Isolation and Cryopreservation of Primary Macaque Hepatocytes.

Primary human hepatocytes (PHHs) are recognized as the "gold standard" for evaluating toxicity of various drugs or chemicals in vitro. However, due to their limited availability, primary hepatocytes isolated from rodents are more commonly used in various experimental studies than PHHs. However, bigger differences in drug metabolism were seen between humans and rats compared to those between human and non-human primates. Here, we describe a method to isolate primary hepatocytes from the liver of rhesus macaques (Macaca mulatta, a species of Old-World monkey) after in situ whole liver perfusion. Techniques for cryopreserving and recovering primary macaque hepatocytes (PMHs) are also described. Given the remarkable physiological and genetic similarity of non-human primates to humans, PMHs isolated using this protocol may serve as a reliable surrogate of PHHs in toxicological research and preclinical studies. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: In situ whole liver perfusion Basic Protocol 2: Primary macaque hepatocyte isolation and cell plating Basic Protocol 3: Cryopreservation and recovery of primary macaque hepatocytes.

Improved functionality of hepatic spheroids cultured in acoustic levitation compared to existing 2D and 3D models.

Hepatic spheroids are of high interest in basic research, drug discovery and cell therapy. Existing methods for spheroid culture present advantages and drawbacks. An alternative technology is explored: the hepatic spheroid formation and culture in an acoustofluidic chip, using HepaRG cell line. Spheroid formation and morphology, cell viability, genetic stability, and hepatic functions are analyzed after 6 days of culture in acoustic levitation. They are compared to 2D culture and non-levitated 3D cultures. Sizes of the 25 spheroids created in a single acoustofluidic microphysiological system are homogeneous. The acoustic parameters in our system do not induce cell mortality nor DNA damage. Spheroids are cohesive and dense. From a functional point of view, hepatic spheroids obtained by acoustic levitation exhibit polarity markers, secrete albumin and express hepatic genes at higher levels compared to 2D and low attachment 3D cultures. In conclusion, this microphysiological system proves not only to be suitable for long-term culture of hepatic spheroids, but also to favor differentiation and functionality within 6 days of culture.

Targeting mitochondrial homeostasis in the treatment of non-alcoholic fatty liver disease: a review.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, and its prevalence is rapidly increasing. Antioxidants, lipid-lowering medications, and lifestyle interventions are the most commonly used treatment options for NAFLD, but their efficacy in inhibiting steatosis progression is limited and their long-term ineffectiveness and adverse effects have been widely reported. Therefore, it is important to gain a deeper understanding of the pathogenesis of NAFLD and to identify more effective therapeutic approaches. Mitochondrial homeostasis governs cellular redox biology, lipid metabolism, and cell death, all of which are crucial to control hepatic function. Recent findings have indicated that disruption of mitochondrial homeostasis occurs in the early stage of NAFLD and mitochondrial dysfunction reinforces disease progression. In this review, we summarize the physical roles of the mitochondria and describe their response and dysfunction in the context of NAFLD. We also discuss the drug targets associated with the mitochondria that are currently in the clinical trial phase of exploration. From our findings, we hope that the mitochondria may be a promising therapeutic target for the treatment of NAFLD.

An Ultrathin Coating of Microcapsules Enhances the Function of Encapsulated Hepatocyte Spheroids.

Maintaining the differentiated phenotype and function of primary hepatocytes in vitro and in vivo represents a distinct challenge. Our paper describes microcapsules comprised of a bioactive polymer and overcoated with an ultrathin film as a means of maintaining the function of entrapped hepatocytes for at least two weeks. We previously demonstrated that heparin (Hep)-based microcapsules improved the function of entrapped primary hepatocytes by capturing and releasing cell-secreted inductive signals, including hepatocyte growth factor (HGF). Further enhancement of hepatic function could be gained by loading exogenous HGF into microcapsules. In this study, we demonstrate that an ultrathin coating of tannic acid (TA) further enhances endogenous HGF signaling for entrapped hepatocytes and increases by 2-fold the rate of uptake of exogenous HGF by Hep microcapsules. Hepatocytes in overcoated microcapsules exhibited better function and hepatic gene expression than in capsules without a TA coating. Our study showcases the potential application of ultrathin coatings to modulate the bioactivity of microcapsules and may enable the use of encapsulated hepatocytes for modeling drug toxicity or treating liver diseases.

Methionine deficiency inhibited pyroptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella): possibly via activating the ROS-AMPK-autophagy axis.

BACKGROUND: Methionine (Met) is the only sulfur-containing amino acid among animal essential amino acids, and methionine deficiency (MD) causes tissue damage and cell death in animals. The common modes of cell death include apoptosis, autophagy, pyroptosis, necroptosis. However, the studies about the major modes of cell death caused by MD have not been reported, which worth further study. METHODS: Primary hepatocytes from grass carp were isolated and treated with different doses of Met (0, 0.5, 1, 1.5, 2, 2.5 mmol/L) to examine the expression of apoptosis, pyroptosis, autophagy and necroptosis-related proteins. Based on this, we subsequently modeled pyroptosis using lipopolysaccharides and nigericin sodium salt, then autophagy inhibitors chloroquine (CQ), AMP-activated protein kinase (AMPK) inhibitors compound C (CC) and reactive oxygen species (ROS) scavengers N-acetyl-L-cysteine (NAC) were further used to examine the expression of proteins related to pyroptosis, autophagy and AMPK pathway in MD-treated cells respectively. RESULTS: MD up-regulated B-cell lymphoma protein 2 (Bax), microtubule-associated protein 1 light chain 3 II (LC3 II), and down-regulated the protein expression levels of B-cell lymphoma-2 (Bcl-2), sequestosome 1 (p62), cleaved-caspase-1, cleaved-interleukin (IL)-1ß, and receptor-interacting protein kinase (RIP) 1 in hepatocytes, while it did not significantly affect RIP3. In addition, MD significantly increased the protein expression of liver kinase B1 (LKB1), p-AMPK, and Unc-51-like kinase 1 (ULK1) without significant effect on p-target of rapamycin. Subsequently, the use of CQ increased the protein expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cleaved-caspase-1, and cleaved-IL-1ß inhibited by MD; the use of CC significantly decreased the protein expression of MD-induced LC3 II and increased the protein expression of MD-suppressed p62; then the use of NAC decreased the MD-induced p-AMPK protein expression. CONCLUSION: MD promoted autophagy and apoptosis, but inhibited pyroptosis and necroptosis. MD inhibited pyroptosis may be related regarding the promotion of autophagy. MD activated AMPK by inducing ROS production which in turn promoted autophagy. These results could provide partial theoretical basis for the possible mechanisms of Met in ensuring the normal structure and function of animal organs. Furthermore, ferroptosis is closely related to redox states, it is worth investigating whether MD affects ferroptosis in hepatocytes.

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-1ß, 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.

ZLN005, a PGC-1α Activator, Protects the Liver against Ischemia-Reperfusion Injury and the Progression of Hepatic Metastases.

BACKGROUND: Exercise can promote sustainable protection against cold and warm liver ischemia-reperfusion injury (IRI) and tumor metastases. We have shown that this protection is by the induction of hepatic mitochondrial biogenesis pathway. In this study, we hypothesize that ZLN005, a PGC-1α activator, can be utilized as an alternative therapeutic strategy. METHODS: Eight-week-old mice were pretreated with ZLN005 and subjected to liver warm IRI. To establish a liver metastatic model, MC38 cancer cells (1 × 106) were injected into the spleen, followed by splenectomy and liver IRI. RESULTS: ZLN005-pretreated mice showed a significant decrease in IRI-induced tissue injury as measured by serum ALT/AST/LDH levels and tissue necrosis. ZLN005 pretreatment decreased ROS generation and cell apoptosis at the site of injury, with a significant decrease in serum pro-inflammatory cytokines, innate immune cells infiltration, and intrahepatic neutrophil extracellular trap (NET) formation. Moreover, mitochondrial mass was significantly upregulated in hepatocytes and maintained after IRI. This was confirmed in murine and human hepatocytes treated with ZLN005 in vitro under normoxic and hypoxic conditions. Additionally, ZLN005 preconditioning significantly attenuated tumor burden and increased the percentage of intratumoral cytotoxic T cells. CONCLUSIONS: Our study highlights the effective protection of ZLN005 pretreatment as a therapeutic alternative in terms of acute liver injury and tumor metastases.

The antifibrotic potential of IMT504: modulation of GLAST + Wnt1 + bone marrow stromal progenitors and hepatic microenvironment.

BACKGROUND: The immunomodulatory oligodeoxynucleotide (ODN) IMT504 might harbor antifibrotic properties within the liver. METHODS: Fibrosis models were induced in mice through thioacetamide (TAA) administration and bile-duct ligation. Cre-loxP mice were utilized to identify GLAST + Wnt1 + bone marrow stromal progenitors (BMSPs) and to examine their contribution with cells in the liver. In vivo and in vitro assays; flow-cytometry, immunohistochemistry, and qPCR were conducted. RESULTS: IMT504 demonstrated significant inhibition of liver fibrogenesis progression and reversal of established fibrosis. Early responses to IMT504 involved the suppression of profibrogenic and proinflammatory markers, coupled with an augmentation of hepatocyte proliferation. Additionally, this ODN stimulated the proliferation and mobilization of GLAST + Wnt1 + BMSPs, likely amplifying their contribution with endothelial- and hepatocytes-like cells. Moreover, IMT504 significantly modulated the expression levels of Wnt ligands and signaling pathway/target genes specifically within GLAST + Wnt1 + BMSPs, with minimal impact on other BMSPs. Intriguingly, both IMT504 and conditioned media from IMT504-pre-treated GLAST + Wnt1 + BMSPs shifted the phenotype of fibrotic macrophages, hepatic stellate cells, and hepatocytes, consistent with the potent antifibrotic effects observed. CONCLUSION: In summary, our findings identify IMT504 as a promising candidate molecule with potent antifibrotic properties, operating through both direct and indirect mechanisms, including the activation of GLAST + Wnt1 + BMSPs.

A two-step strategy to expand primary human hepatocytes in vitro with efficient metabolic and regenerative capacities.

BACKGROUND: Primary human hepatocytes (PHHs) are highly valuable for drug-metabolism evaluation, liver disease modeling and hepatocyte transplantation. However, their availability is significantly restricted due to limited donor sources, alongside their constrained proliferation capabilities and reduced functionality when cultured in vitro. To address this challenge, we aimed to develop a novel method to efficiently expand PHHs in vitro without a loss of function. METHODS: By mimicking the in vivo liver regeneration route, we developed a two-step strategy involving the de-differentiation/expansion and subsequent maturation of PHHs to generate abundant functional hepatocytes in vitro. Initially, we applied SiPer, a prediction algorithm, to identify candidate small molecules capable of activating liver regenerative transcription factors, thereby formulating a novel hepatic expansion medium to de-differentiate PHHs into proliferative human hepatic progenitor-like cells (ProHPLCs). These ProHPLCs were then re-differentiated into functionally mature hepatocytes using a new hepatocyte maturation condition. Additionally, we investigated the underlying mechanism of PHHs expansion under our new conditions. RESULTS: The novel hepatic expansion medium containing hydrocortisone facilitated the de-differentiation of PHHs into ProHPLCs, which exhibited key hepatic progenitor characteristics and demonstrated a marked increase in proliferation capacity compared to cells cultivated in previously established expansion conditions. Remarkably, these subsequent matured hepatocytes rivaled PHHs in terms of transcriptome profiles, drug metabolizing activities and in vivo engraftment capabilities. Importantly, our findings suggest that the enhanced expansion of PHHs by hydrocortisone may be mediated through the PPARα signaling pathway and regenerative transcription factors. CONCLUSIONS: This study presents a two-step strategy that initially induces PHHs into a proliferative state (ProHPLCs) to ensure sufficient cell quantity, followed by the maturation of ProHPLCs into fully functional hepatocytes to guarantee optimal cell quality. This approach offers a promising means of producing large numbers of seeding cells for hepatocyte-based applications.

Airway extracellular LTA4H concentrations are governed by release from liver hepatocytes and changes in lung vascular permeability.

Leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme, with dual activities critical in defining the scale of tissue inflammation and pathology. LTA4H classically operates intracellularly, primarily within myeloid cells, to generate pro-inflammatory leukotriene B4. However, LTA4H also operates extracellularly to degrade the bioactive collagen fragment proline-glycine-proline to limit neutrophilic inflammation and pathological tissue remodeling. While the dichotomous functions of LTA4H are dictated by location, the cellular source of extracellular enzyme remains unknown. We demonstrate that airway extracellular LTA4H concentrations are governed by the level of pulmonary vascular permeability and influx of an abundant repository of blood-borne enzyme. In turn, blood LTA4H originates from liver hepatocytes, being released constitutively but further upregulated during an acute phase response. These findings have implications for our understanding of how inflammation and repair are regulated and how perturbations to the LTA4H axis may manifest in pathologies of chronic diseases.

The AP-1 adaptor complex is essential for intracellular trafficking of the ORF2 capsid protein and assembly of Hepatitis E virus.

Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.

Analysis of host factor networks during hepatitis B virus infection in primary human hepatocytes.

BACKGROUND: Chronic hepatitis B virus (HBV) infection affects around 250 million people worldwide, causing approximately 887,000 deaths annually, primarily owing to cirrhosis and hepatocellular carcinoma (HCC). The current approved treatments for chronic HBV infection, such as interferon and nucleos(t)ide analogs, have certain limitations as they cannot completely eradicate covalently closed circular DNA (cccDNA). Considering that HBV replication relies on host transcription factors, focusing on host factors in the HBV genome may provide insights into new therapeutic targets against HBV. Therefore, understanding the mechanisms underlying viral persistence and hepatocyte pathogenesis, along with the associated host factors, is crucial. In this study, we investigated novel therapeutic targets for HBV infection by identifying gene and pathway networks involved in HBV replication in primary human hepatocytes (PHHs). Importantly, our study utilized cultured primary hepatocytes, allowing transcriptomic profiling in a biologically relevant context and enabling the investigation of early HBV-mediated effects. METHODS: PHHs were infected with HBV virion particles derived from HepAD38 cells at 80 HBV genome equivalents per cell (Geq/cell). For transcriptomic sequencing, PHHs were harvested 1, 2-, 3-, 5-, and 7 days post-infection (dpi). After preparing the libraries, clustering and sequencing were conducted to generate RNA-sequencing data. This data was processed using Bioinformatics tools and software to analyze DEGs and obtain statistically significant results. Furthermore, qRT-PCR was performed to validate the RNA-sequencing results, ensuring consistent findings. RESULTS: We observed significant alterations in the expression patterns of 149 genes from days 1 to 7 following HBV infection (R2 > 0.7, q < 0.05). Functional analysis of these genes identified RNA-binding proteins involved in mRNA metabolism and the regulation of alternative splicing during HBV infection. Results from qRT-PCR experiments and the analysis of two validation datasets suggest that RBM14 and RPL28 may serve as potential biomarkers for HBV-associated HCC. CONCLUSIONS: Transcriptome analysis of gene expression changes during HBV infection in PHHs provided valuable insights into chronic HBV infection. Additionally, understanding the functional involvement of host factor networks in the molecular mechanisms of HBV replication and transcription may facilitate the development of novel strategies for HBV treatment.

Regulation of lipid metabolism in grass carp primary hepatocytes by exosomes derived from fatty hepatocytes though GRP78.

Exosomes regulate lipid metabolism by carrying miRNAs, nucleic acids, and proteins, thereby influencing the function of receptor cells. Glucose-regulated protein 78 (GRP78) is also involved in the regulation of lipid metabolism. However, it remains unclear whether exosomes derived from fatty hepatocytes (OA-Exo) regulate lipid metabolism through the enrichment of GRP78. In this study, we observed the expression of GRP78 was significantly increased in fatty hepatocytes (incubating hepatocytes with oleic acid (OA) for 24 h) and OA-Exo (P < 0.05). In addition, OA-Exo (50 µg/mL) and GRP78 protein (1 µg/mL) significant increased the content of triacylglycerol (TG) and total cholesterol (TC), as well as up-regulated the expression of GRP78 and inositol-requiring enzyme-1alpha (IRE1α) protein (P < 0.05). We further used YUM70 (an inhibitor of GRP78) to inhibit endogenous GRP78, and compared with the YUM70 group, OA-Exo reversed the effect of YUM70 and increased the content of TG, TC, and the expression of GRP78 protein in hepatocytes (P < 0.05). Furthermore, the inhibition of the IRE1α pathway with 4µ8C resulted in a significant decrease in TG content compared to the control group (P < 0.05). However, when compared with the 4µ8C group, OA-Exo and GRP78 reversed the effect of 4µ8C and significantly increased TG content (P < 0.05). Taken together, these results indicated that OA-Exo activated IRE1α to promote lipid accumulation in hepatocytes through the enrichment of GRP78. This study provided a new perspective for further exploration of exosomal lipid metabolism in fish.

MicroRNAs modulate SARS-CoV-2 infection of primary human hepatocytes by regulating the entry factors ACE2 and TMPRSS2.

BACKGROUND AND AIMS: Severe acute respiratory syndrome coronavirus (SARS-CoV-2) preferentially infects the respiratory tract; however, several studies have implicated a multi-organ involvement. Hepatic dysfunctions caused by SARS-CoV-2 infection have been increasingly recognized and described to correlate with disease severity. To elucidate molecular factors that could contribute towards hepatic infection, we concentrated on microRNAs (miRNAs), a class of small non-coding RNAs that modulate various cellular processes and which are reported to be differentially regulated during liver injury. We aimed to study the infection of primary human hepatocytes (PHH) with SARS-CoV-2 and to evaluate the potential of miRNAs for modulating viral infection. METHODS: We analysed liver autopsies from a coronavirus disease 19 (COVID-19)-positive cohort for the presence of viral RNA using Nanopore sequencing. PHH were used for the infection with SARS-CoV-2. The candidate miRNAs targeting angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) were identified using in silico approaches. To discover the potential regulatory mechanism, transfection experiments, qRT-PCRs, western blots and luciferase reporter assays were performed. RESULTS: We could detect SARS-CoV-2 RNA in COVID-19-positive liver autopsies. We show that PHH express ACE2 and TMPRSS2 and can be readily infected with SARS-CoV-2, resulting in robust replication. Transfection of selected miRNA mimics reduced SARS-CoV-2 receptor expression and SARS-CoV-2 burden in PHH. In silico and biochemical analyses supported a potential direct binding of miR-141-3p to the SARS-CoV-2 genome. CONCLUSION: We confirm that PHH are susceptible to SARS-CoV-2 infection and demonstrate selected miRNAs targeting SARS-CoV-2 entry factors and/or the viral genome reduce viral loads. These data provide novel insights into hepatic susceptibility to SARS-CoV-2 and associated dysfunctions in COVID-19.

The pivotal role of dysregulated autophagy in the progression of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a clinicopathologic syndrome characterized by excessive fat deposition in hepatocytes and a major cause of end-stage liver disease. Autophagy is a metabolic pathway responsible for degrading cytoplasmic products and damaged organelles, playing a pivotal role in maintaining the homeostasis and functionality of hepatocytes. Recent studies have shown that pharmacological intervention to activate or restore autophagy provides benefits for liver function recovery by promoting the clearance of lipid droplets (LDs) in hepatocytes, decreasing the production of pro-inflammatory factors, and inhibiting activated hepatic stellate cells (HSCs), thus improving liver fibrosis and slowing down the progression of NAFLD. This article summarizes the physiological process of autophagy, elucidates the close relationship between NAFLD and autophagy, and discusses the effects of drugs on autophagy and signaling pathways from the perspectives of hepatocytes, kupffer cells (KCs), and HSCs to provide assistance in the clinical management of NAFLD.

Sex hormones differently regulate lipid metabolism genes in primary human hepatocytes.

BACKGROUND: Prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is higher in men than in women. Hormonal and genetic causes may account for the sex differences in MASLD. Current human in vitro liver models do not sufficiently take the influence of biological sex and sex hormones into consideration. METHODS: Primary human hepatocytes (PHHs) were isolated from liver specimen of female and male donors and cultured with sex hormones (17ß-estradiol, testosterone and progesterone) for up to 72 h. mRNA expression levels of 8 hepatic lipid metabolism genes were analyzed by RT-qPCR. Sex hormones and their metabolites were determined in cell culture supernatants by LC-MS analyses. RESULTS: A sex-specific expression was observed for LDLR (low density lipoprotein receptor) with higher mRNA levels in male than female PHHs. All three sex hormones were metabolized by PHHs and the effects of hormones on gene expression levels varied depending on hepatocyte sex. Only in female PHHs, 17ß-estradiol treatment affected expression levels of PPARA (peroxisome proliferator-activated receptor alpha), LIPC (hepatic lipase) and APOL2 (apolipoprotein L2). Further changes in mRNA levels of female PHHs were observed for ABCA1 (ATP-binding cassette, sub-family A, member 1) after testosterone and for ABCA1, APOA5 (apolipoprotein A-V) and PPARA after progesterone treatment. Only the male PHHs showed changing mRNA levels for LDLR after 17ß-estradiol and for APOA5 after testosterone treatment. CONCLUSIONS: Male and female PHHs showed differences in their expression levels of hepatic lipid metabolism genes and their responsiveness towards sex hormones. Thus, cellular sex should be considered, especially when investigating the pathophysiological mechanisms of MASLD.

Ontogeny of hepatic Organic Cation Transporter-1 in rat and human.

The organic cation transporter-1 (OCT1) mediates hepatic uptake of cationic endogenous compounds and xenobiotics. To date, limited information exists on how Oct1/OCT1 functionally develops with age in rat and human livers) and how this would affect pharmacokinetics of OCT substrates in children or juvenile animals. The functional ontogeny of rOct/hOCT was profiled in suspended rat (2-57 days old) and human hepatocytes (paediatric liver tissue donors: age 2-12 months) by determining uptake clearance of 4-[4-(dimethylamino)styryl]-N-methylpyridinium iodide (ASP+) as a known rOct/hOCT probe substrate. mRNA expression was determined in rat liver tissue corresponding to rat ages used in the functional studies, while hOCT1 mRNA expressions were determined in the same hepatocyte batches as used for uptake studies. Maturation of rOct/hOCT activity and expression were evaluated by comparing values obtained at the various ages to the adult values. Relative to adult values (at 8 weeks), ASP+ uptake clearance in suspended rat hepatocytes aged 0, 1, 2, 3, 4, 5 and 6 weeks reached 26, 29, 33, 37, 72, 63 and 71%, respectively. Hepatic Oct1 mRNA expression was consistent with Oct activity (correlation coefficient of 0.92). In human hepatocytes, OCT1 activity was age-dependent and also correlated with mRNA levels (correlation coefficient of 0.88). These data show thatOct1/OCT1 activities and expression mature gradually in rat/human liver, thereby mirroring the expression pattern of Organic Anion Transporting Polypeptide (Oatp1b2) in rat. These high-resolution transporter ontogeny profiles will allow for more accurate prediction of the pharmacokinetics of OCT1/Oct1 substrates in paediatric populations and juvenile animals. Significance Statement Organic Cation Transporter-1 (OCT1) represents a major drug uptake transporter in human liver. This study provides high resolution data regarding the age-dependent function of OCT1 in the liver, based on in vitro experiments with rat and human hepatocytes obtained from donors between birth and adulthood. These ontogeny profiles will inform improved age-specific physiologically-based pharmacokinetic (PBPK) models for OCT1 drug substrates in neonates, infants, children and adults.

Hepatocyte-derived tissue extracellular vesicles safeguard liver regeneration and support regenerative therapy.

Tissue-derived extracellular vesicles (EVs) are emerging as pivotal players to maintain organ homeostasis, which show promise as a next-generation candidate for medical use with extensive source. However, the detailed function and therapeutic potential of tissue EVs remain insufficiently studied. Here, through bulk and single-cell RNA sequencing analyses combined with ultrastructural tissue examinations, we first reveal that in situ liver tissue EVs (LT-EVs) contribute to the intricate liver regenerative process after partial hepatectomy (PHx), and that hepatocytes are the primary source of tissue EVs in the regenerating liver. Nanoscale and proteomic profiling further identify that the hepatocyte-specific tissue EVs (Hep-EVs) are strengthened to release with carrying proliferative messages after PHx. Moreover, targeted inhibition of Hep-EV release via AAV-shRab27a in vivo confirms that Hep-EVs are required to orchestrate liver regeneration. Mechanistically, Hep-EVs from the regenerating liver reciprocally stimulate hepatocyte proliferation by promoting cell cycle progression through Cyclin-dependent kinase 1 (Cdk1) activity. Notably, supplementing with Hep-EVs from the regenerating liver demonstrates translational potential and ameliorates insufficient liver regeneration. This study provides a functional and mechanistic framework showing that the release of regenerative Hep-EVs governs rapid liver regeneration, thereby enriching our understanding of physiological and endogenous tissue EVs in organ regeneration and therapy.

Chromium Affects Mitochondrial Function, Leading to Apoptosis and Autophagy in Turtle Primary Hepatocytes.

Hexavalent chromium (Cr(VI)), a pervasive industrial contaminant, is highly toxic to both humans and animals. However, its effects on turtles are largely unexplored. Our study aimed to investigate the toxic effects of Cr(VI) on the Reeves' turtles (Mauremys reevesii) primary hepatocytes. We exposed hepatocytes to two concentrations (25 µM and 50 µM) of Cr(VI) for 24 h. The results showed that compared to controls, Cr(VI)-treated cells showed elevated antioxidant enzyme activity (catalase (CAT) and superoxide dismutase (SOD)) and increased reactive oxygen species (ROS) levels. Adenosine triphosphatae (ATP) levels decreased, indicating mitochondrial dysfunction. Additionally, we found significant changes in mitochondrial dynamics related genes, with downregulation of mitofusin 2 (Mfn2) and silent information regulator 1 (SIRT1) and a decrease in sirtuin 3 (SIRT3) and tumor protein 53 (p53) mRNA levels. Annexin V-FITC fluorescence staining-positive cells increased with higher Cr(VI) concentrations, marked by elevated bcl-2-associated X protein (Bax) and cysteinyl aspartate specific proteinase (Caspase3) mRNA levels and reduced B-cell lymphoma-2 (Bcl2) expression. Autophagy-related genes were also affected, with increased microtubule-associated protein 1 light chain 3 (LC3-I), microtubule-associated protein light chain 3II (LC3-II), unc-51-like autophagy-activating kinase 1 (ULK1), and sequestosome 1 (p62/SQSTM1) mRNA levels and decreased mammalian target of rapamycin (mTOR) and Beclin1 expression. Taken together, Cr(VI) promotes cell apoptosis and autophagy in turtle hepatocytes by inducing oxidative stress and disrupting mitochondrial function. These findings highlight the serious health risks posed by Cr(VI) pollution and emphasize the need for protecting wild turtle populations.

Interleukin-1ß Signaling Contributes to Cell Cycle Arrest and Apoptotic Cell Death by Leptin via Modulation of AKT and p38MAPK in Hepatocytes.

Leptin, an adipose tissue-derived hormone, has exhibited the potent hepatotoxic effects. However, the underlying molecular mechanisms are not fully understood. In this study, we have elucidated the mechanisms by which leptin exerts cytotoxic effects in hepatocytes, particularly focusing on the role of interleukin-1ß (IL-1ß) signaling. Leptin significantly induced maturation and secretion of IL-1ß in cultured rat hepatocytes. Interestingly, inhibition of IL-1ß signaling by pretreatment with an IL-1 receptor antagonist (IL-1Ra) or gene silencing of type I IL-1 receptor (IL-1R1) markedly abrogated leptin-induced cell cycle arrest. The critical role of IL-1ß signaling in leptin-induced cell cycle arrest is mediated via upregulation of p16, which acts as an inhibitor of cyclin-dependent kinase. In addition, leptin-induced apoptotic cell death was relieved by inhibition of IL-1ß signaling, as determined by annexin V/7-AAD binding assay. Mechanistically, IL-1ß signaling contributes to apoptotic cell death and cell cycle arrest by suppressing AKT and activation of p38 mitogen-activated protein kinase (p38MAPK) signaling pathways. Involvement of IL-1ß signaling in cytotoxic effect of leptin was further confirmed in vivo using hepatocyte specific IL-1R1 knock out (IL-1R1 KO) mice. Essentially similar results were obtained in vivo, where leptin administration caused the upregulation of apoptotic markers, dephosphorylation of AKT, and p38MAPK activation were observed in wild type mice liver without significant effects in the livers of IL-1R1 KO mice. Taken together, these results demonstrate that IL-1ß signaling critically contributes to leptin-induced cell cycle arrest and apoptosis, at least in part, by modulating p38MAPK and AKT signaling pathways.