Hepatocyte Function Research Articles

Polydatin: a potential NAFLD therapeutic drug that regulates mitochondrial autophagy through SIRT3-FOXO3-BNIP3 and PINK1-PRKN mechanisms - a network pharmacology and experimental investigation

Non-alcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disorder that is linked to metabolic syndrome, mitochondrial dysfunction and impaired autophagy. Polydatin (PD), a natural polyphenol from Polygonum cuspidatum, exhibits various pharmacological effects and protects against NAFLD. The aim of this study was to reveal the molecular mechanisms and therapeutic potential of PD for NAFLD, with a focus on the role of mitochondrial autophagy mediated by sirtuin 3 (SIRT3), fork-head box O3 (FOXO3) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), and by PTEN-induced putative kinase 1 (PINK1) and parkin (PRKN). We combined network pharmacology analysis, animal models and cell culture experiments to show that PD could regulate the mitochondrial autophagy pathway by modulating several key genes related to mitochondrial function, and ameliorate the liver function, histopathology and mitochondrial biogenesis of NAFLD mice and hepatocytes by activating the SIRT3-FOXO3-BNIP3 axis and the PINK1-PRKN-dependent mechanism of mitochondrial autophagy. We also identified the core targets of PD, including SIRT3, FOXO3A, CASP3, PARKIN, EGFR, STAT3, MMP9 and PINK, and confirmed that silencing SIRT3 could significantly attenuate the beneficial effect of PD. This study provided novel theoretical and experimental support for PD as a promising candidate for NAFLD treatment, and also suggested new avenues and methods for investigating the role of mitochondrial autophagy in the pathogenesis and intervention of NAFLD.

The mitochondrial TSPO ligand Atriol mitigates metabolic-associated steatohepatitis by downregulating CXCL1

Background and aimsThe mitochondrial translocator protein (TSPO, 18 kDa) is pivotal in binding cholesterol and facilitating its transfer from the outer to the inner mitochondrial membrane. Atriol is a TSPO ligand disrupting cholesterol binding by targeting the cholesterol-recognition amino acid consensus domain. Prior research has shown that TSPO deficiency improved metabolic-associated steatohepatitis (MASH). We hypothesized that Atriol may have the potential to alleviate MASH. Methods and resultsIn vitro cell culture studies revealed that Atriol treatment effectively mitigated MASH by restoring mitochondrial function, inhibiting the NF-κB signaling pathway, and reducing hepatic stellate cell (HSC) activation. SD male rats were fed a GAN diet for 10 months to induce MASH. During the final two weeks of feeding, rats received intraperitoneal Atriol administration daily. Atriol treatment significantly ameliorated MASH by reducing lipid accumulation, diminishing hepatic lobular inflammation and fibrosis, decreasing cell death, and inhibiting excessive bile acid synthesis. Moreover, Atriol restored mitochondrial function in primary hepatocytes isolated from MASH rats. In search of the mechanism(s) governing these effects, we found that Atriol downregulated the proinflammatory chemokine CXCL1 through the NF-κB signaling pathway or via myeloperoxidase (MPO) in HSCs and Kupffer cells. Additionally, in vitro, studies further suggested that CXCL1 treatment induced dysfunctional mitochondria, inflammation, HSCs activation, and macrophage migration, whereas Atriol countered these effects. Finally, the mitigating effects of Atriol on MASH were reproduced by pharmacological inhibition of NF-κB or MPO and neutralization of CXCL1. ConclusionAtriol ameliorates MASH both in vitro and in vivo, demonstrating its potential therapeutic benefits in managing MASH.

Indocyanine green fluorescence quantification during normothermic ex situ perfusion for the assessment of porcine liver grafts after circulatory death.

Current graft evaluation during normothermic ex situ liver perfusion lacks real-time parameters for predicting posttransplant hepatocyte and biliary function. Indocyanine green (ICG) imaging has been widely used in liver surgery, enabling the visualization of hepatic uptake and excretion through bile using near-infrared light. In this research, porcine livers under various ischemic conditions were examined during a 5-hour normothermic ex situ liver perfusion procedure, introducing ICG at 1 hour through the hepatic artery. These conditions included livers from heart-beating donors, donation after circulatory death (DCD) with warm ischemic durations of 60 minutes (DCD60) and 120 minutes (DCD120), as well as interventions utilizing tissue plasminogen activator in DCD120 cases (each n = 5). Distinct hepatic fluorescence patterns correlated with different degrees of ischemic injury ( p = 0.01). Low ICG uptake in the parenchyma (less than 40% of maximum intensity) was more prevalent in DCD120 (21.4%) compared to heart-beating donors (6.2%, p = 0.06) and DCD60 (3.0%, p = 0.02). Moreover, ICG clearance from 60 minutes to 240 minutes was significantly higher in heart-beating donors (69.3%) than in DCD60 (17.5%, p < 0.001) and DCD120 (32.1%, p = 0.01). Furthermore, thrombolytic intervention using tissue plasminogen activator in DCD120 resulted in noteworthy outcomes, including significantly reduced ALP levels ( p = 0.04) and improved ICG clearance ( p = 0.02) with a trend toward mitigating fibrin deposition similar to DCD60, as well as enhancements in bile production ( p = 0.09). In conclusion, ICG fluorescence imaging during normothermic ex situ liver perfusion provides real-time classification of hepatic vascular and biliary injuries, offering valuable insights for the more accurate selection and postintervention evaluation of marginal livers in transplantation.

The Effect of Nelumbo nucifera Ethanolic Extract on Gut Microbiota in Hyperlipidemic Rats Induced by a High-Fat Diet

Objective: This study aimed to assess the effect of Nelumbo nucifera alcoholic extract (LLAE) on the intestinal microbiota in high-fat diet-induced hyperlipidemia. Methods: Thirty male-specific pathogen-free Sprague–Dawley rats were randomly divided into 3 groups: the blank control group, the model control group, and the LLAE group. The body weight of the rats was recorded during the treatment period. The serum, liver, and intestinal content samples were collected for further analysis after sacrificing. Results: The results suggest that LLAE-treated hyperlipidemic rats showed a significant reduction in serum triglycerides, total cholesterols, and low-density lipoprotein cholesterol levels. LLAE could improve hepatocyte steatosis and ameliorate hepatocyte injury. The significant variation in gut microbial alpha diversity at multiple levels, including phylum and genus levels occurred throughout treatment in the LLAE group. The intestinal microbiota of hyperlipidemic rats were disordered, and there was a correlation between the amount of intestinal flora and the level of lipids. Conclusion: LLAE may improve hepatocyte function by changing the type and abundance value of intestinal microorganisms, reducing the content of total cholesterol, total triglyceride, and low-density lipoprotein cholesterol, and thus improving hyperlipidemia.

Elevated PINK1/Parkin-Dependent Mitophagy and Boosted Mitochondrial Function Mediate Protection of HepG2 Cells from Excess Palmitic Acid by Hesperetin.

Deregulation of mitochondrial functions in hepatocytes contributes to many liver diseases, such as nonalcoholic fatty liver disease (NAFLD). Lately, it was referred to as MAFLD (metabolism-associated fatty liver disease). Hesperetin (Hst), a bioactive flavonoid constituent of citrus fruit, has been proven to attenuate NAFLD. However, a potential connection between its preventive activities and the modulation of mitochondrial functions remains unclear. Here, our results showed that Hst alleviates palmitic acid (PA)-triggered NLRP3 inflammasome activation and cell death by inhibition of mitochondrial impairment in HepG2 cells. Hst reinstates fatty acid oxidation (FAO) rates measured by seahorse extracellular flux analyzer and intracellular acetyl-CoA levels as well as intracellular tricarboxylic acid cycle metabolites levels including NADH and FADH2 reduced by PA exposure. In addition, Hst protects HepG2 cells against PA-induced abnormal energetic profile, ATP generation reduction, overproduction of mitochondrial reactive oxygen species, and collapsed mitochondrial membrane potential. Furthermore, Hst improves the protein expression involved in PINK1/Parkin-mediated mitophagy. Our results demonstrate that it restores PA-impaired mitochondrial function and sustains cellular homeostasis due to the elevation of PINK1/Parkin-mediated mitophagy and the subsequent disposal of dysfunctional mitochondria. These results provide therapeutic potential for Hst utilization as an effective intervention against fatty liver disease.

Exploring the Pathogenesis of Autoimmune Liver Diseases from the Heterogeneity of Target Cells.

The incidence of autoimmune liver diseases (ALDs) and research on their pathogenesis are increasing annually. However, except for autoimmune hepatitis, which responds well to immunosuppression, primary biliary cholangitis and primary sclerosing cholangitis are insensitive to immunosuppressive therapy. Besides the known effects of the environment, genetics, and immunity on ALDs, the heterogeneity of target cells provides new insights into their pathogenesis. This review started by exploring the heterogeneity in the development, structures, and functions of hepatocytes and epithelial cells of the small and large bile ducts. For example, cytokeratin (CK) 8 and CK18 are primarily expressed in hepatocytes, while CK7 and CK19 are primarily expressed in intrahepatic cholangiocytes. Additionally, emerging technologies of single-cell RNA sequencing and spatial transcriptomic are being applied to study ALDs. This review offered a new perspective on understanding the pathogenic mechanisms and potential treatment strategies for ALDs.

Identifying novel mechanisms of per- and polyfluoroalkyl substance-induced hepatotoxicity using FRG humanized mice.

Per- and polyfluoroalkyl substances (PFAS) such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) and perfluoro-2-methyl-3-oxahexanoic acid (GenX), the new replacement PFAS, are major environmental contaminants. In rodents, these PFAS induce several adverse effects on the liver, including increased proliferation, hepatomegaly, steatosis, hypercholesterolemia, nonalcoholic fatty liver disease and liver cancers. Activation of peroxisome proliferator receptor alpha by PFAS is considered the primary mechanism of action in rodent hepatocyte-induced proliferation. However, the human relevance of this mechanism is uncertain. We investigated human-relevant mechanisms of PFAS-induced adverse hepatic effects using FRG liver-chimeric humanized mice with livers repopulated with functional human hepatocytes. Male FRG humanized mice were treated with 0.067mg/L of PFOA, 0.145mg/L of PFOS, or 1mg/L of GenX in drinking water for 28days. PFOS caused a significant decrease in total serum cholesterol and LDL/VLDL, whereas GenX caused a significant elevation in LDL/VLDL with no change in total cholesterol and HDL. All three PFAS induced significant hepatocyte proliferation. RNA-sequencing with alignment to the human genome showed a total of 240, 162, and 619 differentially expressed genes after PFOA, PFOS, and GenX exposure, respectively. Upstream regulator analysis revealed that all three PFAS induced activation of p53 and inhibition of androgen receptor and NR1D1, a transcriptional repressor important in circadian rhythm. Further biochemical studies confirmed NR1D1 inhibition and in silico modeling indicated potential interaction of all three PFAS with the DNA-binding domain of NR1D1. In conclusion, our studies using FRG humanized mice have revealed new human-relevant molecular mechanisms of PFAS including their previously unknown effect on circadian rhythm.

Evaluation and Optimization of a Microcavity Plate-Based Human Hepatocyte Spheroid Model for Predicting Clearance of Slowly Metabolized Drug Candidates.

In vitro clearance assays are routinely conducted in drug discovery to predict in vivo clearance, but low metabolic turnover compounds are often difficult to evaluate. Hepatocyte spheroids can be cultured for days, achieving higher drug turnover, but have been hindered by limitations on cell number per well. Corning Elplasia microcavity 96-well microplates enable the culture of 79 hepatocyte spheroids per well. In this study, microcavity spheroid properties (size, hepatocyte function, longevity, culturing techniques) were assessed and optimized for clearance assays, which were then compared with microsomes, hepatocyte suspensions, two-dimensional-plated hepatocytes, and macrowell spheroids cultured as one per well. Higher enzyme activity coupled with greater hepatocyte concentrations in microcavity spheroids enabled measurable turnover of all 17 test compounds, unlike the other models that exhibited less drug turnover. Microcavity spheroids also predicted intrinsic clearance (CLint) and blood clearance (CLb) within threefold for 53% [9/17; average absolute fold error (AAFE), 3.9] and 82% (14/17; AAFE, 2.6) of compounds using a linear regression correction model, respectively. An alternate method incorporating mechanistic modeling that accounts for mass transport (permeability and diffusion) within spheroids demonstrated improved predictivity for CLint (12/17; AAFE, 4.0) and CLb (14/17; AAFE, 2.1) without the need for empirical scaling factors. The estimated fraction of drug metabolized by cytochrome P450 3A4 (fm,CYP3A4) using 3 μM itraconazole was within 25% of observed values for 6 of 8 compounds, with 5 of 8 compounds within 10%. In sum, spheroid cultures in microcavity plates permit the ability to test and predict clearance as well as fm,CYP3A4 of low metabolic turnover compounds and represent a valuable complement to conventional in vitro clearance assays. SIGNIFICANCE STATEMENT: Culturing multiple spheroids in ultralow attachment microcavities permits accurate quantitation of metabolically stable compounds in substrate depletion assays, overcoming limitations with singly cultured spheroids. In turn, this permits robust estimates of intrinsic clearance, which is improved with the consideration of mass transport within the spheroid. Incubations with 3 μM itraconazole enabled assessments of CYP3A4 involvement in hepatic clearance.

Direct reprogramming of fibroblasts into functional hepatocytes via CRISPRa activation of endogenous Gata4 and Foxa3.

The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments. Clustered regularly interspaced short palindromic repeats (CRISPR) activator (CRISPRa) is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously, providing a new strategy for cell fate determination. The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis. The differentiation of mouse embryonic fibroblasts (MEFs) into functional induced hepatocyte-like cells (iHeps) was achieved by utilizing the CRISPRa synergistic activation mediator (SAM) system, which drove the combined expression of three endogenous transcription factors- Gata4, Foxa3 , and Hnf1a -or alternatively, the expression of two transcription factors, Gata4 and Foxa3 . In vivo , we injected adeno-associated virus serotype 6 (AAV6) carrying the CRISPRa SAM system into liver fibrotic Col1a1-CreER ; Cas9fl/fl mice, effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts. The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction (RT-qPCR) and RNA-seq, and the morphology and characteristics of the induced hepatocytes were observed through microscopy. The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining, while the improvement of liver fibrosis is evaluated through Sirius red staining, alpha-smooth muscle actin (α-SMA) immunofluorescence staining, and blood alanine aminotransferase (ALT) examination. Activation of only two factors, Gata4 and Foxa3 , via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps. These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes, such as drug metabolism and glycogen storage. Additionally, AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis. After 8 weeks of induction, the reprogrammed hepatocytes comprised 0.87% of the total hepatocyte population in the mice, significantly reducing liver fibrosis. CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.

Functional mouse hepatocytes derived from interspecies chimeric livers effectively mitigate chronic liver fibrosis

Liver disease is a major global health challenge. There is a shortage of liver donors worldwide, and hepatocyte transplantation (HT) may be an effective treatment to overcome this problem. However, the present approaches for generation of hepatocytes are associated with challenges, and interspecies chimera-derived hepatocytes produced by interspecies blastocyst complementation (IBC) may be promising donor hepatocytes because of their more comprehensive hepatic functions. In this study, we isolated mouse hepatocytes from mouse-rat chimeric livers using IBC and found that interspecies chimera-derived hepatocytes exhibited mature hepatic functions in terms of lipid accumulation, glycogen storage, and urea synthesis. Meanwhile, they were more similar to endogenous hepatocytes than hepatocytes derived invitro. Interspecies chimera-derived hepatocytes could relieve chronic liver fibrosis and reside in the injured liver after transplantation. Our results suggest that interspecies chimera-derived hepatocytes are a potentially reliable source of hepatocytes and can be applied as a therapeutic approach for HT.

Digital PCR as a novel way to assess chimerism of humanized immune system mice

Abstract Humanized immune system (HIS) mice contain human and murine immune cells. Analysis of chimerism in HIS mice is typically accomplished via flow cytometry, but this requires collecting a 75 µl blood sample and access to a flow cytometer. Repeated bleeding can negatively affect the health of HIS mice, limiting serial analysis. Digital PCR offers a novel method for chimerism analysis, which uses much smaller blood volumes and can be performed on clotted or frozen samples, sample types unsuitable for flow cytometry. A digital PCR assay directly comparing the presence of three human genes and one murine gene was developed. This assay was validated in the huNOG-EXL HIS mouse engrafted with CD34+ hematopoetic stem cells from 3 unique donors via blind comparison with chimerism data from flow cytometry. At 10 weeks post engraftment, the digital PCR assay showed excellent correlation for all three human genes against chimerism as measured by flow cytometry of the peripheral blood. This assay represents a new, user-friendly, rapid tool for the analysis of HIS mice and may permit serial chimerism measurement using 10 μL whole blood. This assay may be useful to investigate other mouse-human chimera models, including mice harboring functional human hepatocytes, tumors, and other cells.

Utility of gadoxetate disodium-enhanced magnetic resonance imaging in evaluating liver failure risk after major hepatic resection.

Post-hepatectomy liver failure (PHLF) is still a predominant cause of hepatectomy-related mortality. However, it is difficult to evaluate the remnant liver functional reserve accurately before surgery to prevent PHLF. In this study, we aimed to explore the role of gadoxetate disodium-enhanced magnetic resonance imaging (MRI) in evaluating remnant liver functional reserve. For this cross-sectional study, the sample retrospectively included 56 patients undergoing liver resections of at least three segments between June 2019 and September 2022 at The General Hospital of the Western Theater Command. Pre-surgery assessments involved liver computer tomography (CT), an indocyanine green (ICG) clearance test, the Child-Pugh scoring system, and liver function serum biochemical indicators. Each patient underwent a gadoxetate disodium-enhanced MRI before the hepatectomy, and we measured the remnant hepatocellular uptake index (rHUI) as well as the standard remnant hepatocellular uptake index (SrHUI). We examined the diagnostic utility of rHUI, SrHUI, indocyanine green retention rate of 15 minutes (ICG R15), and Albumin for PHLF. Receiver operating characteristics (ROC) analyses were used to measure the preoperative liver function parameters (namely, rHUI, SrHUI, ICG R15, and Albumin) for predicting PHLF. The areas under the curve (AUCs) were calculated and compared between different preoperative liver function parameters using the Wilson/Brown method. The Pearson or Spearman correlation coefficient was used for correlation analysis between ICG R15, Albumin, and rHUI and between ICG R15, Albumin, and SrHUI, respectively. Twelve patients (21.43%) had complications of PHLF. We found significant differences in rHUI, SrHUI, ICG R15, and Albumin between the non-PHLF and PHLF groups. The pooled r between ICG R15 and rHUI was -0.591 [95% confidence interval (CI): -0.740 to -0.389, P<0.001], and between ICG R15 and SrHUI was -0.534 (95% CI: -0.703 to -0.308, P<0.001). The area under the curve (AUC) values of rHUI, SrHUI, ICG R15, and Ablumin were 0.871 (sensitivity 81.82%; specificity 91.67%), 0.878 (sensitivity 79.55%; specificity 83.33%), 0.835 (sensitivity 99.73%; specificity 66.67%), and 0.782 (sensitivity 88.64%; specificity 58.33%), respectively. We found that the rHUI and SrHUI calculated using the gadoxetate disodium-enhanced MRI reflected a combination of remnant hepatocyte function and liver volume, and these were useful as a quantitative assessment indicator of remnant liver functional reserve and can be a better predictor of PHLF after major hepatic resection.

Current Insights into the Effects of Dietary α-Linolenic Acid Focusing on Alterations of Polyunsaturated Fatty Acid Profiles in Metabolic Syndrome.

The plant-derived α-linolenic acid (ALA) is an essential n-3 acid highly susceptible to oxidation, present in oils of flaxseeds, walnuts, canola, perilla, soy, and chia. After ingestion, it can be incorporated in to body lipid pools (particularly triglycerides and phospholipid membranes), and then endogenously metabolized through desaturation, elongation, and peroxisome oxidation to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), with a very limited efficiency (particularly for DHA), beta-oxidized as an energy source, or directly metabolized to C18-oxilipins. At this moment, data in the literature about the effects of ALA supplementation on metabolic syndrome (MetS) in humans are inconsistent, indicating no effects or some positive effects on all MetS components (abdominal obesity, dyslipidemia, impaired insulin sensitivity and glucoregulation, blood pressure, and liver steatosis). The major effects of ALA on MetS seem to be through its conversion to more potent EPA and DHA, the impact on the n-3/n-6 ratio, and the consecutive effects on the formation of oxylipins and endocannabinoids, inflammation, insulin sensitivity, and insulin secretion, as well as adipocyte and hepatocytes function. It is important to distinguish the direct effects of ALA from the effects of EPA and DHA metabolites. This review summarizes the most recent findings on this topic and discusses the possible mechanisms.

Emerging Roles of Spatial Transcriptomics in Liver Research.

Spatial transcriptomics, leveraging sequencing- and imaging-based techniques, has emerged as a groundbreaking technology for mapping gene expression within the complex architectures of tissues. This approach provides an in-depth understanding of cellular and molecular dynamics across various states of healthy and diseased livers. Through the integration of sophisticated bioinformatics strategies, it enables detailed exploration of cellular heterogeneity, transitions in cell states, and intricate cell-cell interactions with remarkable precision. In liver research, spatial transcriptomics has been particularly revelatory, identifying distinct zonated functions of hepatocytes that are crucial for understanding the metabolic and detoxification processes of the liver. Moreover, this technology has unveiled new insights into the pathogenesis of liver diseases, such as the role of lipid-associated macrophages in steatosis and endothelial cell signals in liver regeneration and repair. In the domain of liver cancer, spatial transcriptomics has proven instrumental in delineating intratumor heterogeneity, identifying supportive microenvironmental niches and revealing the complex interplay between tumor cells and the immune system as well as susceptibility to immune checkpoint inhibitors. In conclusion, spatial transcriptomics represents a significant advance in hepatology, promising to enhance our understanding and treatment of liver diseases.

Vascular damage and excessive proliferation compromise liver function after extended hepatectomy in mice.

Surgical resection remains the gold standard for liver tumor treatment, yet the emergence of post-operative liver failure, known as the small for size syndrome (SFSS), poses a significant challenge. The activation of hypoxia sensors in a SFSS liver remnant initiated early angiogenesis, improving vascular architecture, safeguarding against liver failure and reducing mortality. The study aimed to elucidate vascular remodeling mechanisms in SFSS, its impact on hepatocyte function and subsequent liver failure. Mice underwent extended partial hepatectomy to induce SFSS, with a subset exposed to hypoxia immediately after surgery. Hypoxia bolstered post-hepatectomy survival rates. Early proliferation of liver sinusoidal cells, coupled with recruitment of putative endothelial progenitor cells (EPC), increased vascular density, improved lobular perfusion, and limited hemorrhagic events in the regenerating liver under hypoxia. Administration of granulocyte colony stimulating factor (G-CSF) in hepatectomized mice mimicked the effects of hypoxia on vascular remodeling and EPC recruitment, but failed to rescue survival. Compared to normoxia, hypoxia favored hepatocyte function over proliferation, promoting functional preservation in the regenerating remnant. Injection of AAV8-TBG-HNF4α virus for hepatocyte-specific overexpression of HNF4α, the master regulator of hepatocyte function, enforced functionality in proliferating hepatocytes but did not rescue survival. The combination of HNF4α overexpression and G-CSF treatment rescued survival after SFSS-setting hepatectomy. In summary, SFSS arises from an imbalance and desynchronized interplay between functional regeneration and vascular restructuring. To improve survival following SFSS-hepatectomy, it is essential to adopt a two-pronged strategy aimed at preserving the function of proliferating parenchymal cells and simultaneously attenuating vascular damage.

MicroRNA‐Modified DNA Hexahedron‐Induced Hepatocyte‐Like Cells Integrating 3D Printed Scaffold for Acute Liver Failure Therapy

AbstractAcute liver failure (ALF) involves extensive necrosis of liver cells and severe impairment of liver function. Hepatocyte transplantation holds promise for treating ALF by swiftly supporting liver functions and promoting liver regeneration. However, the scarcity of suitable cell sources requires strategies to obtain enough functional hepatocyte‐like cells (HLCs) and optimize their in vivo transplantation efficiency. A DNA hexahedral nanostructure (DHN) is developed loaded with microRNA‐122 to efficiently induce hepatic differentiation of human adipose‐derived mesenchymal stem cells into HLCs. These HLCs can serve as alternative hepatocyte sources, as confirmed by expression of liver‐specific genes and proteins, and the restoration of liver functions. To enhance in vivo survival efficiency of HLCs, a versatile scaffold is also created by 3D printing the calcium‐cross‐linked mixture bioink composed of sodium alginate, gelatin, and silk fibroin with excellent ROS scavenging capabilities. The scaffold is infused with chitosan‐DHN hydrogel containing HLCs for tissue engineering orthotopic transplantation in CCl4‐induced ALF mice. The transplanted composite scaffold‐HLCs successfully repair tissue necrosis in the liver injury area of mice and regulate expressions of genes and proteins associated with inflammation, oxidative stress, and hepatocyte function. Collectively, this study offers a novel approach and strategy for identifying alternative hepatocyte sources and treating ALF.

A comprehensive review of advances in hepatocyte microencapsulation: selecting materials and preserving cell viability.

Liver failure represents a critical medical condition with a traditionally grim prognosis, where treatment options have been notably limited. Historically, liver transplantation has stood as the sole definitive cure, yet the stark disparity between the limited availability of liver donations and the high demand for such organs has significantly hampered its feasibility. This discrepancy has necessitated the exploration of hepatocyte transplantation as a temporary, supportive intervention. In light of this, our review delves into the burgeoning field of hepatocyte transplantation, with a focus on the latest advancements in maintaining hepatocyte function, co-microencapsulation techniques, xenogeneic hepatocyte transplantation, and the selection of materials for microencapsulation. Our examination of hepatocyte microencapsulation research highlights that, to date, most studies have been conducted in vitro or using liver failure mouse models, with a notable paucity of experiments on larger mammals. The functionality of microencapsulated hepatocytes is primarily inferred through indirect measures such as urea and albumin production and the rate of ammonia clearance. Furthermore, research on the mechanisms underlying hepatocyte co-microencapsulation remains limited, and the practicality of xenogeneic hepatocyte transplantation requires further validation. The potential of hepatocyte microencapsulation extends beyond the current scope of application, suggesting a promising horizon for liver failure treatment modalities. Innovations in encapsulation materials and techniques aim to enhance cell viability and function, indicating a need for comprehensive studies that bridge the gap between small-scale laboratory success and clinical applicability. Moreover, the integration of bioengineering and regenerative medicine offers novel pathways to refine hepatocyte transplantation, potentially overcoming the challenges of immune rejection and ensuring the long-term functionality of transplanted cells. In conclusion, while hepatocyte microencapsulation and transplantation herald a new era in liver failure therapy, significant strides must be made to translate these experimental approaches into viable clinical solutions. Future research should aim to expand the experimental models to include larger mammals, thereby providing a clearer understanding of the clinical potential of these therapies. Additionally, a deeper exploration into the mechanisms of cell survival and function within microcapsules, alongside the development of innovative encapsulation materials, will be critical in advancing the field and offering new hope to patients with liver failure.

Notch1 regulates hepatic thrombopoietin production

AbstractNotch signaling regulates cell-fate decisions in several developmental processes and cell functions. However, the role of Notch in hepatic thrombopoietin (TPO) production remains unclear. We noted thrombocytopenia in mice with hepatic Notch1 deficiency and so investigated TPO production and other features of platelets in these mice. We found that the liver ultrastructure and hepatocyte function were comparable between control and Notch1-deficient mice. However, the Notch1-deficient mice had significantly lower plasma TPO and hepatic TPO messenger RNA levels, concomitant with lower numbers of platelets and impaired megakaryocyte differentiation and maturation, which were rescued by addition of exogenous TPO. Additionally, JAK2/STAT3 phosphorylation was significantly inhibited in Notch1-deficient hepatocytes, consistent with the RNA-sequencing analysis. JAK2/STAT3 phosphorylation and TPO production was also impaired in cultured Notch1-deficient hepatocytes after treatment with desialylated platelets. Consistently, hepatocyte-specific Notch1 deletion inhibited JAK2/STAT3 phosphorylation and hepatic TPO production induced by administration of desialylated platelets in vivo. Interestingly, Notch1 deficiency downregulated the expression of HES5 but not HES1. Moreover, desialylated platelets promoted the binding of HES5 to JAK2/STAT3, leading to JAK2/STAT3 phosphorylation and pathway activation in hepatocytes. Hepatocyte Ashwell-Morell receptor (AMR), a heterodimer of asialoglycoprotein receptor 1 [ASGR1] and ASGR2, physically associates with Notch1, and inhibition of AMR impaired Notch1 signaling activation and hepatic TPO production. Furthermore, blockage of Delta-like 4 on desialylated platelets inhibited hepatocyte Notch1 activation and HES5 expression, JAK2/STAT3 phosphorylation, and subsequent TPO production. In conclusion, our study identifies a novel regulatory role of Notch1 in hepatic TPO production, indicating that it might be a target for modulating TPO level.

Pathophysiologic Mapping of Chronic Liver Diseases With Longitudinal Multiparametric MRI in Animal Models.

Chronic liver diseases (CLDs) have diverse etiologies. To better classify CLDs, we explored the ability of longitudinal multiparametric MRI (magnetic resonance imaging) in depicting alterations in liver morphology, inflammation, and hepatocyte and macrophage activity in murine high-fat diet (HFD)- and carbon tetrachloride (CCl 4 )-induced CLD models. Mice were either untreated, fed an HFD for 24 weeks, or injected with CCl 4 for 8 weeks. Longitudinal multiparametric MRI was performed every 4 weeks using a 7 T MRI scanner, including T1/T2 relaxometry, morphological T1/T2-weighted imaging, and fat-selective imaging. Diffusion-weighted imaging was applied to assess fibrotic remodeling and T1-weighted and T2*-weighted dynamic contrast-enhanced MRI and dynamic susceptibility contrast MRI using gadoxetic acid and ferucarbotran to target hepatocytes and the mononuclear phagocyte system, respectively. Imaging data were associated with histopathological and serological analyses. Principal component analysis and clustering were used to reveal underlying disease patterns. The MRI parameters significantly correlated with histologically confirmed steatosis, fibrosis, and liver damage, with varying importance. No single MRI parameter exclusively correlated with 1 pathophysiological feature, underscoring the necessity for using parameter patterns. Clustering revealed early-stage, model-specific patterns. Although the HFD model exhibited pronounced liver fat content and fibrosis, the CCl 4 model indicated reduced liver fat content and impaired hepatocyte and macrophage function. In both models, MRI biomarkers of inflammation were elevated. Multiparametric MRI patterns can be assigned to pathophysiological processes and used for murine CLD classification and progression tracking. These MRI biomarker patterns can directly be explored clinically to improve early CLD detection and differentiation and to refine treatments.

Amelioration of NAFLD by sleeve gastrectomy-triggered hepatocyte regeneration in mice - experimental research.

Sleeve gastrectomy (SG) is known to alleviate non-alcoholic fatty liver disease (NAFLD) and restore liver function; however, its underlying mechanism remains unclear. We investigated the effect of SG on the metabolic phenotype of diet-induced obese (DIO) mice. Postoperative stained liver images were analyzed to determine the hepatocyte proliferation phenotype. Single-cell RNA sequencing was used to characterize the regeneration signals of the liver after SG in DIO mice, and qRT PCR was performed to verify the above results. We employed Olink proteomics to capture serum element changes and investigated the role of Yes1 protein in liver regeneration and carcinogenesis through the Hippo-YAP pathway. DIO mice were treatment with YAP inhibitor verteporfin after SG mice to clarify whether SG-induced liver regeneration is related to the YAP signaling pathway. SG significantly reduced NAFLD-associated dysfunction in hepatocytes and replaced them with fully functional hepatocytes, which have a high regenerative capacity across the entire liver. SG also enhanced the hepatic regenerative capacity, as demonstrated by SG combined with hepatic lobectomy in healthy mice. Yes1 protein was identified as the signaling molecule most closely related to classical regeneration signals. Our study showed that SG-enhanced proliferation and improved metabolism did not depend on YAP signaling. SG can enhance hepatic regenerative capacity and improve liver metabolism. This study provides a better understanding of the mechanisms underlying SG-induced metabolic improvements.