.

Defective efferocytosis by aged macrophages promotes STING signaling mediated inflammatory liver injury

Aged livers have shown aggravated liver ischemia and reperfusion (IR) injury. Timely efferocytosis of apoptotic cells is a key mechanism for avoiding excessive inflammation and tissue injury. Here, we investigated the alteration of efferocytosis by aged macrophages and its role in regulating macrophage STING (stimulator of interferon genes) signaling and liver IR injury. Aged and young mice were subjected to liver partial IR model. Liver injury and inflammation were measured. Efferocytosis by aged macrophages and the underlying regulatory mechanism were analyzed as well. Aged macrophages exhibited impaired efferocytosis with decreased MerTK (c-mer proto-oncogene tyrosine kinase) activation, which was reversed by treatment of the MerTK CRISPR activation plasmid. Increased MerTK cleavage by ADAM17 (a disintegrin and metalloproteinase 17) due to enhanced ROS (reactive oxygen species) levels contributed to defective efferocytosis by aged macrophages. MerTK activation by suppressing ADAM17 or ROS improved aged macrophage efferocytosis, leading to reduced inflammatory liver injury. Moreover, increased apoptotic hepatocytes, DNA accumulation, and macrophage STING activation were observed in aged ischemic livers. Improvement in efferocytosis by aged macrophages via MerTK activation suppressed STING activation and inflammatory liver injury. Our study demonstrates that aging suppresses MerTK- mediated macrophage efferocytosis to promote macrophage STING activation and inflammatory liver IR injury, suggesting a new mechanism and potential therapy to promote inflammation resolution and efferocytosis in aged livers.

Deletion of Cyclic GMP-AMP Synthase Aggravates Concanavalin A-Induced Acute Hepatic Injury by Facilitating Leukocyte Chemotaxis

Growing evidence demonstrates that cyclic GMP-AMP synthase (cGAS), as a cytosolic DNA sensor, is essential for activating innate immunity and regulating inflammatory response against cellular damage. However, its role in immune-mediated hepatitis remains unclear. Here by challenging the cGAS knockout (KO) and their littermate wide-type (WT) mice with intravenous ConA injection to induce acute immune-mediated liver injury, we found that lack of cGAS drastically aggravated liver damage post ConA treatment for 24h, reflected by increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and amplified hepatic necrosis. The number of apoptotic hepatocytes was also significantly increased in the KO mice. RNA-sequencing analysis revealed that leukocyte chemotaxis and migration-related genes were remarkably upregulated in the KO livers. Consistently, immunofluorescence assays illustrated that the infiltrating F4/80-positive macrophages, Ly6G-positive neutrophils, and CD3-positive T cells were all significantly increased in the KO liver sections. The hepatic expression of the pro-inflammatory genes was elevated as well. Supporting the in vivo findings, the knockdown of cGAS in cultured macrophages showed promoted migration potential and enhanced pro-inflammatory gene expression. These results collectively demonstrated that deletion of cGAS could aggravate ConA-induced acute liver injury, at least at the 24-h time point, and its mechanism might be related to facilitating leukocyte chemotaxis and promoting liver inflammatory response.