Hepatocyte Cell Research Articles

Study on the correlation between mineral bone metabolism and CRP in patients with SHPT during perioperative period.

This study mainly observes changes in perioperative mineral bone metabolism-related indicators and inflammatory factors in patients with secondary hyperparathyroidism (SHPT), and analyzed the correlation between mineral bone metabolism-related indicators and inflammatory factors. Clinical data were collected. The study detects mineral bone metabolism-related indicators and inflammatory factor of perioperative patients with SHPT before and 4 days after operation. The production of high-sensitivity c-reactive protein (hs-CRP) in human hepatocytes cells (LO2 cells) stimulated by different concentrations of parathyroid hormone-associated protein was detected by enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction(RT-PCR), and western blot. The levels of mineral bone metabolism-related indicators and hs-CRP in SHPT group were significantly higher than those of control group. After operation, serum calcium, serum phosphorus, iPTH, FGF-23 decreased, and the level of osteoblast active biomarkers increased, while the level of osteoclast active biomarkers decreased. The levels of hs-CRP decreased significantly after operation. With the increase of PTHrP concentration, hs-CRP level in supernatant of LO2 cells decreased first and then increased. RT-PCR and western blot shows the same trend. Parathyroidectomy can significantly improve bone resorption and inflammation in SHPT patients. We speculate that there may be an optimal range of PTH concentrations to minimize inflammation in the body.

Aryl hydrocarbon receptor maintains hepatic mitochondrial homeostasis in mice

ObjectiveMitophagy removes damaged mitochondria to maintain cellular homeostasis. Aryl hydrocarbon receptor (AhR) expression in the liver plays a crucial role in supporting normal liver functions, but its impact on mitochondrial function is unclear. Here, we identified a new role of AhR in the regulation of mitophagy to control hepatic energy homeostasis. MethodsIn this study, we utilized primary hepatocytes from AhR knockout (KO) mice and AhR knockdown AML12 hepatocytes. An endogenous AhR ligand, kynurenine (Kyn), was used to activate AhR in AML12 hepatocytes. Mitochondrial function and mitophagy process were comprehensively assessed by MitoSOX and mt-Keima fluorescence imaging, Seahorse XF-based oxygen consumption rate measurement, and Mitoplate S-1 mitochondrial substrate utilization analysis. ResultsTranscriptomic analysis indicated that mitochondria-related gene sets were dysregulated in AhR KO liver. In both primary mouse hepatocytes and AML12 hepatocyte cell lines, AhR inhibition strongly suppressed mitochondrial respiration rate and substrate utilization. AhR inhibition also blunted the fasting response of several essential autophagy genes and the mitophagy process. We further identified BCL2 interacting protein 3 (BNIP3), a mitophagy receptor that senses nutrient stress, as an AhR target gene. AhR is directly recruited to the Bnip3 genomic locus, and Bnip3 transcription was enhanced by AhR endogenous ligand treatment in wild-type liver and abolished entirely in AhR KO liver. Mechanistically, overexpression of Bnip3 in AhR knockdown cells mitigated the production of mitochondrial reactive oxygen species (ROS) and restored functional mitophagy. ConclusionsAhR regulation of the mitophagy receptor BNIP3 coordinates hepatic mitochondrial function. Loss of AhR induces mitochondrial ROS production and impairs mitochondrial respiration. These findings provide new insight into how endogenous AhR governs hepatic mitochondrial homeostasis.

Directed evolution of AAV capsids for improved efficacy and specificity of delivery to preclinical models of human liver

Introduction and objectiveRecombinant vectors derived from adeno-associated viruses (rAAVs) are the leading platform in human gene therapy applications, with high-profile examples targeting diseases of the central nervous system, eye and liver. The liver, quite likely a natural host organ for wild type AAV2, is a particularly attractive target for the development of AAV-mediated gene therapies. Despite large number of AAV variants current at various stages of development as carriers of liver therapeutics, thus far no liver-directed AAV-based therapy has obtained market authorization. Strong preclinical data is the cornerstone of any translational program, and while AAV bioengineering is commonly applied to try to develop novel human-tropic vectors for clinical applications, due to species-to-species differences, dedicated vectors to support preclinical work may need to be developed. Here we applied AAV directed evolution and <i>in vitro</i> selection to identify AAV capsids that target human liver cells <i>in vitro</i>.Material and methodsUsing DNA shuffling technology, we have generated a capsid gene library based on natural parental serotypes (AAV1 through AAV12). Shuffled capsid library was selected in a preclinical model of human liver.ResultsThe AAV variants enriched based on their improved efficiency of transduction of a human hepatocyte cell line were vectorized and subsequently functionally characterized on human cell lines. This directed evolution method enabled us to select novel AAV variant, AAV-CH4.2. While the selected variant did not exceed the parental serotype in terms of transduction efficiency, it was substantially more efficient at packaging than its closest homolog, serotypes AAV6.ConclusionsBased on its strong transduction profile and manufacturability, we believe that AAV-CH4.2 is a strong candidate for further evaluation and as a potential novel gene therapy vector for preclinical studies in human liver applications.

Polysaccharide Nanofiber-Stabilized Pickering Emulsion Microparticles Induce Pyroptotic Cell Death in Hepatocytes and Kupffer Cells.

The intracellular uptake and interaction behavior of emulsion microparticles in liver cells critical to host defense and inflammation is significant to understanding their potential cytotoxicity and biomedical applications. In this study, the cell death responses of fibroblastic, hepatocyte, and Kupffer cells (KCs) induced by four types of emulsion particles that are stabilized by polysaccharide nanofibers (cellulose or chitin), an inorganic nanoparticle (β-tricalcium phosphate), or surfactants are compared. Pickering emulsion (PE) microparticles stabilized by polysaccharide nanofibers or inorganic nanoparticles have a droplet size of 1-3µm, while the surfactant-stabilized emulsion has a diameter of ≈190nm. Polysaccharide nanofiber-stabilized PEs (PPEs) markedly induce lactate dehydrogenase release in all cell types. Additionally, characteristic pyroptotic cell death, which is accompanied by cell swelling, membrane blebbing, and caspase-1 activation, occurs in hepatocytes and KCs. These PE microparticles are co-cultured with lipopolysaccharide-primed KCs associated with cytokine interleukin-1β release, and the PPEs demonstrate biological activity as a mediator of the inflammation response. Well-designed PPE microparticles induce pyroptosis of liver cells, which may provide new insight into regulating inflammation-related diseases for designing potent anticancer drugs and vaccine adjuvants.

Roles of viable but non-culturable state in the survival of Campylobacter jejuni.

Despite being considered fragile and fastidious, Campylobacter jejuni is the most prevalent cause of foodborne bacterial gastroenteritis, and chicken meat is considered the main vehicle of transmission to humans. This agent can survive adverse conditions in the form of biofilms, but extreme stress (nutritional, oxidative and thermal) promotes the acquisition of a state called viable but not culturable (VBNC). The emergence of this pathogen worldwide and the recent international requirements in its control instigated us to qualitatively and quantitatively estimate the time required for the acquisition of the VBNC form in 27 strains of C. jejuni, characterize morphological aspects, determine its adaptive and invasive potential and perform comparative metabolomic evaluation. Extreme stress promoted the complete acquisition of the VBNC form in a mean time of 26 days. Starting from an average initial count of 7.8 log CFU/mL, the first four days determined the greatest average reduction of the culturable form of 3.2 log CFU/mL. The scanning and transmission image analyses showed a transition from the typical viable form (VT) to the VBNC form, with initial acquisition of the straight rod shape, followed by loss of the flagella and subdivision into two to 11 imperfect cocci arranged in a chain and rich in cellular content, until their individual release. RT-PCR identified the presence of ciaB and p19 transcripts in the 27 cultivable C. jejuni strains, a character maintained in the VBNC form only for p19 and in 59.3% (16/27) of the VBNC strains for the ciaB gene. The average inoculation of 1.8 log CFU/mL of C. jejuni VBNC into primary chicken embryo hepatocyte cells promoted the occurrence of apoptosis processes significantly after 24 hours of contact by one of the strains tested. In C. jejuni VBNC, we detected higher expression of metabolites linked to protective and adaptation mechanisms and of volatile organic precursor compounds indicative of metabolism interruption. The oscillations in the time of acquisition of the VBNC form together with the presence of transcripts for ciaB and p19, the identification of cell lysis and metabolites that ensure the maintenance of the pathogen alert to the fact that C. jejuni VBNC remains virulent and adapted to stress, which makes evident the potential danger of this latent form, which is not detectable by official methodologies.

The Protective Potential eff ect of Metformin during Acetaminophen Hepatotoxicity through Nrf2 Activation

Acetaminophen (N-acetyl-para-aminophenol [APAP]) is the most commonly used medication for the relief of pain and fever around the world. Although APAP is safe and eff ective at a therapeutic level, acute overdose causes hepatotoxicity and severe liver damage. The hepatotoxicity induced by APAP is a persistent global problem that results in hepatotoxicity cases; acute liver failure and even death worldwide. This toxicity is characterized by extensive oxidant stress which consequently causes hepatocyte cell death. On the other hand, scientifi c studies have proven that MET shows hepatoprotective eff ect against hepatotoxicity induced by APAP through numerous mechanisms, e.g., antioxidant activity that alleviate the hepatotoxicity by activating Nrf2 pathway. The activation of Nrf2 pathway is anticipated to protect cells from oxidative stress that forms during hepatotoxicity. The benefi cial of using MET to protect against hepatotoxicity after APAP has been performed in an in-vitro experiment using Hep2G cell culture. However, up to our knowledge non, an in-vivo study (experimental animal) has been used to approve the benefi t of MET. Justifi cation: Nrf2 pathway activation by MET is one of the most important issues that need to be explained and followed up in laboratory animals since it has been previously studied in-vitro. Our study focuses on studying Nrf2 pathway in-vivo, since the results of in-vivo study are more relevant and similar to that of human beings. Aim of the study: To examine the possible stimulant eff ect of MET on Nrf2 pathway in experimental animals and its role in protecting the liver from oxidative stress that formed during APAP-induced hepatotoxicity. Methodology: Twenty-four wistar rats were divided randomly into four groups (six rats/Grp). Grp1; normal saline orally, Grp2; toxic dose of APAP (1000 mg/kg) orally; Grp3; 200 mg/kg of MET ip after 1-hour of APAP (1000 mg/kg) orally, Grp 4; 200 mg/kg MET ip for 24 hours. Then sera from experimental animals were collected for subsequent assessments of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzyme activities. Liver tissue was harvested to detect the expression of keap1 which is the negative regulator of Nrf2, and HO-1 and GST A1, which are related to Nrf2 pathway, by western blotting technique. Results: The results were showed a signifi cant elevation in ALT and AST activities in APAP treated group while MET normalized these biomarkers (AST and ALT). Western blotting assay showed that keap1 expression increased in APAP-treated animals while MET showed a signifi cant decrement in keap1 expression. The expression of antioxidant proteins HO-1 and GST A1 are decreased signifi cantly in APAP-treated animals while increased signifi cantly by MET treatment. Conclusion: The present results demonstrated that MET has a hepatoprotective eff ect in experimental animals against hepatotoxicity induced by APAP through activating Nrf2 pathway.

Effect of L-Dopa on Carboplatin Induced Hepatotoxicity

Carboplatin is an alkylating antineoplastic drug. Although carboplatin induces apoptosis in rapidly dividing cells like cisplatin, it has fewer side effects. The use of platinum compounds as antineoplastic agents at high doses causes hepatotoxicity, which is important in dose regulation. Levodopa is used as a precursor for dopamine synthesis in the brain in the treatment of Parkinson's disease. Dopamine synthesized in the liver from levodopa cannot cross the blood-brain barrier. There are studies suggesting that dopamine may have a protective effect on hepatocytes. In our study, we aimed to evaluate the cytoprotective effect of levodopa in cell damage induced by carboplatin in the liver cell line. Cell viability was evaluated by MTT test by exposing AML-12 hepatocyte cells to the combination of Carboplatin, L-DOPA and Carboplatin + L-DOPA for 24 hours (0, 3.13, 6.25, 12.5, 25, 50 ,100 µM) has been left. The LD50 value of carboplatin was determined as 62.65 µM. The LD50 values of the combination of L-DOPA and carboplatin + L-DOPA were higher in the tested dose range. The cytotoxic effect of carboplatin on the AML-12 cell line was determined by the increasing LD50 value, which was statistically significantly reduced when combined with levodopa. Further studies are needed to explain the mechanism of action.

ETNPPL modulates hyperinsulinemia-induced insulin resistance through the SIK1/ROS-mediated inactivation of the PI3K/AKT signaling pathway in hepatocytes.

Hyperinsulinemia is a critical risk factor for the pathogenesis of insulin resistance (IR) in metabolic tissues, including the liver. Ethanolamine phosphate phospholyase (ETNPPL), a newly discovered metabolic enzyme that converts phosphoethanolamine (PEA) to ammonia, inorganic phosphate, and acetaldehyde, is abundantly expressed in liver tissue. Whether it plays a role in the regulation of hyperinsulinemia-induced IR in hepatocytes remains elusive. Here, we established an in vitro hyperinsulinemia-induced IR model in the HepG2 human liver cancer cell line and primary mouse hepatocyte via a high dose of insulin treatment. Next, we overexpressed ETNPPL by using lentivirus-mediated ectopic to investigate the effects of ETNPPL per se on IR without insulin stimulation. To explore the underlying mechanism of ETNPPL mediating hyperinsulinemia-induced IR in HepG2, we performed genome-wide transcriptional analysis using RNA sequencing (RNA-seq) to identify the downstream target gene of ETNPPL. The results showed that ETNPPL expression levels in both mRNA and protein were significantly upregulated in hyperinsulinemia-induced IR in HepG2 and primary mouse hepatocytes. Upon silencing ETNPPL, hyperinsulinemia-induced IR was ameliorated. Under normal conditions without IR in hepatocytes, overexpressing ETNPPL promotes IR, reactive oxygen species (ROS) generation, and AKT inactivation. Transcriptome analysis revealed that salt-inducible kinase 1 (SIK1) is markedly downregulated in the ETNPPL knockdown HepG2 cells. Moreover, disrupting SIK1 prevents ETNPPL-induced ROS accumulation, damage to the PI3K/AKT pathway and IR. Our study reveals that ETNPPL mediates hyperinsulinemia-induced IR through the SIK1/ROS-mediated inactivation of the PI3K/AKT signaling pathway in hepatocyte cells. Targeting ETNPPL may present a potential strategy for hyperinsulinemia-associated metabolic disorders such as type 2 diabetes.

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis

Carboxylesterases (CEs) are a class of enzymes that catalyze the hydrolysis of esters in a variety of endogenous and exogenous molecules. CEs play an important role in drug metabolism, in the onset and progression of disease, and can be harnessed for prodrug activation strategies. As such, the regulation of CEs is an important clinical and pharmaceutical consideration. Here, we report the first ratiometric sensor for CE activity using Raman spectroscopy based on a bisarylbutadiyne scaffold. The sensor was shown to be highly sensitive and specific for CE detection and had low cellular cytotoxicity. In hepatocyte cells, the ratiometric detection of esterase activity was possible, and the result was validated by multimodal imaging with standard viability stains used for fluorescence microscopy within the same cell population. In addition, we show that the detection of localized ultraviolet damage in a mixed cell population was possible using stimulated Raman scattering microscopy coupled with spectral phasor analysis. This sensor demonstrates the practical advantages of low molecular weight sensors that are detected using ratiometric Raman imaging and will have applications in drug discovery and biomedical research.

Orthotopic Transplantation of Functional Bioengineered Livers in Rats.

Functional bioengineered livers (FBLs) are promising alternatives to orthotopic liver transplantation. However, orthotopic transplantation of FBLs has not yet been reported. This study aimed to perform the orthotopic transplantation of FBLs in rats subjected to complete hepatectomy. FBLs were developed using rat whole decellularized liver scaffolds (DLSs) with human umbilical vein endothelial cells implanted via the portal vein, and human bone marrow mesenchymal stem cells (hBMSCs) and mouse hepatocyte cell line implanted via the bile duct. FBLs were evaluated in terms of endothelial barrier function, biosynthesis, and metabolism and orthotopically transplanted into rats to determine the survival benefit. The FBLs with well-organized vascular structures exhibited endothelial barrier function, with reduced blood cell leakage. The implanted hBMSCs and hepatocyte cell line were well aligned in the parenchyma of the FBLs. The high levels of urea, albumin, and glycogen in the FBLs indicated biosynthesis and metabolism. Orthotopic transplantation of FBLs achieved a survival time of 81.38 ± 4.263 min in rats (n = 8) subjected to complete hepatectomy, whereas control animals (n = 4) died within 30 min (p < 0.001). After transplantation, CD90-positive hBMSCs and the albumin-positive hepatocyte cell line were scattered throughout the parenchyma, and blood cells were limited within the vascular lumen of the FBLs. In contrast, the parenchyma and vessels were filled with blood cells in the control grafts. Thus, orthotopic transplantation of whole DLS-based FBLs can effectively prolong the survival of rats subjected to complete hepatectomy. In summary, this work was the first to perform the orthotopic transplantation of FBLs, with limited survival benefits, which still has important value for the advancement of bioengineered livers.

Hypothermic Oxygenated Machine Perfusion Promotes Mitophagy Flux against Hypoxia-Ischemic Injury in Rat DCD Liver

Hypothermic oxygenated machine perfusion (HOPE) can enhance organ preservation and protect mitochondria from hypoxia-ischemic injury; however, an understanding of the underlying HOPE mechanism that protects mitochondria is somewhat lacking. We hypothesized that mitophagy may play an important role in HOPE mitochondria protection. Experimental rat liver grafts were exposed to 30 min of in situ warm ischemia. Then, grafts were procured, followed by cold storage for 3 or 4 h to mimic the conventional preservation and transportation time in donation after circulatory death (DCD) in clinical contexts. Next, the grafts underwent hypothermic machine perfusion (HMP) or HOPE for 1 h through portal vein only perfusion. The HOPE-treated group showed a better preservation capacity compared with cold storage and HMP, preventing hepatocyte damage, nuclear injury, and cell death. HOPE can increase mitophagy marker expression, promote mitophagy flux via the PINK1/Parkin pathway to maintain mitochondrial function, and reduce oxygen free radical generation, while the inhibition of autophagy by 3-methyladenine and chloroquine could reverse the protective effect. HOPE-treated DCD liver also demonstrated more changes in the expression of genes responsible for bile metabolism, mitochondrial dynamics, cell survival, and oxidative stress. Overall, HOPE attenuates hypoxia-ischemic injury in DCD liver by promoting mitophagy flux to maintain mitochondrial function and protect hepatocytes. Mitophagy could pave the way for a protective approach against hypoxia-ischemic injury in DCD liver.

ROS mediated pyroptosis-M1 polarization crosstalk participates in inflammation of chicken liver induced by bisphenol A and selenium deficiency

The earth's natural environmental factors and man-made industrial pollution often lead to the co-occurrence of environmental pathogenic factors and malnutrition. Bisphenol A (BPA) is a serious environmental endocrine disruptor, and its exposure can cause liver tissue damage. Selenium (Se) deficiency is a worldwide problem that afflicts thousands of people, and Se deficiency can cause M1/M2 imbalance. In addition, the crosstalk between hepatocyte and immune cell is closely related to the occurrence of hepatitis. Therefore, this study found for the first time that the combined exposure of BPA and Se deficiency caused liver pyroptosis and M1 polarization through ROS, and the crosstalk between pyroptosis and M1 polarization aggravated liver inflammation in chicken. In this study, the BPA or/and Se deficiency chicken liver, single and co-culture model of LMH and HD11 cells were established. The results displayed that BPA or Se deficiency induced liver inflammation accompanied by pyroptosis and M1 polarization through oxidative stress, and increased expressions of chemokines (CCL4, CCL17, CCL19, and MIF) and inflammatory factors (IL-1β and TNF-α). The vitro experiments further verified the above changes and showed that LMH pyroptosis promoted M1 polarization of HD11 cells, and vice versa. NAC counteracted pyroptosis and M1 polarization caused by BPA and low-Se, reducing the release of inflammatory factors. In brief, BPA and Se deficiency treatment can exacerbate liver inflammation by increasing oxidative stress to induce pyroptosis and M1 polarization.

Phytochemical and in-vitro Biological Investigation of Indian Traditional Medicinal Plants for their Cytotoxicity and Hepatoprotective Potential.

This study aimed to select 16 medicinal plants based on their folklore remedy for treating various diseases like inflammation, cancer, etc., and scientifically validate their potency. Five among them, namely Centella asiatica (CA), Myristica fragrans (MF), Trichosanthes palmata (TP), Woodfordia fruticosa (WF), and Curculigo orchioides (CO), were scientifically confirmed through the extraction and in-vitro cytotoxic and hepatoprotective evaluation. Based on the cytotoxic and hepatoprotective results, the various fractions of CO were chosen for an in-depth phytochemical study to isolate and characterize active compounds by GC-MS. The results showed promising cytotoxic activity (i.e., IC50=<100 μg/ml) against HeLa cell lines and significant hepatoprotective activity in a dose-dependent manner on CCl4 intoxicated isolated hepatocyte cells. The present study confirmed the scientific evidence regarding the effectiveness of selected medicinal plants in HeLa and hepatocyte cells. Furthermore, a detailed study on their mechanism of action and clinical application is suggested.

Single-cell transcriptomic dissection of the cellular and molecular events underlying the triclosan-induced liver fibrosis in mice

BackgroundTriclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage.MethodsC57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing (scRNA-seq) was then carried out on TCS- or mock-treated mouse livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor (TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the molecular and cellular events after TCS exposure. To verify the TCS-induced liver fibrosis, the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson’s trichrome and Sirius red staining. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies.ResultsWe established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control group profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells (HSCs) were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition, other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interaction-mediated cellular communication in promoting liver fibrosis.ConclusionsTCS modulates the cellular activities and fates of several specific cell types (including hepatocytes, HSCs, endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis. Overall, we provide the first comprehensive single-cell atlas of mouse livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.

Ultrasound Histotripsy on a Viable Perfused Whole Porcine Liver: Histological Aspects, Including 3D Reconstruction of the Histotripsy Site.

Non-invasive therapeutic-focused ultrasound (US) can be used for the mechanical dissociation of tissue and is described as histotripsy. We have performed US histotripsy in viable perfused ex vivo porcine livers as a step in the development of a novel approach to hepatocyte cell transplantation. The histotripsy nidus was created with a 2 MHz single-element focused US transducer, producing 50 pulses of 10 ms duration, with peak positive and negative pressure values of P+ = 77.7 MPa and P- = -13.7 MPaat focus, respectively, and a duty cycle of 1%. Here, we present the histological analysis, including 3D reconstruction of histotripsy sites. Five whole porcine livers were retrieved fresh from the abattoir using human transplant retrieval and cold static preservation techniques and were then perfused using an organ preservation circuit. Whilst under perfusion, histotripsy was performed to randomly selected sites on the live. Fifteen lesional sites were formalin-fixed and paraffin-embedded. Sections were stained with Haematoxylin and Eosin and picro-Sirius red, and they were also stained for reticulin. Additionally, two lesion sites were used for 3D reconstruction. The core of the typical lesion consisted of eosinophilic material associated with reticulin loss, collagen damage including loss of birefringence to fibrous septa, and perilesional portal tracts, including large portal vein branches, but intact peri-lesional hepatic plates. The 3D reconstruction of two histotripsy sites was successful and confirmed the feasibility of this approach to investigate the effects of histotripsy on tissue in detail.

Effect of HepG2 cell 3D cultivation on the metabolism of the anabolic androgenic steroid metandienone.

The elucidation of the metabolic fate of prohibited substances is crucial for the abuse detection. The human hepatocyte cell line HepG2 can be used to study biotransformation. In order to improve this in vitro model system, we compared the HepG2 spheroid generation using three different techniques: a forced floating, a scaffold-free and a scaffold-based method. We characterized the spheroids with regard to the expression levels of the proliferation marker Mki67, the liver-specific marker albumin and biotransformation enzymes. Moreover, the metandienone metabolite pattern was comparatively analysed by high-performance liquid chromatography mass spectrometry. With all three techniques, HepG2 spheroids were generated showing a degree of differentiation. The forced floating method resulted in very large spheroids (1mm in diameter) showing signs of necrosis in the centre and a very low metandienone conversion rate. The spheroids formed by the two other techniques were comparable in size with 0.5mm in diameter on average. Among the three different 3D cultivation methods, the HepG2 spheroids formed on Matrigel® as extracellular matrix were the most promising regarding biotransformation studies on anabolic androgenic steroids. Prospectively, HepG2 spheroids are a promising in vitro model system to study multidrug setups, drug-drug interactions and the biotransformation of other substance classes.

Piezo1 alleviates acetaminophen-induced acute liver injury by activating Nrf2 and reducing mitochondrial reactive oxygen species

Acetaminophen (APAP) overdose is the most common cause for acute liver failure (ALF) in the developed countries, with limited treatment options. Piezo1 is a mechanosensitive cation channel. We found that APAP caused upregulation of Piezo1 in both an APAP-induced acute liver injury (ALI) animal model and a mouse hepatocyte cell line AML12. Activation of Piezo1 by its activator Yoda1 reduced APAP-induced hepatotoxicity and ROS level. Mechanistically, activation of Piezo1 led to accumulation of the antioxidant regulator Nrf2 and upregulation of its target genes Nqo1 and Gsta1, while knockdown of Piezo1 downregulated this pathway. Finally, injection of Yoda1 decreased serum AST and ALT levels, reduced cell death and rescued liver injury in the APAP-induced ALI mouse model. Our findings suggested a previously undiscovered protective role of Piezo1 in APAP-induced ALI, which might shed light on a new therapeutic target for this disease.

Hepatocyte SGK1 activated by hepatic ischemia-reperfusion promotes the recurrence of liver metastasis via IL-6/STAT3

BackgroundLiver metastasis is the leading cause of death in patients with colorectal cancer (CRC). Surgical resection of the liver metastases increases the incidence of long-term survival in patients with colorectal liver metastasis (CRLM). However, many patients experience CRLM recurrence after the initial liver resection. As an unavoidable pathophysiological process in liver surgery, liver ischemia-reperfusion (IR) injury increases the risk of tumor recurrence and metastasis.MethodsColorectal liver metastasis (CRLM) mouse models and mouse liver partial warm ischemia models were constructed. The levels of lipid peroxidation were detected in cells or tissues. Western Blot, qPCR, elisa, immunofluorescence, immunohistochemistry, scanning electron microscope, flow cytometry analysis were conducted to evaluate the changes of multiple signaling pathways during CRLM recurrence under liver ischemia-reperfusion (IR) background, including SGK1/IL-6/STAT3, neutrophil extracellular traps (NETs) formation, polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) infiltration.ResultsHepatocyte serum/glucocorticoid regulated kinase 1 (SGK1) was activated in response to hepatic ischemia-reperfusion injury to pass hepatocyte STAT3 phosphorylation and serum amyloid A (SAA) hyperactivation signals in CRLM-IR mice, such regulation is dependent on SGK-activated IL-6 autocrine. Administration of the SGK1 inhibitor GSK-650394 further reduced ERK-related neutrophil extracellular traps (NETs) formation and polymorphonucler myeloid-derived suppressor cells (PMN-MDSC) infiltration compared with targeting hepatocyte SGK1 alone, thereby alleviating CRLM in the context of IR.ConclusionsOur study demonstrates that hepatocyte and immune cell SGK1 synergistically promote postoperative CRLM recurrence in response to hepatic IR stress, and identifies SGK1 as a translational target that may improve postoperative CRLM recurrence.

Efficient transfection of Atlantic salmon primary hepatocyte cells for functional assays and gene editing.

The expansion of genomic resources for Atlantic salmon over the past half decade has enabled efficient interrogation of genetic traits by large-scale correlation of genotype to phenotype. Moving from correlation to causation will require genotype-phenotype relationships to be tested experimentally in a cost-efficient and cell context-relevant manner. To enable such future experiments, we have developed a method for the isolation and genetic manipulation of primary hepatocytes from Atlantic salmon for use in heterologous expression, reporter assay, and gene editing experiments. We chose the liver as the tissue of interest because it is the metabolic hub and many current Atlantic salmon research projects focus on understanding metabolic processes to improve traits such as the growth rate, total fat content, and omega-3 content. We find that isolated primary hepatocytes are optimally transfected with both plasmid and ribonucleoprotein using a Neon electroporator at 1,400 V, 10 ms, and 2 pulses. Transfection efficiency with plasmid and cutting efficiency with ribonucleoprotein were optimally 46% and 60%, respectively. We also demonstrate a 26 times increase in luciferase expression under the promoter of the key liver metabolic gene, elovl5b, compared to an empty vector, in line with expected liver-specific expression. Taken together, this work provides a valuable resource enabling transfection and gene editing experiments in a context-relevant and cost-effective system.

Mechanisms of Arachidonic Acid In Vitro Tumoricidal Impact.

To promote the potential of arachidonic acid (ARA) for cancer prevention and management, experiments were implemented to disclose the mechanisms of its tumoricidal action. Hepatocellular, lung, and breast carcinoma and normal hepatocytes cell lines were exposed to 0 or 50 μM ARA for 30 min and then assessed for proliferative capacity, surface membrane-associated sphingomyelin (SM) content, neutral sphingomyelinase (nSMase) activity, beta 2 microglobulin (β2 m) expression, and ceramide (Cer) levels. Reactive oxygen species (ROS) content and caspase 3/7 activity were evaluated. Exposure to ARA for 30 min led to impairment of the tumor cells' proliferative capacity and revealed that the different cell lines display remarkably similar surface membrane SM content but diverse responses to ARA treatment. Arachidonic acid tumoricidal impact was shown to be associated with nSMase activation, exposure of cell surface membrane β2 m to antibody binding, and hydrolysis of SM to Cer, which accumulated on the cell surface and in the cytosol. The ARA and Cer-mediated inhibition of tumor cell viability appeared to be independent of ROS generation or caspase 3/7 activation. The data were compared and contrasted to findings reported in the literature on ARA tumoricidal mechanisms.