Liver Tissue Hepatocytes Research Articles

SEMA6B induces macrophage-mediated inflammation and hepatocyte apoptosis in hepatitis B virus-related acute-on-chronic liver failure.

Rationale: Patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) have a high short-term mortality rate. Semaphorin-6B (SEMA6B) plays a crucial role in the pathogenesis of HBV-ACLF, but its molecular basis remains unclear. This study aimed to elucidate the mechanisms of SEMA6B in HBV-ACLF progression. Methods: A total of 321 subjects with HBV-ACLF, liver cirrhosis (LC), chronic hepatitis B (CHB), and normal controls (NC) from a prospective multicenter cohort were studied. 84 subjects (HBV-ACLF, n = 50; LC, n = 10; CHB, n = 10; NC, n = 14) among them underwent mRNA sequencing using peripheral blood mononuclear cells (PBMCs) to clarify the mechanisms of SEMA6B in HBV-ACLF. These mechanisms were validated through in vitro studies with hepatocytes and macrophages, as well as in vivo using SEMA6B knockout mice and mice treated with synthetic SEMA6B siRNA. Results: Transcriptome analysis of PBMCs showed that SEMA6B was among the most differentially expressed genes when comparing patients with HBV-ACLF to those with LC, CHB, or NC. ROC analysis demonstrated the reliable diagnostic value of SEMA6B for HBV-ACLF in both the sequencing cohort and an external validation cohort (AUROC = 0.9788 and 0.9026, respectively). SEMA6B levels were significantly higher in the HBV-ACLF patients, especially in non-survivors, with high expression mainly observed in macrophages and hepatocytes in liver tissue. Genes significantly associated with highly expressed SEMA6B were enriched in inflammation and apoptosis pathways in HBV-ACLF non-survivors. Overexpression of SEMA6B in macrophages activated systemic inflammatory responses, while its overexpression in hepatocytes inhibited proliferation through G0/G1 cell cycle arrest and induced apoptosis. Knocking out SEMA6B rescued mice with liver failure by improving liver functions, reducing inflammatory responses, and decreasing hepatocyte apoptosis. Transcriptome analysis of liver tissue showed that SEMA6B knockout significantly ameliorated the liver failure signature, significantly downregulating inflammation-related pathways. Importantly, therapeutic delivery of synthetic SEMA6B siRNA also improved liver function, and reduced both inflammation and hepatocyte apoptosis in mice with liver failure. Conclusion: SEMA6B, a potential diagnostic biomarker for HBV-ACLF, exacerbates liver failure through macrophage-mediated systemic inflammation and hepatocyte apoptosis. These findings highlight SEMA6B as a promising early treatment target for HBV-ACLF patients.

Idiosyncratic liver injury induced by bolus combination treatment with emodin and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucopyranoside in rats.

Polygoni Multiflori Radix (PMR) is a commonly used traditional Chinese medicine in clinical practice, while adverse effects of hepatotoxicity related to PMR have been frequently reported. The clinical case reports indicated that PMR hepatotoxicity could occur under both overdose medication/long-term exposure and low doses with short-duration (idiosyncratic) conditions. The combination treatment with emodin and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucopyranoside (TSG), two major PMR components, was reported to contribute to PMR hepatotoxicity after long-term treatment. However, the role of the combination treatment of these two components in PMR-induced idiosyncratic liver injury has not been clearly clarified. In this study, the LPS-mediated inflammatory stress model rats were adopted to explore the idiosyncratic liver injury induced by the bolus combination treatment with emodin and TSG. After a bolus oral administration with TSG (165 mg/kg), emodin (5 mg/kg) or their combination in both normal and LPS-mediated inflammatory stress model rats, the systemic/hepatic concentrations of emodin, emodin glucuronides and bile acids were determined; the hepatotoxicity assessments were conducted via monitoring histopathological changes and liver injury biomarkers (ALT and AST). Moreover, the protein expressions of bile acid homeostasis- and apoptosis-related proteins were examined. No liver damage was observed in the normal rats after a bolus dose with the individual or combination treatment, while the bolus combination treatment with emodin and TSG induced liver injury in the LPS-mediated inflammatory stress model rats, evidenced by the elevated plasma levels of alanine aminotransferase (∼66%) and aspartate aminotransferase (∼72%) accompanied by severe inflammatory cell infiltration and apoptotic hepatocytes in liver tissue. Moreover, such combination treatment at a bolus dose in the LPS-mediated inflammatory stress model rats could significantly elevate the hepatic TBA levels by about 45% via up-regulating the hepatic protein expression levels of bile acid synthesis enzymes and inhibiting that of bile acid efflux transporters and the expression levels of apoptosis-related proteins. Our study for the first time proved the major contribution of the combination treatment with emodin and TSG in PMR-induced idiosyncratic liver injury.

Toxicological impact of gasoline generator emissions using rat models

Nigeria has been identified as one of the largest active users of gasoline generators globally. Due to the widespread incessant use of gasoline generators, the present study assessed the exposure to gasoline generator emission (GGE) and some toxicity endpoints. This study employed a laboratory-based experimental design. An emission chamber was designed using clear thermoplastic to house the rats during exposure to 0.5KVA GGEs. Three experimental groups, each with eight rats respectively, were subjected to 5, 10, 15 minutes of of daily GGE exposure for fourteen days for fourteen days, while the fourth was a control group. Selected air quality parameters were measured using the appropriate samplers. Assays like liver function tests, antioxidant enzyme activity, and micronucleus frequency were conducted after the experiment. Micronucleus frequency significantly increased with time of GGE exposure (p=0.000), while the liver tissue antioxidant activity of the test groups was significantly higher than that of the control group (p<0.05). Moreover, histopathological examinations revealed lesions like sinusoidal congestion and vacuolar degeneration of the hepatocytes in liver tissues of the exposed rats. However, the liver function test showed a non-significant association with their level of GGE exposure. The result of this study suggested that exposure to GGEs at a non-lethal concentration in a sub-acute or chronic manner has deleterious effects on mammalian biochemical systems. Therefore, the use of exhaust filters and improvement in the ventilating systems of houses are encouraged to reduce exposure rates in developing countries like Nigeria, where GGEs are widespread due to erratic electricity supply.

Abstract LB200: A transcriptionally distinct subpopulation of healthy acinar cells exhibit features of pancreatic progenitors and pancreatic ductal adeno carcinoma

Abstract The stochasticity of transcription leads to phenotypic variation within a clonal population. This means that within a healthy tissue, a fraction of cells could assume an “edge” transcriptional state that is primed for a transition toward malignancy. We find such an edge transcriptional state amongst acinar cells in a publicly available PDAC single-cell RNA-seq dataset. These acinar edge cells (AE) cells have transcriptomes that are highly diverged from a typical normal acinar cell, and show transcriptional similarities to pancreatic ductal adenocarcinoma (PDAC). The AE state can be found in several single-cell datasets from healthy human pancreatic tissue. The AE state is not mutationally-driven as AE cells in healthy tissues do not possess any known oncogenic driver mutations, nor are they clonally derived. The transition from a non-edge acinar to an AE state recapitulates key aspects of pancreatic biology; genes associated with this transition are enriched for known markers of acinar de-differentiation and acinar-ductal metaplasia. Further, the AE state represents a partial reversion to a SOX9+PTF1A+ multi-potent embryonic pancreatic progenitor state, likely through the activation of KLF5, HES1 and RBPJ targets. Remarkably, the promoters of genes upregulated in the AE state are significantly hypomethylated in PDAC relative to normal pancreas, while the genes up-regulated in the AE state are up-regulated in PDAC samples in the Cancer Genome Atlas (TCGA) database. It is thus likely that transcriptomic changes in the edge state are fixed epigenetically during tumor initiation and progression. A gene signature derived from the AE cells shows strong association with survival among PDAC patients. Remarkably, we also find that the fraction of AE cells in the healthy pancreas increases with age, which points to a strong non-genetic and transcriptomic component to the known increase in PDAC risk with age. We also observed edge-like transcriptomic heterogeneity among alveolar, ciliated and club cells in the human lung, and amongst hepatocytes in liver tissues. This suggests that, across tissues, gene expression heterogeneity can lead to sub-populations of healthy cells across tissues existing in pre-malignant states. Citation Format: Vishaka Gopalan, Arashdeep Singh, Farid Rashidi, Li Wang, Eytan Ruppin, Efsun Arda, Sridhar Hannenhalli. A transcriptionally distinct subpopulation of healthy acinar cells exhibit features of pancreatic progenitors and pancreatic ductal adeno carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB200.

Cytotoxic Screening and Transcriptomics Reveal Insights into the Molecular Mechanisms of Trihexyl Phosphate-Triggered Hepatotoxicity.

Mounting evidence shows that organophosphate flame retardants (OPFRs), especially aryl- and halogenated-OPFRs, exert various adverse health effects on living organisms. This study evaluated the hepatotoxic effect of trihexyl phosphate (THP) as a long-chain alkyl-OPFR on human hepatocyte cells (LO2) and mouse hepatocyte cells (AML12) by performing screening of cytotoxicity in vitro. In combination with transcriptomic analysis, toxicological mechanisms in vitro were further investigated. Results showed that THP triggered hepatotoxicity in vitro by altering four signaling pathways: endoplasmic reticulum (ER) stress, apoptosis, cell cycle, and the glycolysis signaling pathway. Exposure of LO2 and AML12 liver cells to THP (25 μg/mL) significantly induced ER stress-mediated apoptosis and cell cycle arrest. Meanwhile, downregulation of glycolysis caused the blockage of energy metabolism. Furthermore, the high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS) revealed that much of THP was absorbed into the cells and displayed stability in the two liver cell lines. In vivo assays using a mouse model demonstrated that exposure to THP at 400 mg/kg induced the ballooning degeneration of hepatocytes in liver tissue, whereas exposure to THP at 800 mg/kg caused acute liver injury with high alanine aminotransferase levels. This study provides novel insights into the impact of THP on hepatotoxicity in vitro and in vivo and uncovers the underlying toxicological mechanisms, which may serve as a guide for further ecological risk assessment and reasonable application of alkyl-OPFRs.

Dynamic Regulation of miRNA Expression by Functionally Enhanced Placental Mesenchymal Stem Cells PromotesHepatic Regeneration in a Rat Model with Bile Duct Ligation.

Placenta-derived mesenchymal stem cells (PD-MSCs) were highlighted as therapeutic sources in several degenerative diseases. Recently, microRNAs (miRNAs)were found to mediate one of the therapeutic mechanisms of PD-MSCs in regenerative medicine. To enhance the therapeutic effects of PD-MSCs, we established functionally enhanced PD-MSCs with phosphatase of regenerating liver-1 overexpression (PRL-1(+)). However, the profile and functions of miRNAs induced by PRL-1(+) PD-MSCs in a rat model with hepatic failure prepared by bile duct ligation (BDL) remained unclear. Hence, the objectives of the present study were to analyze the expression of miRNAs and investigate their therapeutic mechanisms for hepatic regeneration via PRL-1(+) in a rat model with BDL. We selected candidate miRNAs based on microarray analysis. Under hypoxic conditions, compared with migrated naïve PD-MSCs, migrated PRL-1(+) PD-MSCs showed improved integrin-dependent migration abilitythrough Ras homolog (RHO) family-targeted miRNA expression (e.g., hsa-miR-30a-5p, 340-5p, and 146a-3p). Moreover, rno-miR-30a-5p and 340-5p regulated engraftment into injured rat liver by transplantedPRL-1(+) PD-MSCs through the integrin family. Additionally, an increase inplatelet-derived growth factor receptor A (PDGFRA) by suppressing rno-miR-27a-3p improved vascular structure in rat liver tissues after PRL-1(+) PD-MSC transplantation. Furthermore, decreased rno-miR-122-5p was significantly correlated with increased proliferation of hepatocytes in liver tissues by PRL-1(+) PD-MSCs byactivating the interleukin-6 (IL-6) signaling pathway through the repression of rno-miR-21-5p. Taken together, these findings improve the understandingof therapeutic mechanisms based on miRNA-mediated stem-cell therapy in liver diseases.

Modulations of Histone Deacetylase 2 Offer a Protective Effect through the Mitochondrial Apoptosis Pathway in Acute Liver Failure.

The purpose of this study was to investigate the modulation of histone deacetylase 2 (HDAC2) on mitochondrial apoptosis in acute liver failure (ALF). The cellular model was established with LO2 cells stimulated by tumor necrosis factor alpha (TNF-α)/D-galactosamine (D-gal). Rats were administrated by lipopolysaccharide (LPS)/D-gal as animal model. The cell and animal models were then treated by HDAC2 inhibitor CAY10683. HDAC2 was regulated up or down by lentiviral vector transfection in LO2 cells. The mRNA levels of bcl2 and bax were detected by real-time PCR. The protein levels of HDAC2, bcl2, bax, cytochrome c (cyt c) in mitochondrion and cytosol, apoptosis protease activating factor 1 (apaf1), caspase 3, cleaved-caspase 3, caspase 9, cleaved-caspase 9, acetylated histone H3 (AH3), and histone H3 (H3) were assayed by western blot. Apoptosis was detected by flow cytometry. The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) levels were also assayed. The openness degree of the mitochondrial permeability transition pore (MPTP) was detected by ultraviolet spectrophotometry. The apoptosis of hepatocytes in liver tissues was determined by tunnel staining. The liver tissue pathology was detected by hematoxylin eosin (HE) staining. The ultrastructure of liver tissue was observed by electron microscopy. Compared with cell and rat model groups, the bax mRNA level was decreased, and bcl2 mRNA was increased in the CAY10683 treatment group. The protein levels of HDAC2, bax, cyt c in cytosol, apaf1, cleaved-caspase 3, and cleaved-caspase 9 were decreased, and the apoptosis rate was decreased (P < 0.05), whereas the protein level of bcl2 and cyt c in the mitochondrion was elevated (P < 0.05) in the CAY10683 treatment group. In the HDAC2 down- or upregulated LO2 cells, the mitochondrial apoptosis pathway was inhibited or activated, respectively. After being treated with TNF-α/D-gal in HDAC2 down- or upregulated LO2 cells, the mitochondrial apoptosis pathway was further suppressed or activated, respectively. The MPTP value was elevated in CAY10683-treated groups compared with the rat model group (P < 0.05). Liver tissue pathological damage and apoptotic index in the CAY10683-treated group were significantly reduced. In addition, AH3 was elevated in both cell and animal model groups (P < 0.05). Downregulated or overexpressed HDAC2 could accordingly increase or decrease the AH3 level, and TNF-α/D-gal could enhance the acetylation effect. These results suggested that modulations of histone deacetylase 2 offer a protective effect through the mitochondrial apoptosis pathway in acute liver failure.

Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis

Wilson disease (WD) is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in the liver and brain. It is caused by mutations in the adenosine triphosphatase copper transporting β gene (ATP7B), which encodes a protein that transports copper from hepatocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to decrease copper toxicity in patients with WD. We used RNA-seq to compare gene expression patterns between wild-type and ATP7B-knockout HepG2 cells exposed to copper. We collected blood and liver tissues from Atp7b-/- and Atp7b+/- (control) rats (LPP) and mice; some mice were given 5 daily injections of an autophagy inhibitor (spautin-1) or vehicle. We obtained liver biopsies from 2 patients with WD in Italy and liver tissues from patients without WD (control). Liver tissues were analyzed by immunohistochemistry, immunofluorescence, cell viability, apoptosis assays, and electron and confocal microscopy. Proteins were knocked down in cell lines using small interfering RNAs. Levels of copper were measured in cell lysates, blood samples, liver homogenates, and subcellular fractions by spectroscopy. After exposure to copper, ATP7B-knockout cells had significant increases in the expression of 103 genes that regulate autophagy (including MAP1LC3A, known as LC3) compared with wild-type cells. Electron and confocal microscopy visualized more autophagic structures in the cytoplasm of ATP7B-knockout cells than wild-type cells after copper exposure. Hepatocytes in liver tissues from patients with WD and from Atp7b-/- mice and rats (but not controls) had multiple autophagosomes. In ATP7B-knockout cells, mammalian target of rapamycin (mTOR) had decreased activity and was dissociated from lysosomes; this resulted in translocation of the mTOR substrate transcription factor EB to the nucleus and activation of autophagy-related genes. In wild-type HepG2 cells (but not ATP7B-knockout cells), exposure to copper and amino acids induced recruitment of mTOR to lysosomes. Pharmacologic inhibitors of autophagy or knockdown of autophagy proteins ATG7 and ATG13 induced and accelerated the death of ATP7B-knockout HepG2 cells compared with wild-type cells. Autophagy protected ATP7B-knockout cells from copper-induced death. ATP7B-deficient hepatocytes, such as in those in patients with WD, activate autophagy in response to copper overload to prevent copper-induced apoptosis. Agents designed to activate this autophagic pathway might decrease copper toxicity in patients with WD.

Physiological and biochemical effects of nickel on rainbow trout (Oncorhynchus mykiss) tissues: Assessment of nuclear factor kappa B activation, oxidative stress and histopathological changes

We investigated changes in nuclear factor kappa B (NFkB) activity, antioxidant responses and histopathological effects in the liver, gill and kidney tissues of rainbow trout exposed to nickel chloride (Ni). Two different concentrations (1 mg/L and 2 mg/L) were administrated to fish for 21 days. Tissues were taken from all fish for NFkB activity, histopathological examination and determination of superoxide dismutase (SOD), catalase (CAT) enzyme activity and of lipid peroxidation (LPO), and glutathione (GSH) levels. The findings of this study indicated that Ni exposure led to a significant increase in LPO indicating peroxidative damage and antioxidant enzymes SOD and CAT activity in tissues (p < 0.05), but 2 mg/Ni concentration caused a significant decrease in CAT activity in kidney tissues (p < 0.05). One of mechanism in the antioxidant defense system seems to be GSH, which increased in gill and kidney tissues of fish exposed to Ni (p < 0.05). NFkB immunopositivity was detected in all tissues. Ni exposure caused lamellar thickening, cellular infiltration in gill tissues, hydropic degeneration of hepatocytes in liver tissues, hyalinous accumulation within the glomeruli and tubular degeneration in kidney tissues. Our results suggested that Ni toxicity may disturb the biochemical and physiological functions of fish by causing changes in NFkB activity and oxidative and histopathological damage in the tissues of rainbow trout. This study can provide useful information for understanding of Ni-induced toxicity.

Association analysis of toll-like receptor 4 polymorphisms in Japanese primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is characterized by portal inflammation and immune-mediated destruction of intrahepatic bile ducts that often result in liver failure. Toll-like receptor (TLR) 4 recognizes lipopolysaccharides of Gram-negative bacteria. Infectious agents have been suspected to play a crucial role in PBC pathogenesis since TLR4 expression was found in bile duct epithelial cells and periportal hepatocytes in liver tissues of PBC. To assess the potential contribution of TLR4 SNPs to the development of this disease, we genotyped five SNPs in TLR4 in 261 PBC patients and 359 controls using a TaqMan assay. No significant positive associations with either PBC susceptibility or progression were uncovered. These results indicate that TLR4 polymorphisms do not play a prominent role in the development of PBC in Japanese patients.

Granulocyte colony-stimulating factor treatment ameliorates liver injury and improves survival in rats with d-galactosamine-induced acute liver failure

Only liver transplantation is currently available therapy for the patients with acute liver failure (ALF). This study was designed to determine whether administration of granulocyte colony-stimulating factor (G-CSF) has therapeutic efficacy in animals with ALF. Female Sprague-Dawley (SD) rats were intraperitoneally injected with a single dose of d-galactosamine ( d-GalN, 1.4 g/kg) to induce ALF. After 2 h, the rats were randomized to receive G-CSF (50 μg/kg/day), or saline vehicle injection for 5 days. Rats were observed for survival and assessed for liver injury by serum alanine transaminase (ALT) measurement and histological analysis. CD34+ cells in bone marrow were assessed by flow cytometry. CD34+ cells and Ki-67+ hepatocytes in liver tissue were evaluated by immunohistochemistry. In the ALF model, 5-day survival after d-GalN injection was 33.3% (10/30), while G-CSF administration following d-GalN resulted in 53.3% (16/30) survival ( p = 0.027). G-CSF treated rats had lower ALT level and less hepatic injury compared with saline vehicle rats. The increases of CD34+ cells in bone marrow and liver tissue and Ki-67+ cells in liver tissue in G-CSF treated rats were higher than those in saline rats. No correlation was observed between CD34+ cells and Ki-67+ hepatocytes in liver tissue in both G-CSF and vehicle rats. It is suggested that G-CSF increases survival rate, decreases liver injury and enhances hepatocyte proliferation in rats with d-GalN-induced ALF possibly through actions including but not limiting to CD34+ cell mobilization, and that G-CSF may be of potential value in treating ALF.

Hepatitis C virus receptor expression in normal and diseased liver tissue

The principal site of hepatitis C virus (HCV) replication is the liver. HCV pseudoparticles infect human liver derived cell lines and this suggests that liver-specific receptors contribute to defining HCV hepatotropism. At least three host cell molecules have been reported to be important for HCV entry: the tetraspanin CD81, scavenger receptor class B member I (SR-BI), and the tight junction (TJ) protein Claudin 1 (CLDN1). Hepatocytes in liver tissue coexpress CD81, SR-BI, and CLDN1, consistent with their ability to support HCV entry. CLDN1 localized at the apical-canalicular TJ region and at basolateral-sinusoidal hepatocyte surfaces in normal tissue and colocalized with CD81 at both sites. In contrast, CLDN1 appeared to colocalize with SR-BI at the basolateral-sinusoidal surface. CLDN1 expression was increased on basolateral hepatocyte membranes in HCV-infected and other chronically inflamed liver tissue compared with normal liver. In contrast, CLDN4 hepatocellular staining was comparable in normal and diseased liver tissue. HCV infection of Huh-7.5 hepatoma cells in vitro significantly increased CLDN1 expression levels, consistent with a direct modulation of CLDN1 by virus infection. In HCV infected livers, immunohistochemical studies revealed focal patterns of CLDN1 staining, suggesting localized areas of increased CLDN1 expression in vivo which may potentiate local viral spread within the liver.

The protease inhibitor leupeptin induces several signs of aging in brain, retina and internal organs of young rats

The protease inhibitor leupeptin was administered to brain, retina and internal organs of young rats for up to two weeks in order to determine if specifically decreased proteolysis could cause symptoms of cellular aging in a variety of tissues. Electron microscopy showed that leupeptin induced the formation of dense substances with fine morphologies similar to and, in many cases, apparently identical with those of natural lipofuscin from aged tissues. Leupeptin also caused increased immunoreactivity to ubiquitin in cerebellar Purkinje cells and presumed Bergmann glia perikarya of brain tissue as well as in hepatocytes of liver tissue. Both of these effects were found in aged tissues as well. Finally, both leupeptin treatment and normal aging led to the onset of immunoreactivity in Purkinje cells to antibodies to the abnormal tau molecule of paired helical filaments from Alzheimer's disease brain. Together, these results indicate that inhibition of thiol (and possibly some serine) proteases by leupeptin is sufficient to cause obvious morphological manifestations of aging in several tissues, and are thus consistent with the hypothesis that lipofuscinogenesis as well as a build-up of ubiquilinated proteins with age is caused by decreased or defective proteolysis. These effects are likely secondary to the mechanism(s) interfering with proteolysis itself.