Group IVA Phospholipase A2 in Collagen-Producing Cells Promotes High-Fat Diet-Induced Infiltration of Inflammatory Cells into the Liver by Upregulating the Expression of MCP-1.

Nonalcoholic steatohepatitis (NASH) is characterized by hepatic inflammation and fibrosis due to excessive fat accumulation. Monocyte chemoattractant protein-1 (MCP-1) is a key chemokine that infiltrates inflammatory cells into the liver during the development of NASH. Our previous studies demonstrated that a systemic deficiency of group IVA phospholipase A2 (IVA-PLA2), an enzyme that contributes to the production of lipid inflammatory mediators, protects mice against high-fat diet-induced hepatic fibrosis and markedly suppresses the CCl4-induced expression of MCP-1 in the liver. However, it remains unclear which cell types harboring IVA-PLA2 are involved in the elevated production of MCP-1. Hence, the present study assessed the types of cells responsible for IVA-PLA2-mediated production of MCP-1 using cultured hepatic stellate cells, endothelial cells, macrophages, and hepatocytes, as well as cell-type specific IVA-PLA2 deficient mice fed a high-fat diet. A relatively specific inhibitor of IVA-PLA2 markedly suppressed the expression of MCP-1 mRNA in cultured hepatic stellate cells, but the suppression of MCP-1 expression was partial in endothelial cells and not observed in monocytes/macrophages or hepatocytes. In contrast, a deficiency of IVA-PLA2 in collagen-producing cells (hepatic stellate cells), but not in other types of cells, reduced the high-fat diet-induced expression of MCP-1 and inflammatory cell infiltration in the liver. Our results suggest that IVA-PLA2 in hepatic stellate cells is critical for hepatic inflammation in the high-fat diet-induced development of NASH. This supports a potential therapeutic approach for NASH using a IVA-PLA2 inhibitor targeting hepatic stellate cells.

Hepatocyte-specific Selenoi deficiency predisposes mice to hepatic steatosis and obesity.

Selenoprotein I (Selenoi) is highly expressed in liver and plays a key role in lipid metabolism as a phosphatidylethanolamine (PE) synthase. However, the precise function of Selenoi in the liver remains elusive. In the study, we generated hepatocyte-specific Selenoi conditional knockout (cKO) mice on a high-fat diet to identify the physiological function of Selenoi. The cKO group exhibited a significant increase in body weight, with a 15.6% and 13.7% increase in fat accumulation in white adipose tissue (WAT) and the liver, respectively. Downregulation of the lipolysis-related protein (p-Hsl) and upregulation of the adipogenesis-related protein (Fasn) were observed in the liver of cKO mice. The cKO group also showed decreased oxygen consumption (VO2), carbon dioxide production (VCO2), and energy expenditure (p < .05). Moreover, various metabolites of the steroid hormone synthesis pathway were affected in the liver of cKO mice. A potential cascade of Selenoi-phosphatidylethanolamine-steroid hormone synthesis might serve as a core mechanism that links hepatocyte-specific Selenoi cKO to biochemical and molecular reactions. In conclusion, we revealed that Selenoi inhibits body fat accumulation and hepatic steatosis and elevates energy consumption; this protein could also be considered a therapeutic target for such related diseases.

Chaos and bifurcations of a two-dimensional hepatitis C virus model with hepatocyte homeostasis.

In this paper, we delve into the intricate local dynamics at equilibria within a two-dimensional model of hepatitis C virus (HCV) alongside hepatocyte homeostasis. The study investigates the existence of bifurcation sets and conducts a comprehensive bifurcation analysis to elucidate the system's behavior under varying conditions. A significant focus lies on understanding how changes in parameters can lead to bifurcations, which are pivotal points where the qualitative behavior of the system undergoes fundamental transformations. Moreover, the paper introduces and employs hybrid control feedback and Ott-Grebogi-Yorke strategies as tools to manage and mitigate chaos inherent within the HCV model. This chaos arises due to the presence of flip and Neimark-Sacker bifurcations, which can induce erratic behavior in the system. Through the implementation of these control strategies, the study aims to stabilize the system and restore it to a more manageable and predictable state. Furthermore, to validate the theoretical findings and the efficacy of the proposed control strategies, extensive numerical simulations are conducted. These simulations serve as a means of confirming the theoretical predictions and provide insight into the practical implications of the proposed control methodologies. By combining theoretical analysis with computational simulations, the paper offers a comprehensive understanding of the dynamics of the HCV model and provides valuable insights into potential strategies for controlling and managing chaos in such complex biological systems.

Myocardial infarction accelerates the progression of MASH by triggering immunoinflammatory response and induction of periosti.

Patients with metabolic dysfunction-associated steatotic liver disease (MASLD), especially advanced metabolic dysfunction-associated steatohepatitis (MASH), have an increased risk of cardiovascular diseases (CVDs). Whether CVD events will, in turn, influence the pathogenesis of MASLD remains unknown. Here, we show that myocardial infarction (MI) accelerates hepatic pathological progression of MASLD. Patients with MASLD who experience CVD events after their diagnosis exhibit accelerated liver fibrosis progression. MI promotes hepatic fibrosis in mice with MASH, accompanied by elevated circulating Ly6Chi monocytes and their recruitment to damaged liver tissues. These adverse effects are significantly abrogated when deleting these cells. Meanwhile, MI substantially increases circulating and cardiac periostin levels, which act on hepatocytes and stellate cells to promote hepatic lipid accumulation and fibrosis, finally exacerbating hepatic pathological progression of MASH. These preclinical and clinical results demonstrate that MI alters systemic homeostasis and upregulates pro-fibrotic factor production, triggering cross-disease communication that accelerates hepatic pathological progression of MASLD.

Exosomes target HBV-host interactions to remodel the hepatic immune microenvironment.

Chronic hepatitis B poses a significant global burden, modulating immune cells, leading to chronic inflammation and long-term damage. Due to its hepatotropism, the hepatitis B virus (HBV) cannot infect other cells. The mechanisms underlying the intercellular communication among different liver cells in HBV-infected individuals and the immune microenvironment imbalance remain elusive. Exosomes, as important intercellular communication and cargo transportation tools between HBV-infected hepatocytes and immune cells, have been shown to assist in HBV cargo transportation and regulate the immune microenvironment. However, the role of exosomes in hepatitis B has only gradually received attention in recent years. Minimal literature has systematically elaborated on the role of exosomes in reshaping the immune microenvironment of the liver. This review unfolds sequentially based on the biological processes of exosomes: exosomes' biogenesis, release, transport, uptake by recipient cells, and their impact on recipient cells. We delineate how HBV influences the biogenesis of exosomes, utilizing exosomal covert transmission, and reshapes the hepatic immune microenvironment. And based on the characteristics and functions of exosomes, potential applications of exosomes in hepatitis B are summarized and predicted.

Dissection of molecular mechanisms of liver injury induced by microcystin-leucine arginine via single-cell RNA-sequencing.

The occurrence of poisoning incidents caused by cyanobacterial blooms has aroused wide public concern. Microcystin-leucine arginine (MC-LR) is a well-established toxin produced by cyanobacterial blooms, which is widely distributed in eutrophic waters. MC-LR is not only hazardous to the water environment but also exerts multiple toxic effects including liver toxicity in both humans and animals. However, the underlying mechanisms of MC-LR-induced liver toxicity are unclear. Herein, we used advanced single-cell RNA sequencing technology to characterize MC-LR-induced liver injury in mice. We established the first single-cell atlas of mouse livers in response to MC-LR. Our results showed that the differentially expressed genes and pathways in diverse cell types of liver tissues of mice treated with MC-LR are highly heterogeneous. Deep analysis showed that MC-LR induced an increase in a subpopulation of hepatocytes that highly express Gstm3, which potentially contributed to hepatocyte apoptosis in response to MC-LR. Moreover, MC-LR increased the proportion and multiple subtypes of Kupffer cells with M1 phenotypes and highly expressed proinflammatory genes. Furthermore, the MC-LR increased several subtypes of CD8+ T cells with highly expressed multiple cytokines and chemokines. Overall, apart from directly inducing hepatocytes apoptosis, MC-LR activated proinflammatory Kupffer cell and CD8+ T cells, and their interaction may constitute a hostile microenvironment that contributes to liver injury. Our findings not only present novel insight into underlying molecular mechanisms but also provide a valuable resource and foundation for additional discovery of MC-LR-induced liver toxicity.

Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism.

Background: Acute liver injury (ALI), which is a type of inflammation-mediated hepatocellular injury, is a clinical syndrome that results from hepatocellular apoptosis and hemorrhagic necrosis. Apoptosis stimulating protein of p53-2 (ASPP2) is a proapoptotic member of the p53 binding protein family. However, the role of ASPP2 in the pathogenesis of ALI and its regulatory mechanisms remain unclear. Methods: The expression of ASPP2 were compared between liver biopsies derived from patients with CHB, patients with ALI, and normal controls. Acute liver injury was modelled in mice by administration of D-GalN/LPS. Liver injury was demonstrated by serum transaminases and histological assessment of liver sections. ASPP2-knockdown mice (ASPP2+/-) were used to determine its role in acute liver injury. Mouse bone marrow macrophages (BMMs) were isolated from wildtype and ASPP2+/- mice and stimulated with LPS, and the supernatant was collected to incubate with the primary hepatocytes. Quantitative real-time PCR and western blot were used to analyze the expression level of target. Results: The expression of ASPP2 was significantly upregulated in the liver tissue of ALI patients and acute liver injury mice. ASPP2+/- mice significantly relieved liver injury through reducing liver inflammation and decreasing hepatocyte apoptosis. Moreover, the conditioned medium (CM) of ASPP2+/- bone marrow-derived macrophages (BMMs) protected hepatocytes against apoptosis. Mechanistically, we revealed that ASPP2 deficiency in BMMs specifically upregulated IL-6 through autophagy activation, which decreased the level of TNF-α to reduce hepatocytes apoptosis. Furthermore, up-regulation of ASPP2 sensitizes hepatocytes to TNF-α-induced apoptosis. Conclusion: Our novel findings show the critical role of ASPP2 in inflammatory immunoregulatory mechanism of ALI and provide a rationale to target ASPP2 as a refined therapeutic strategy to ameliorate acute liver injury.

Beneficial Effects of Capybara Oil Supplementation on Steatosis and Liver Apoptosis in Obese Mice.

Obesity is a complex chronic disease characterized by excess body fat (adipose) that is harmful to health and has been a major global health problem. It may be associated with several diseases, such as nonalcoholic fatty liver disease (NAFLD). Polyunsaturated fatty acids (PUFA) are lipid mediators that have anti-inflammatory characteristics and can be found in animals and plants, with capybara oil (CO) being a promising source. So, we intend to evaluate the hepatic pathophysiological alterations in C57Bl/6 mice with NAFLD, caused by obesity, and the possible beneficial effects of OC in the treatment of this disease. Eighteen 3-month-old male C57Bl/6 mice received a control or high-fat diet for 18 weeks. From the 15th to the 18th week, the animals received treatment-through orogastric gavage-with placebo or free capybara oil (5 g/kg). Parameters inherent to body mass, glucose tolerance, evaluation of liver enzymes, percentage of hepatic steatosis, oxidative stress, the process of cell death with the apoptotic biomarkers (Bax, Bcl2, and Cytochrome C), and the ultrastructure of hepatocytes were analyzed. Even though the treatment with CO was not able to disassemble the effects on the physiological parameters, it proved to be beneficial in reversing the morphological and ultrastructural damage present in the hepatocytes. Thus, demonstrating that CO has beneficial effects in reducing steatosis and the apoptotic pathway, it is a promising treatment for NAFLD.

The adiponectin-derived peptide ALY688 protects against the development of metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is the severe form of non-alcoholic fatty liver disease which has a high potential to progress to cirrhosis and hepatocellular carcinoma, yet adequate effective therapies are lacking. Hypoadiponectinemia is causally involved in the pathogenesis of MASH. This study investigated the pharmacological effects of adiponectin replacement therapy with the adiponectin-derived peptide ALY688 (ALY688-SR) in a mouse model of MASH. Human induced pluripotent stem (iPS) cell-derived hepatocytes were used to test cytotoxicity and signaling of unmodified ALY688 in vitro. High-fat diet with low methionine and no added choline (CDAHF) was used to induce MASH and test the effects of ALY688-SR in vivo. Histological MASH activity score (NAS) and fibrosis score were determined to assess the effect of ALY688-SR. Transcriptional characterization of mice through RNA sequencing was performed to indicate potential molecular mechanisms involved. In cultured hepatocytes, ALY688 efficiently induced adiponectin-like signaling, including the AMP-activated protein kinase and p38 mitogen-activated protein kinase pathways, and did not elicit cytotoxicity. Administration of ALY688-SR in mice did not influence body weight but significantly ameliorated CDAHF-induced hepatic steatosis, inflammation, and fibrosis, therefore effectively preventing the development and progression of MASH. Mechanistically, ALY688-SR treatment markedly induced hepatic expression of genes involved in fatty acid oxidation, whereas it significantly suppressed the expression of pro-inflammatory and pro-fibrotic genes as demonstrated by transcriptomic analysis. ALY688-SR may represent an effective approach in MASH treatment. Its mode of action involves inhibition of hepatic steatosis, inflammation, and fibrosis, possibly via canonical adiponectin-mediated signaling.

Targeted protein degradation systems to enhance Wnt signaling.

Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETS) to drive tissue-specific degradation of ZNRF3/RNF43 E3 ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody-RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1- and ASGR1/2-specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate-binding site. Both antibodies enhanced Wnt activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.

Hepatoprotective effects of baicalein against liver ischemia-reperfusion injury and partial hepatectomy in a rat model.

BACKGROUND: Baicalein is the main active flavonoid in Scutellariae Radix and is included in shosaikoto, a Kampo formula used for treating hepatitis and jaundice. However, little is known about its hepatoprotective effects against hepatic ischemia-reperfusion injury (HIRI), a severe clinical condition directly caused by interventional procedures. We aimed to investigate the hepatoprotective effects of baicalein against HIRI and partial hepatectomy (HIRI + PH) and its potential underlying mechanisms. METHODS AND RESULTS: Male Sprague-Dawley rats received either baicalein (5 mg/kg) or saline intraperitoneally and underwent a 70% hepatectomy 15 min after hepatic ischemia. After reperfusion, liver and blood samples were collected. Survival was monitored 30 min after hepatic ischemia and hepatectomy. In interleukin 1ß (IL-1ß)-treated primary cultured rat hepatocytes, the influence of baicalein on inflammatory mediator production and the associated signaling pathway was analyzed. Baicalein suppressed apoptosis and neutrophil infiltration, which are the features of HIRI + PH treatment-induced histological injury. Baicalein also reduced the mRNA expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α). In addition, HIRI + PH treatment induced liver enzyme deviations in the serum and hypertrophy of the remnant liver, which were suppressed by baicalein. In the lethal HIRI + PH treatment group, baicalein significantly reduced mortality. In IL-1ß-treated rat hepatocytes, baicalein suppressed TNF-α and chemokine mRNA expression as well as the activation of nuclear factor-kappa B (NF-κB) and Akt. CONCLUSIONS: Baicalein treatment attenuates HIRI + PH-induced liver injury and may promote survival. This potential hepatoprotection may be partly related to suppressing inflammatory gene induction through the inhibition of NF-κB activity and Akt signaling in hepatocytes.

Pap12-6: A host defense peptide with potent immunomodulatory activity in a chicken hepatic cell culture.

In the fight against antimicrobial resistance, host defense peptides (HDPs) are increasingly referred to as promising molecules for the design of new antimicrobial agents. In terms of their future clinical use, particularly small, synthetic HDPs offer several advantages, based on which their application as feed additives has aroused great interest in the poultry sector. However, given their complex mechanism of action and the limited data about the cellular effects in production animals, their investigation is of great importance in these species. The present study aimed to examine the immunomodulatory activity of the synthetic HDP Pap12-6 (PAP) solely and in inflammatory environments evoked by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (Poly I:C), in a primary chicken hepatocyte-non-parenchymal cell co-culture. Based on the investigation of the extracellular lactate dehydrogenase (LDH) activity, PAP seemed to exert no cytotoxicity on hepatic cells, suggesting its safe application. Moreover, PAP was able to influence the immune response, reflected by the decreased production of interleukin (IL)-6, IL-8, and "regulated on activation, normal T cell expressed and secreted"(RANTES), as well as the reduced IL-6/IL-10 ratio in Poly I:C-induced inflammation. PAP also diminished the levels of extracellular H2O2 and nuclear factor erythroid 2-related factor 2 (Nrf2) when applied together with Poly I:C and in both inflammatory conditions, respectively. Consequently, PAP appeared to display potent immunomodulatory activity, preferring to act towards the cellular anti-inflammatory and antioxidant processes. These findings confirm that PAP might be a promising alternative for designing novel antimicrobial immunomodulatory agents for chickens, thereby contributing to the reduction of the use of conventional antibiotics.

A patient-based iPSC-derived hepatocyte model of alcohol-associated cirrhosis reveals bioenergetic insights into disease pathogenesis.

Only ~20% of heavy drinkers develop alcohol cirrhosis (AC). While differences in metabolism, inflammation, signaling, microbiome signatures and genetic variations have been tied to the pathogenesis of AC, the key underlying mechanisms for this interindividual variability, remain to be fully elucidated. Induced pluripotent stem cell-derived hepatocytes (iHLCs) from patients with AC and healthy controls differ transcriptomically, bioenergetically and histologically. They include a greater number of lipid droplets (LDs) and LD-associated mitochondria compared to control cells. These pre-pathologic indicators are effectively reversed by Aramchol, an inhibitor of stearoyl-CoA desaturase. Bioenergetically, AC iHLCs have lower spare capacity, slower ATP production and their mitochondrial fuel flexibility towards fatty acids and glutamate is weakened. MARC1 and PNPLA3, genes implicated by GWAS in alcohol cirrhosis, show to correlate with lipid droplet-associated and mitochondria-mediated oxidative damage in AC iHLCs. Knockdown of PNPLA3 expression exacerbates mitochondrial deficits and leads to lipid droplets alterations. These findings suggest that differences in mitochondrial bioenergetics and lipid droplet formation are intrinsic to AC hepatocytes and can play a role in its pathogenesis.

Bystander Effects and Profibrotic Interactions in Hepatic Stellate Cells during HIV and HCV Coinfection.

This study aims to explore the influence of coinfection with HCV and HIV on hepatic fibrosis. A coculture system was set up to actively replicate both viruses, incorporating CD4 T lymphocytes (Jurkat), hepatic stellate cells (LX-2), and hepatocytes (Huh7.5). LX-2 cells' susceptibility to HIV infection was assessed through measurements of HIV receptor expression, exposure to cell-free virus, and cell-to-cell contact with HIV-infected Jurkat cells. The study evaluated profibrotic parameters, including programed cell death, ROS imbalance, cytokines (IL-6, TGF-ß, and TNF-α), and extracellular matrix components (collagen, α-SMA, and MMP-9). The impact of HCV infection on LX-2/HIV-Jurkat was examined using soluble factors released from HCV-infected hepatocytes. Despite LX-2 cells being nonsusceptible to direct HIV infection, bystander effects were observed, leading to increased oxidative stress and dysregulated profibrotic cytokine release. Coculture with HIV-infected Jurkat cells intensified hepatic fibrosis, redox imbalance, expression of profibrotic cytokines, and extracellular matrix production. Conversely, HCV-infected Huh7.5 cells exhibited elevated profibrotic gene transcriptions but without measurable effects on the LX-2/HIV-Jurkat coculture. This study highlights how HIV-infected lymphocytes worsen hepatic fibrosis during HCV/HIV coinfection. They increase oxidative stress, profibrotic cytokine levels, and extracellular matrix production in hepatic stellate cells through direct contact and soluble factors. These insights offer valuable potential therapies for coinfected individuals.

Lipotoxic hepatocyte derived LIMA1 enriched small extracellular vesicles promote hepatic stellate cells activation via inhibiting mitophagy.

BACKGROUND: Hepatic stellate cells (HSCs) play a crucial role in the development of fibrosis in non-alcoholic fatty liver disease (NAFLD). Small extracellular vesicles (sEV) act as mediators for intercellular information transfer, delivering various fibrotic factors that impact the function of HSCs in liver fibrosis. In this study, we investigated the role of lipotoxic hepatocyte derived sEV (LTH-sEV) in HSCs activation and its intrinsic mechanisms. METHODS: High-fat diet (HFD) mice model was constructed to confirm the expression of LIMA1. The relationship between LIMA1-enriched LTH-sEV and LX2 activation was evaluated by measurement of fibrotic markers and related genes. Levels of mitophagy were detected using mt-keima lentivirus. The interaction between LIMA1 and PINK1 was discovered through database prediction and molecular docking. Finally, sEV was injected to investigate whether LIMA1 can accelerate HFD induced liver fibrosis in mice. RESULTS: LIMA1 expression was upregulated in lipotoxic hepatocytes and was found to be positively associated with the expression of the HSCs activation marker α-SMA. Lipotoxicity induced by OPA led to an increase in both the level of LIMA1 protein in LTH-sEV and the release of LTH-sEV. When HSCs were treated with LTH-sEV, LIMA1 was observed to hinder LX2 mitophagy while facilitating LX2 activation. Further investigation revealed that LIMA1 derived from LTH-sEV may inhibit PINK1-Parkin-mediated mitophagy, consequently promoting HSCs activation. Knocking down LIMA1 significantly attenuates the inhibitory effects of LTH-sEV on mitophagy and the promotion of HSCs activation. CONCLUSIONS: Lipotoxic hepatocyte-derived LIMA1-enriched sEVs play a crucial role in promoting HSCs activation in NAFLD-related liver fibrosis by negatively regulating PINK1 mediated mitophagy. These findings provide new insights into the pathological mechanisms involved in the development of fibrosis in NAFLD.

Hepatitis B: Model Systems and Therapeutic Approaches.

Hepatitis B virus (HBV) infection is a major global health issue and ranks among the top causes of liver cirrhosis and hepatocellular carcinoma. Although current antiviral medications, including nucleot(s)ide analogs and interferons, could inhibit the replication of HBV and alleviate the disease, HBV cannot be fully eradicated. The development of cellular and animal models for HBV infection plays an important role in exploring effective anti-HBV medicine. During the past decades, advancements in several cell culture systems, such as HepG2.2.15, HepAD38, HepaRG, hepatocyte-like cells, and primary human hepatocytes, have propelled the research in inhibiting HBV replication and expression and thus enriched our comprehension of the viral life cycle and enhancing antiviral drug evaluation efficacy. Mouse models, in particular, have emerged as the most extensively studied HBV animal models. Additionally, the present landscape of HBV therapeutics research now encompasses a comprehensive assessment of the virus's life cycle, targeting numerous facets and employing a variety of immunomodulatory approaches, including entry inhibitors, strategies aimed at cccDNA, RNA interference technologies, toll-like receptor agonists, and, notably, traditional Chinese medicine (TCM). This review describes the attributes and limitations of existing HBV model systems and surveys novel advancements in HBV treatment modalities, which will offer deeper insights toward discovering potentially efficacious pharmaceutical interventions.

CBX7 silencing promoted liver regeneration by interacting with BMI1 and activating the Nrf2/ARE signaling pathway.

Multiple studies have shown knockdown of chromobox 7 (CBX7) promotes the regenerative capacity of various cells or tissues. We examined the effect of CBX7 on hepatocyte proliferation and liver regeneration after 2/3 hepatectomy in a mouse model. For in vitro experiments, NCTC 1469 and BNL CL.2 hepatocytes were co-transfected with siRNA-CBX7-1 (si-CBX7-1), siRNA-CBX7-2 (si-CBX7-2), pcDNA-CBX7, si-BMI1-1, si-BMI1-2, pcDNA-BMI1, or their negative control. For in vivo experiments, mice were injected intraperitoneally with lentivirus-packaged shRNA and shRNA CBX7 before hepatectomy. Our results showed that CBX7 was rapidly induced in the early stage of liver regeneration. CBX7 regulated hepatocyte proliferation, cell cycle, and apoptosis of NCTC 1469 and BNL CL.2 hepatocytes. CBX7 interacted with BMI1 and inhibited BMI1 expression in hepatocytes. Silencing BMI1 aggregated the inhibitory effect of CBX7 overexpression on hepatocyte viability and the promotion of apoptosis. Furthermore, silencing BMI1 enhanced the regulatory effect of CBX7 on Nrf2/ARE signaling in HGF-induced hepatocytes. In vivo, CBX7 silencing enhanced liver/body weight ratio in PH mice. CBX7 silencing promoted the Ki67-positive cell count and decreased the Tunel-positive cell count after hepatectomy, and also increased the expression of nuclear Nrf2, HO-1, and NQO-1. Our results suggest that CBX7 silencing may increase survival following hepatectomy by promoting liver regeneration.

Hepatic recruitment of myeloid-derived suppressor cells upon liver injury promotes both liver regeneration and fibrosis.

BACKGROUND: The liver regeneration is a highly complicated process depending on the close cooperations between the hepatocytes and non-parenchymal cells involving various inflammatory cells. Here, we explored the role of myeloid-derived suppressor cells (MDSCs) in the processes of liver regeneration and liver fibrosis after liver injury. METHODS: We established four liver injury models of mice including CCl4-induced liver injury model, bile duct ligation (BDL) model, concanavalin A (Con A)-induced hepatitis model, and lipopolysaccharide (LPS)-induced hepatitis model. The intrahepatic levels of MDSCs (CD11b+Gr-1+) after the liver injury were detected by flow cytometry. The effects of MDSCs on liver tissues were analyzed in the transwell co-culture system, in which the MDSCs cytokines including IL-10, VEGF, and TGF-ß were measured by ELISA assay and followed by being blocked with specific antibodies. RESULTS: The intrahepatic infiltrations of MDSCs with surface marker of CD11b+Gr-1+ remarkably increased after the establishment of four liver injury models. The blood served as the primary reservoir for hepatic recruitment of MDSCs during the liver injury, while the bone marrow appeared play a compensated role in increasing the number of MDSCs at the late stage of the inflammation. The recruited MDSCs in injured liver were mainly the M-MDSCs (CD11b+Ly6G-Ly6Chigh) featured by high expression levels of cytokines including IL-10, VEGF, and TGF-ß. Co-culture of the liver tissues with MDSCs significantly promoted the proliferation of both hepatocytes and hepatic stellate cells (HSCs). CONCLUSIONS: The dramatically and quickly infiltrated CD11b+Gr-1+ MDSCs in injured liver not only exerted pro-proliferative effects on hepatocytes, but also accounted for the activation of profibrotic HSCs.

Hepatocytes differentiate into intestinal epithelial cells through a hybrid epithelial/mesenchymal cell state in culture.

Hepatocytes play important roles in the liver, but in culture, they immediately lose function and dedifferentiate into progenitor-like cells. Although this unique feature is well-known, the dynamics and mechanisms of hepatocyte dedifferentiation and the differentiation potential of dedifferentiated hepatocytes (dediHeps) require further investigation. Here, we employ a culture system specifically established for hepatic progenitor cells to study hepatocyte dedifferentiation. We found that hepatocytes dedifferentiate with a hybrid epithelial/mesenchymal phenotype, which is required for the induction and maintenance of dediHeps, and exhibit Vimentin-dependent propagation, upon inhibition of the Hippo signaling pathway. The dediHeps re-differentiate into mature hepatocytes by forming aggregates, enabling reconstitution of hepatic tissues in vivo. Moreover, dediHeps have an unexpected differentiation potential into intestinal epithelial cells that can form organoids in three-dimensional culture and reconstitute colonic epithelia after transplantation. This remarkable plasticity will be useful in the study and treatment of intestinal metaplasia and related diseases in the liver.

A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel host-pathogen interactions.

BACKGROUND: Sporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host-pathogen protein-protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host-pathogen protein-protein interactions involved are poorly understood. METHODS: To gain a better understanding of the protein-protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites. RESULTS: A total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified. Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes. CONCLUSION: Pf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.