In vitro immuno-prevention of nitration/dysfunction of myogenic stem cell activator HGF, towards developing a strategy for age-related muscle atrophy.

In response to peroxynitrite (ONOO-) generation, myogenic stem satellite cell activator HGF (hepatocyte growth factor) undergoes nitration of tyrosine residues (Y198 and Y250) predominantly on fast IIa and IIx myofibers to lose its binding to the signaling receptor c-met, thereby disturbing muscle homeostasis during aging. Here we show that rat anti-HGF monoclonal antibody (mAb) 1H41C10, which was raised in-house against a synthetic peptide FTSNPEVRnitroY198EV, a site well-conserved in mammals, functions to confer resistance to nitration dysfunction on HGF. 1H41C10 was characterized by recognizing both nitrated and non-nitrated HGF with different affinities as revealed by Western blotting, indicating that the paratope of 1H41C10 may bind to the immediate vicinity of Y198. Subsequent experiments showed that 1H41C10-bound HGF resists peroxynitrite-induced nitration of Y198. A companion mAb-1H42F4 presented similar immuno-reactivity, but did not protect Y198 nitration, and thus served as the control. Importantly, 1H41C10-HGF also withstood Y250 nitration to retain c-met binding and satellite cell activation functions in culture. The Fab region of 1H41C10 exerts resistivity to Y250 nitration possibly due to its localization in the immediate vicinity to Y250, as supported by an additional set of experiments showing that the 1H41C10-Fab confers Y250-nitration resistance which the Fc segment does not. Findings highlight the in vitro preventive impact of 1H41C10 on HGF nitration-dysfunction that strongly impairs myogenic stem cell dynamics, potentially pioneering cogent strategies for counteracting or treating age-related muscle atrophy with fibrosis (including sarcopenia and frailty) and the therapeutic application of investigational HGF drugs.

The Progress and Promise of Lineage Reprogramming Strategies for Liver Regeneration.

The liver exhibits remarkable regenerative capacity. However, the limited ability of primary human hepatocytes to proliferate in vitro, combined with a compromised regenerative capacity induced by pathological conditions in vivo, presents significant obstacles to effective liver regeneration following liver injuries and diseases. Developing strategies to compensate for the loss of endogenous hepatocytes is crucial for overcoming these challenges, and this remains an active area of investigation. Lineage reprogramming, the process of directly converting one cell type into another bypassing the intermediate pluripotent state, has emerged as a promising method for generating specific cell types for therapeutic purposes in regenerative medicine. Here, we discuss the recent progress and emergent technologies in lineage reprogramming into hepatic cells, and their potential applications in enhancing liver regeneration or treating liver disease models. We also address controversies and challenges that confront this field.

Drosophila HNF4 acts in distinct tissues to direct a switch between lipid storage and export in the gut.

Nutrient digestion, absorption, and export must be coordinated in the gut to meet the nutritional needs of the organism. We used the Drosophila intestine to characterize the mechanisms that coordinate the fate of dietary lipids. We identified enterocytes specialized in absorbing and exporting lipids to peripheral organs. Distinct hepatocyte-like cells, called oenocytes, communicate with these enterocytes to adjust intestinal lipid storage and export. A single transcription factor, Drosophila hepatocyte nuclear factor 4 (dHNF4), supports this gut-liver axis. In enterocytes, dHNF4 maximizes dietary lipid export by preventing their sequestration in cytoplasmic lipid droplets. In oenocytes, dHNF4 promotes the expression of the insulin antagonist ImpL2 to activate Foxo and suppress lipid retention in enterocytes. Disruption of this switch between lipid storage and export is associated with intestinal inflammation, suggesting a lipidic origin for inflammatory bowel diseases. These studies establish dHNF4 as a central regulator of intestinal metabolism and inter-organ lipid trafficking.

TERT upregulation promotes cell proliferation via degradation of p21 and increases carcinogenic potential.

Telomerase reverse transcriptase (TERT) gene aberration is detectable in >80% of cases with hepatocellular carcinoma (HCC). TERT reactivation is essential for cellular immortalization because it stabilizes telomere length, although the role of TERT in hepatocarcinogenesis remains unelucidated. To elucidate the significance of aberrant TERT expression in hepatocytes in inflammation-associated hepatocarcinogenesis, we generated Alb-Cre;TertTg mice, which overexpress TERT in the liver and examined their phenotype during chronic inflammation. Based on transcriptome data from the liver tissue of Alb-Cre;TertTg mice, we examined the role of TERT in hepatocarcinogenesis in vitro. We also evaluated the relationship between TERT and cell-cycle-related molecules, including p21, in HCC samples. The liver tumor development rate was increased by TERT overexpression during chronic inflammation, especially in the absence of p53 function. Gene set enrichment analysis of liver tissues revealed that gene sets related to TNF-NFκB signaling, cell cycle, and apoptosis were upregulated in Alb-Cre;TertTg liver. A luciferase reporter assay and immunoprecipitation revealed that TERT interacted with NFκB p65 and enhanced NFKB1 promoter activity. On the other hand, TERT formed protein complexes with p21, cyclin A2, and cyclin E and promoted ubiquitin-mediated degradation of p21, specifically in the G1 phase. In the clinical HCC samples, TERT was highly expressed but p21 was conversely downregulated, and TERT expression was associated with the upregulation of molecules related to the cell cycle. Taken together, the aberrant upregulation of TERT increased NFKB1 promoter activity and promoted cell cycle progression via p21 ubiquitination, leading to hepatocarcinogenesis. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Pretreatment with aqueous Moringa oleifera Lam. leaf extract prevents cadmium-induced hepatotoxicity by improving cellular antioxidant machinery and reducing cadmium accumulation.

Cadmium (Cd) is a highly harmful pollutant that poses a serious threat to human health. The liver is the primary organ for Cd accumulation, and Cd-induced hepatotoxicity has been shown to be strongly correlated with an oxidative imbalance in hepatocytes. Our previous studies in the eukaryotic model organism Saccharomyces cerevisiae revealed that not only co-treatment but also pretreatment with aqueous Moringa oleifera Lam. leaf extract (AMOLE) effectively mitigated Cd toxicity by reducing intracellular Cd accumulation and Cd-mediated oxidative stress. In this study, we therefore investigated the preventive effect of AMOLE against Cd toxicity in human HepG2 hepatocytes. The results showed that, similar to the case of the yeast model, pretreatment with AMOLE prior to Cd exposure also significantly inhibited Cd-induced oxidative stress in HepG2 cells. Untargeted LC-MS/MS-based metabolomic analysis of AMOLE revealed that its major phytochemical constituents were organic acids, particularly phenolic acids and carboxylic acids. Additionally, DPPH-HPTLC fingerprints suggested that quercetin and other flavonoids possibly contribute to the antioxidant activities of AMOLE. Based on our findings, it appears that pretreatment with AMOLE prevented Cd-induced hepatotoxicity via three possible mechanisms: i) direct elimination of free radicals by AMOLE antioxidant compounds; ii) upregulation of antioxidant defensive machinery (GPx1, and HO-1) via Nrf2 signaling cascade to improve cellular antioxidant capacity; and iii) reduction of intracellular Cd accumulation, probably by suppressing Cd uptake. These data strongly suggest the high potential of AMOLE for clinical utility in the prevention of Cd toxicity.

L-Glutamine mitigates bile acid-induced inhibition of growth factor activity in rat hepatocyte cultures.

Bile acid-induced hepatotoxicity is inevitable in Cholestasis pathogenesis and L-Glutamine (L-Gln) has been reported to prevent total parenteral nutrition (TPN)-induced cholestasis in premature neonates. While mechanisms remain unknown, we hypothesize that bile acids impair growth factor (GF) function in hepatocytes which L-glutamine prevents through NAPDH oxidase (NOX) modulation. Glycochenodeoxycholic acid (GCDC, 0-100 µM) when added to primary hepatocyte cultures significantly (p < 0.01) decreased the FBS-induced BrdU incorporation, however inhibition of Fibroblast Growth factor (FGF)- or Hepatocyte growth factor (HGF)-induced DNA synthesis was more pronounced (p < 0.001). L-Gln markedly attenuated GCDC-mediated inhibition of DNA synthesis in both FBS and GF-treated cells. GCDC significantly increased the NADPH oxidase activity and NOX-1 protein expression that were markedly reduced by L-Gln and protein kinase c (PKC) inhibitor, LY-333531. Apocynin (APCN) and diphenyliodonium (DPI) significantly blocked the GCDC-mediated inhibition of GF-induced DNA synthesis. This study demonstrates that bile acid-induced hepatotoxicity involves dysfunction of certain growth factors via protein kinase c (PKC)- mediated NOX modulation which can be corrected, at least partly, by L-glutamine.

Effect of controlled release of HGF on extracellular vesicle secretion by urine-derived stem cells.

Introduction: The hepatic growth factor (HGF) stimulates DNA synthesis and cell proliferation and plays a role in tissue protection and regeneration. In this study, we have examined the effect of incubation of HGF with urine-derived stem cells (USCs) on the secretion of small extracellular vesicles (sEV) by the cells. Materials and Methods: HGF in the incubation medium was either a bolus administration or a controlled release of an equivalent amount from microbeads within the size range of 50-200 µm made with ultrapurified low-viscosity high-guluronic acid (UP-LVG) alginate. USCs were incubated with or without HGF for 3 days or 7 days before removal of the incubation media, followed by harvesting sEV by the precipitation method. The protein content of isolated sEV was measured by bicinchoninic acid assay (BCA) for these three groups: control (no HGF beads), bolus HGF, and HGF beads. We also performed nanoparticle tracking analysis (NTA), Western blot assay, and ELISA for the HGF content of samples. Results: We found a significantly higher concentration of proteins in the HGF microbead group (control release group) compared to the bolus group and the control group after 7 days (p < 0.0017). The NTA data aligned with the BCA; they showed a significantly higher concentration of particles within the size range of sEV (<200 nm) in the group treated with HGF beads compared to the two other groups on day 7 (p < 0.0001). Conclusion: We found that administration of HGF to USCs by controlled release of the growth factor significantly enhances the levels of sEV secretion during 7 days of incubation.

Salmon Milt Extract Suppresses Glucose Uptake by Downregulating SGLT1 and GLUT2 Expression in Caco-2 Cells.

Salmon milt extract (SME) is rich in nucleotides, especially deoxyribonucleoside monophosphates (dNMPs), which has the potential to exert anti-obesity effects. Sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) are responsible for absorbing sugar from the small intestine. The purpose of this study was to examine the effects of SME on the functions of SGLT1 and GLUT2 and elucidate the mechanisms underlying the inhibition of glucose absorption by SME. We investigated the effect of SME on the expression and function of intestinal glucose transporters, using differentiated Caco-2 cells. SME treatment decreased the expression SGLT1 and GLUT2 mRNA and protein in Caco-2 cells. [14C]-Labelled methyl-α-D-glucopyranoside and [3H]-labelled 2-deoxy-D-glucose (DG) uptake into Caco-2 cells was significantly reduced by SME treatment. Similarly, the dNMP mixture containing the four mononucleotides 2'-deoxyadenosine 5'-monophosphate (dAMP), 2'-deoxyguanosine 5'-monophosphate (dGMP), 2'-deoxycytidine 5'-monophosphate (dCMP), and 2'-deoxythymidine 5'-monophosphate (dTMP) decreased SGLT1 and GLUT2 expression. dNMP mixture-induced reduction in the mRNA expression of these transporters was suppressed when exposed to the mixture without dTMP. Furthermore, dNMP mixture-induced alterations in the expression of hepatocyte nuclear factor (HNF)-1α and HNF1ß, which have been characterized as modulators of both transporters also showed a similar trend. dTMP treatment alone decreased GLUT2 expression, resulting in reduced [3H] DG uptake by Caco-2 cells. SME decreased the expression of HNF1α, HNF1ß, and its targets SGLT1 and GLUT2, resulting in reduced glucose uptake by Caco-2 cells. In addition, our results revealed that dTMP plays an important role in suppressing the expression of intestinal glucose transporters.

Dual effects of targeting neuropilin-1 in lenvatinib-resistant hepatocellular carcinoma: inhibition of tumor growth and angiogenesis.

In the era of immunotherapy, lenvatinib (LEN) still holds an important position in the sequential treatment of advanced hepatocellular carcinoma (HCC). However, the sustained therapeutic effect of LEN is not sufficient, and there is a need to address the development of resistance. Neuropilin-1 (NRP1) is known to act as a co-receptor for epidermal growth factor receptor (EGFR), Met, and vascular endothelial growth factor receptor 2 (VEGFR2), which have been reported to be involved in LEN resistance. In this study, we used cell culture and in vivo transplantation models to evaluate the contribution of NRP1 in the acquisition of LEN resistance in HCC as well as the potential of NRP1 as a therapeutic target. LEN resistance increased EGF/EGFR and hepatocyte growth factor (HGF)/Met signaling in liver cancer cells and VEGFA/VEGFR2 and HGF/Met signaling in vascular endothelial cells, thereby promoting cell proliferation, cell migration, and angiogenesis. We found that activation of NRP1 is essential for the enhancement of these signaling. In addition, NRP1 inhibition combined with LEN therapy synergistically improved the antitumor effects against LEN-resistant HCC, indicating that NRP1 is an attractive therapeutic target.

Microvesicles derived from mesenchymal stem cells inhibit acute respiratory distress syndrome-related pulmonary fibrosis in mouse partly through hepatocyte growth factor.

BACKGROUND: Pulmonary fibrosis is one of the main reasons for the high mortality rate among acute respiratory distress syndrome (ARDS) patients. Mesenchymal stromal cell-derived microvesicles (MSC-MVs) have been shown to exert antifibrotic effects in lung diseases. AIM: To investigate the effects and mechanisms of MSC-MVs on pulmonary fibrosis in ARDS mouse models. METHODS: MSC-MVs with low hepatocyte growth factor (HGF) expression (siHGF-MSC-MVs) were obtained via lentivirus transfection and used to establish the ARDS pulmonary fibrosis mouse model. Following intubation, respiratory mechanics-related indicators were measured via an experimental small animal lung function tester. Homing of MSC-MVs in lung tissues was investigated by near-infrared live imaging. Immunohistochemical, western blotting, ELISA and other methods were used to detect expression of pulmonary fibrosis-related proteins and to compare effects on pulmonary fibrosis and fibrosis-related indicators. RESULTS: The MSC-MVs gradually migrated and homed to damaged lung tissues in the ARDS model mice. Treatment with MSC-MVs significantly reduced lung injury and pulmonary fibrosis scores. However, low expression of HGF (siHGF-MSC-MVs) significantly inhibited the effects of MSC-MVs (P < 0.05). Compared with the ARDS pulmonary fibrosis group, the MSC-MVs group exhibited suppressed expression of type I collagen antigen, type III collagen antigen, and the proteins transforming growth factor-ß and α-smooth muscle actin, whereas the siHGF-MVs group exhibited significantly increased expression of these proteins. In addition, pulmonary compliance and the pressure of oxygen/oxygen inhalation ratio were significantly lower in the MSC-MVs group, and the effects of the MSC-MVs were significantly inhibited by low HGF expression (all P < 0.05). CONCLUSION: MSC-MVs improved lung ventilation functions and inhibited pulmonary fibrosis in ARDS mice partly via HGF mRNA transfer.

Hepatocyte-Specific Yap1 Knockout Maintained the Liver Homeostasis of Lipid Metabolism in Mice.

Introduction: Yes-associated protein 1 (YAP1) is a crucial molecule in the Hippo pathway. The impact of hepatocyte-specific Yap1 knockout (Yap1 LKO) on hepatic lipid droplets (LD) and pePLIN2 in metabolic fatty liver has not been reported. This study aims to explore whether Yap1 LKO could offer a protective effect in a liver injury model. Methods: Three-week-old Yap1 LKO and Yap1 Flox mice were given aristolochic acid I (AAI) combined carbon tetrachloride (CCL4) establish liver injury model. Eight-week-old Yap1 LKO and Yap1 Flox mice were fed with a high-fat diet for 18 weeks to establish obesity-related liver injury model. Further biochemical, histomorphological, immunohistochemical, and lipidomic analyses were performed on serum and liver tissues of these mice to elucidate the effects of hepatocyte-specific Yap1 knockout on hepatic lipid metabolism. Results: Yap1 LKO reduced triglyceride (TG) content and PLIN2 expression level in the liver during the intervention of AAI combined CCl4. Moreover, Yap1 LKO improved lipid metabolism homeostasis in the liver by increasing the beneficial lipid molecules and reducing the harmful lipid molecules through lipidomics. Finally, Yap1 LKO reduced TG content in the serum and liver, hepatic vacuolar degeneration, and hepatic PLIN2 expression level in mice fed with a high-fat diet (HFD). Conclusion: Yap1 LKO is protective in regulating liver and blood TG when induced with toxic substances AAI combined CCl4 and a high-fat diet.

Sini san regulates intestinal flora and short-chain fatty acids to ameliorate hepatocyte apoptosis and relieve CCl4-induced liver fibrosis in mice.

Introduction: Si-Ni-San (SNS), a traditional Chinese medicine, is effective in treating liver fibrosis with an unclear mechanism. Although disturbance of intestinal flora and the subsequent secretion of short-chain fatty acids (SCFAs) is suggested to be involved in the progression of liver fibrosis, whether SNS produces the anti-fibrosis effect through the regulation of intestinal flora and SCFAs remains unclear. Methods: In the current study, carbon tetrachloride (CCl4)-treated mice were dosed with SNS to examine the anti-fibrotic effects and the involved mechanism. Biochemical parameters, histological staining, and analyses of fibrotic gene expression were used to evaluate the anti-fibrotic effect of SNS, while intestinal flora and SCFA content were determined by 16S rRNA and LC-MS to evaluate the mechanism. Results: In vivo results showed that SNS improved liver function, reduced hepatocyte apoptosis and FFAR2/3 expression, and restored intestinal dysbiosis and reduced PA, BA, and IsA levels. In vitro experiments showed that PA, BA, and IsA exacerbated TNF-α-induced HepG2 apoptosis. Notably, the protective effects of SNS were compromised in pseudo-sterile mice. Discussion: In conclusion, our experimental results suggest that the disturbance in intestinal flora results in elevated SCFA levels, which further exacerbates hepatocyte apoptosis in liver fibrosis, while SNS suppresses CCl4-induced liver fibrosis at least partially by reinstating intestinal flora homeostasis and reducing SCFA levels.

HGF Facilitates the Repair of Spinal Cord Injuries by Driving the Chemotactic Migration of MSCs Through the ß-Catenin/TCF4/Nedd9 Signaling Pathway.

Transplanted mesenchymal stem cells (MSCs) can significantly aid in repairing spinal cord injuries (SCI) by migrating to and settling at the injury site. However, this process is typically inefficient, as only a small fraction of MSCs successfully reach the target lesion area. During SCI, the increased expression and secretion of hepatocyte growth factor (HGF) act as a chemoattractant that guides MSC migration. Nonetheless, the precise mechanisms by which HGF influences MSC migration are not fully understood. This study focused on unraveling the molecular pathways that drive MSC migration towards the SCI site in response to HGF. It was found that HGF can activate ß-catenin signaling in MSCs either by phosphorylating LRP6 or by suppressing GSK3ß phosphorylation through the AKT and ERK1/2 pathways, or by enhancing the expression and nuclear translocation of TCF4. This activation leads to elevated Nedd9 expression, which promotes focal adhesion formation and F-actin polymerization, facilitating chemotactic migration. Transplanting MSCs during peak HGF expression in injured tissues substantially improves nerve regeneration, reduces scarring, and enhances hind limb mobility. Additionally, prolonging HGF release can further boost MSC migration and engraftment, thereby amplifying regenerative outcomes. However, inhibiting HGF/Met or interfering with ß-catenin or Nedd9 signaling significantly impairs MSC engraftment, obstructing tissue repair and functional recovery. Together, these findings provide a theoretical basis and practical strategy for MSC transplantation therapy in SCI, highlighting the specific molecular mechanisms by which HGF regulates ß-catenin signaling in MSCs, ultimately triggering their chemotactic migration.

Small Molecule-Mediated Stage-Specific Reprogramming of MSCs to Hepatocyte-Like Cells and Hepatic Tissue for Liver Injury Treatment.

BACKGROUND: Derivation of hepatocytes from stem cells has been established through various protocols involving growth factor (GF) and small molecule (SM) agents, among others. However, mesenchymal stem cell-based derivation of hepatocytes still remains expensive due to the use of a cocktail of growth factors, and a long duration of differentiation is needed, thus limiting its potential clinical application. METHODS: In this study, we developed a chemically defined differentiation strategy that is exclusively based on SM and takes 14 days, while the GF-based protocol requires 23-28 days. RESULTS: We optimized a stage-specific differentiation protocol for the differentiation of rat bone marrow-derived mesenchymal stem cells (MSCs) into functional hepatocyte-like cells (dHeps) that involved four stages, i.e., definitive endoderm (DE), hepatic competence (HC), hepatic specification (HS) and hepatic differentiation and growth. We further generated hepatic tissue using human decellularized liver extracellular matrix and compared it with hepatic tissue derived from the growth factor-based protocol at the transcriptional level. dHep, upon transplantation in a rat model of acute liver injury (ALI), was capable of ameliorating liver injury in rats and improving liver function and tissue damage compared to those in the ALI model. CONCLUSIONS: In summary, this is the first study in which hepatocytes and hepatic tissue were derived from MSCs utilizing a stage-specific strategy by exclusively using SM as a differentiation factor.

Macrophage-Hepatocyte Circuits Mediated by Grancalcin Aggravate the Progression of Metabolic Dysfunction Associated Steatohepatitis.

The dynamic interplay between parenchymal hepatocytes and non-parenchymal cells (NPCs), such as macrophages, is an important mechanism for liver metabolic homeostasis. Although numerous endeavors have been made to identify the mediators of metabolic dysfunction associated steatohepatitis (MASH), the molecular underpinnings of MASH progression remain poorly understood, and therapies to arrest MASH progression remain elusive. Herein, it is revealed that the expression of grancalcin (GCA) is upregulated in the macrophages of patients and rodents with MASH and correlates with MASH progression. Notably, the administration of recombinant GCA aggravates the development of MASH, whereas, Gca deletion in myeloid cells blunts liver steatosis and inflammation in multiple MASH murine models. Mechanistically, GCA activates macrophages via TLR9-NF-κB signaling, and the activated macrophages promote hepatocyte lipid accumulation and apoptosis via secretion of Interleukin-6(IL-6), Tumor Necrosis Factor α (TNFα), and Interleukin-1ß(IL-1ß), thereby leading to hepatic steatosis and inflammation. Finally, the therapeutic administration of antibody blocking GCA effectively halts the progression of MASH. Collectively, these findings implicate GCA as a crucial mediator of MASH and clarify a new metabolic signaling axis between the hepatocytes and macrophages, implying that GCA can emerge as a particularly interesting putative therapeutic target for reversing MASH progression.

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.

Sappanone A enhances hepatocyte proliferation in lipopolysaccharide-induced acute liver injury in mice by promoting injured hepatocyte apoptosis and regulating macrophage polarization.

OBJECTIVES: Lipopolysaccharide (LPS), also known as endotoxin, is the main toxic component of the cell wall of gram negative bacteria, which is released after bacterial death and widely exists in the living environment. Human exposure to endotoxin may cause sepsis. The occurrence of septic liver injury is a prominent factor contributing to mortality in patients with sepsis. The purpose of this study is to explore the role of Sappanone A (SA), a homoisoflavonoid isolated from the heartwood of Caesalpinia sappan Linn., in LPS-induced acute liver injury (ALI). METHODS: An LPS-induced ALI mouse model was used to evaluate the effects of SA on septic ALI, and murine cells were treated with LPS to explore the mechanisms underlying SA-provided effects. RESULTS: Treating SA substantially improved LPS-induced ALI. We also performed in silico prediction and RNA-seq analysis to elucidate SA's potential mechanisms of action. The terms generated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of predicted target proteins of SA include inflammation, oxidative stress, and apoptosis; protein-protein interaction network (PPI) analysis indicated that fas binding protein 1 (Fbf1) has the strongest correlation with SA. Consistently, RNA-seq analysis displayed that SA administration regulates cell apoptosis and inflammatory responses, which was further confirmed by checking related markers in livers of mice and murine cells challenged with LPS. Of note, SA significantly decreased the expression of Fbf1 in mouse livers, and promoted apoptosis of injured hepatocytes and hepatocyte proliferation, which were substantially abolished by Fbf1 knockdown in AML12 cells. Besides, SA could increase M2 phenotype polarization but inhibit M1 macrophage polarization in LPS-induced ALI in mice. CONCLUSION: SA enhances hepatocyte proliferation and liver repair in LPS-induced ALI in mcie by promoting injured hepatocyte apoptosis through Fbf1 inhibition and regulating macrophage polarization.

Banxia Xiexin Tang attenuates high glucose-induced hepatocyte injury by activating SOD2 to scavenge ROS via PGC-1α/IGFBP1.

This study aimed to explore the protective mechanism of Banxia Xiexin Tang (BXXXT) on liver cell damage caused by high glucose (H-G) and to clarify its molecular regulatory pathways. First, the main components in BXXXT-containing serum were analyzed by high-performance liquid chromatography (HPLC) to provide basic data for subsequent experiments. Subsequently, the effect of BXXXT on high glucose (H-G)-induced hepatocyte activity was evaluated through screening of the optimal concentration of drug-containing serum. Experimental results showed that BXXXT significantly reduced the loss of cell activity caused by high glucose. Further research focuses on the regulatory effect of BXXXT on high glucose-induced hepatocyte apoptosis, especially its effect on the PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α) pathway. Experimental results showed that BXXXT reduced high-glucose-induced hepatocyte apoptosis and exerted its protective effect by upregulating the activity of the PGC-1α pathway. BXXXT significantly increased the expression level of IGFBP1 (insulin-like growth factor-binding proteins) in hepatocytes under a high-glucose environment. It cleared mitochondrial ROS (reactive oxygen species) by enhancing SOD2 (superoxide dismutase) enzyme activity and maintained the survival of hepatocytes under a high-glucose environment. Finally, the regulation of PGC-1α by BXXXT is indeed involved in the regulation of IGFBP1 expression in hepatocytes and its downstream SOD2 effector signaling. Taken together, this study provides an in-depth explanation of the protective mechanism of BXXXT on hepatocytes in a high-glucose environment, focusing on regulating the expression of the PGC-1α pathway and IGFBP1, and reducing cell damage by scavenging ROS. This provides an experimental basis for further exploring the potential of BXXXT in the treatment of diabetes-related liver injury. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04060-0.

An hepatitis B and D virus infection model using human pluripotent stem cell-derived hepatocytes.

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study, we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg, HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV, thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection, we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients, we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions.

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.