Hypoxia preconditioning of human amniotic mesenchymal stem cells enhances proliferation and migration and promotes their homing via the HGF/C-MET signaling axis to augment the repair of acute liver failure.

BACKGROUND: Transplantation of mesenchymal stem cells (MSCs) is a newly developed strategy for treating acute liver failure (ALF). Nonetheless, the low survival rate of MSCs after transplantation and their poor homing to damaged tissues limit the clinical application of MSCs. The research assessed whether hypoxic preconditioning (HPC) can improve the biological activity of human amniotic mesenchymal stem cells (hA-MSCs), promote their homing ability to the liver of mice with ALF, and influence liver tissue repair. METHODS: Flow cytometry, CCK8, Transwell, and Western blotting assays were conducted to assess the effects of hypoxic preconditioning on the phenotype, proliferation, and migration of hA-MSCs and the changes in the c-Met and CXCR4 gene expression levels were studied. To evaluate the effects of the transplantation of hypoxic preconditioning of hA-MSCs on the homing and repair of D-galactosamine (D-GalN)/LPS-induced ALF, the mechanism was elucidated by adding c-Met, CXCR4-specific blockers (SU11274 and AMD3100). RESULTS: After hypoxia pretreatment (1% oxygen volume fraction), hA-MSCs maintained the morphological characteristics of adherence and vortex colony growth and showed high CD44, CD90, and CD105 and low CD31, CD34, and CD45 expression levels. Hypoxic preconditioning of hA-MSCs significantly increased their proliferation and migration and highly expressed the c-Met and CXCR4 genes. In vivo and in vitro, this migration-promoting effect was suppressed by the c-Met specific blocker SU11274. In the acute liver failure mouse model, the HGF expression level was considerably elevated in the liver than that in the serum, lungs and kidneys. The transplantation of hypoxic preconditioned hA-MSCs introduced a remarkable improvement in the liver function and survival rate of mice with ALF and enhanced the anti-apoptosis ability of liver cells. The anti-apoptotic enhancing effect of hypoxic preconditioning was suppressed by the c-Met specific blocker SU11274. Hypoxic hA-MSCs administration was observed to have considerably increased the fluorescent cells in the liver than that recorded after administering normal oxygen-hA-MSCs. The number of hepatic fluorescent cells decreased remarkably after adding the c-Met inhibitor SU11274, compared to that recorded after hypoxic pretreatment, whereas the effect of c-Met inhibitor SU11274 on normal oxygen-hA-MSCs was not significant. CONCLUSIONS: Hypoxic preconditioning depicted no impact on the morphology and phenotype features of the human amniotic mesenchymal stem cells, but it can promote their proliferation, migration, anti-apoptotic effect, and homing rate and improve the repair of acute liver failure, which might be mediated by the HGF/c-Met signaling axis.

miRNA­21 promotes the progression of acute liver failure via the KLF6/autophagy/IL­23 signaling pathway.

Acute liver failure (ALF) is a complex syndrome characterized by overactivation of innate immunity, and the recruitment and differentiation of immune cells at inflammatory sites. The present study aimed to explore the role of microRNA (miRNA/miR)­21 and its potential mechanisms underlying inflammatory responses in ALF. Baseline serum miR­21 was analyzed in patients with ALF and healthy controls. In addition, miR­21 antagomir was injected via the tail vein into C57BL/6 mice, and lipopolysaccharide/D­galactosamine (LPS/GalN) was injected into mice after 48 h. The expression levels of miR­21, Krüppel­like­factor­6 (KLF6), autophagy­related proteins and interleukin (IL)­23, and hepatic pathology were then assessed in the liver tissue. Furthermore, THP­1­derived macrophages were transfected with a miRNA negative control, miR­21 inhibitor, miR­21 mimics or KLF6 overexpression plasmid, followed by treatment with or without rapamycin, and the expression levels of miR­21, KLF6, autophagy­related proteins and IL­23 were evaluated. The results revealed that baseline serum miR­21 levels were significantly upregulated in patients with ALF. In addition, LPS/GalN­induced ALF was attenuated in the antagomir­21 mouse group. KLF6 was identified as a target of miR­21­5p with one putative seed match site identified by TargetScan. A subsequent luciferase activity assay demonstrated a direct interaction between miR­21­5p and the 3'­UTR of KLF6 mRNA. Further experiments suggested that miR­21 promoted the expression of IL­23 via inhibiting KLF6, which regulated autophagy. In conclusion, in the present study, baseline serum miR­21 levels were highly upregulated in patients with ALF, antagomir­21 attenuated LPS/GalN­induced ALF in a mouse model, and miR­21 could promote the expression of IL­23 via inhibiting KLF6.

Modulation of the crosstalk between Keap1/Nrf2/HO-1 and NF-κB signaling pathways by Tomatidine protects against inflammation/oxidative stress-driven fulminant hepatic failure in mice.

Fulminant hepatic failure (FHF) is the terminal phase of acute liver injury, which is characterized by massive hepatocyte necrosis and rapid hepatic dysfunction in patients without preexisting liver disease. There are currently no therapeutic options for such a life-threatening hepatic failure except liver transplantation; therefore, the terminal phase of the underlying acute liver injury should be avoided. Tomatidine (TOM), asteroidal alkaloid, may have different biological activities, including antioxidant and anti-inflammatory effects. Herein, the lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced FHF mouse model was established to explore the protective potential of TOM and the underlying mechanisms of action. TOM pretreatment significantly inhibited hepatocyte necrosis and decreased serum aminotransferase activities in LPS/D-GalN-stimulated mice. TOM further increased the level of different antioxidant enzymes while reducing lipid peroxidation biomarkers in the liver. These beneficial effects of TOM were shown to be associated with targeting of NF-κB signaling pathways, where TOM repressed NF-κB activation and decreased LPS/D-GalN-induced TNF-α, IL-6, IL-1ß, and iNOS production. Moreover, TOM prevented LPS/D-GalN-induced upregulation of Keap1 expression and downregulation of Nrf2 and HO-1 expression, leading to increased Nrf2-binding activity and HO-1 levels. Besides, TOM pretreatment repressed LPS/D-GalN-induced upregulation of proliferating cell nuclear antigen (PCNA) expression, which spared the hepatocytes from damage and subsequent repair following the LPS/D-GalN challenge. Collectively, our findings revealed that TOM has a protective effect on LPS/D-GalN-induced FHF in mice, showing powerful antioxidant and anti-inflammatory effects, primarily mediated via modulating Keap1/Nrf2/HO-1 and NF-κB/TNF-α/IL-6/IL-1ß/iNOS signaling pathways.

IDH1/MDH1 deacetylation promotes acute liver failure by regulating NETosis.

BACKGROUND: Acute liver failure (ALF) is a life-threatening disease, but its pathogenesis is not fully understood. NETosis is a novel mode of cell death. Although the formation of neutrophil extracellular traps (NETs) has been found in various liver diseases, the specific mechanism by which NETosis regulates the development of ALF is unclear. In this article, we explore the role and mechanism of NETosis in the pathogenesis of ALF. METHODS: Clinically, we evaluated NETs-related markers in the liver and peripheral neutrophils of patients with ALF. In in vitro experiments, HL-60 cells were first induced to differentiate into neutrophil-like cells (dHL-60 cells) with dimethyl sulfoxide (DMSO). NETs were formed by inducing dHL-60 cells with PMA. In in vivo experiments, the ALF model in mice was established with LPS/D-gal, and the release of NETs was detected by immunofluorescence staining and western blotting. Finally, the acetylation levels of IDH1 and MDH1 were detected in dHL-60 cells and liver samples by immunoprecipitation. RESULTS: Clinically, increased release of NETs in liver tissue was observed in patients with ALF, and NETs formation was detected in neutrophils from patients with liver failure. In dHL-60 cells, mutations at IDH1-K93 and MDH1-K118 deacetylate IDH1 and MDH1, which promotes the formation of NETs. In a mouse model of ALF, deacetylation of IDH1 and MDH1 resulted in NETosis and promoted the progression of acute liver failure. CONCLUSIONS: Deacetylation of IDH1 and MDH1 reduces their activity and promotes the formation of NETs. This change aggravates the progression of acute liver failure.

The mitochondrial biliverdin exporter ABCB10 in hepatocytes mitigates neutrophilic inflammation in alcoholic hepatitis.

Acute liver failure caused by alcoholic hepatitis (AH) is only effectively treated with liver transplantation. Livers of patients with AH show a unique molecular signature characterized by defective hepatocellular redox metabolism, concurrent to hepatic infiltration of neutrophils that express myeloperoxidase (MPO) and form neutrophil extracellular traps (NETs). Exacerbated NET formation and MPO activity contribute to liver damage in mice with AH and predicts poor prognosis in AH patients. The identification of pathways that maladaptively exacerbate neutrophilic activity in liver could inform of novel therapeutic approaches to treat AH. Whether the redox defects of hepatocytes in AH directly exacerbate neutrophilic inflammation and NET formation is unclear. Here we identify that the protein content of the mitochondrial biliverdin exporter ABCB10, which increases hepatocyte-autonomous synthesis of the ROS-scavenger bilirubin, is decreased in livers from humans and mice with AH. Increasing ABCB10 expression selectively in hepatocytes of mice with AH is sufficient to decrease MPO gene expression and histone H3 citrullination, a specific marker of NET formation. These anti-inflammatory effects can be explained by ABCB10 function reducing ROS-mediated actions in liver. Accordingly, ABCB10 gain-of-function selectively increased the mitochondrial GSH/GSSG ratio and decreased hepatic 4-HNE protein adducts, without elevating mitochondrial fat expenditure capacity, nor mitigating steatosis and hepatocyte death. Thus, our study supports that ABCB10 function regulating ROS-mediated actions within surviving hepatocytes mitigates the maladaptive activation of infiltrated neutrophils in AH. Consequently, ABCB10 gain-of-function in human hepatocytes could potentially decrease acute liver failure by decreasing the inflammatory flare caused by excessive neutrophil activity.

Oral magnesium prevents acetaminophen-induced acute liver injury by modulating microbial metabolism.

Acetaminophen overuse is a common cause of acute liver failure (ALF). During ALF, toxins are metabolized by enzymes such as CYP2E1 and transformed into reactive species, leading to oxidative damage and liver failure. Here, we found that oral magnesium (Mg) alleviated acetaminophen-induced ALF through metabolic changes in gut microbiota that inhibit CYP2E1. The gut microbiota from Mg-supplemented humans prevented acetaminophen-induced ALF in mice. Mg exposure modulated Bifidobacterium metabolism and enriched indole-3-carboxylic acid (I3C) levels. Formate C-acetyltransferase (pflB) was identified as a key Bifidobacterium enzyme involved in I3C generation. Accordingly, a Bifidobacterium pflB knockout showed diminished I3C generation and reduced the beneficial effects of Mg. Conversely, treatment with I3C or an engineered bacteria overexpressing Bifidobacterium pflB protected against ALF. Mechanistically, I3C bound and inactivated CYP2E1, thus suppressing formation of harmful reactive intermediates and diminishing hepatocyte oxidative damage. These findings highlight how interactions between Mg and gut microbiota may help combat ALF.

Silencing of GDF11 suppresses hepatocyte apoptosis to relieve LPS/D-GalN acute liver failure.

In this paper, we generated a short hairpin RNA growth differentiation factor-11 (sh-GDF11) and evaluated the effects of sh-GDF11 on the pathogenesis of acute liver failure (ALF) in vitro and in vivo. Through bioinformatics study, the key gene related to ALF was assayed. Lipopolysaccharide (LPS) and D-galactoamine (D-GalN) were applied to establish the mouse model of LPS/D-GalN-induced liver injury, and TNF-α and D-Gal were used to construct an in vitro cell model, followed by treatment of sh-GDF11 for analysis of liver cell proliferation. Bioinformatics analysis showed that the protective effect of sh-GDF11 on ALF may be mediated by phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. The results of in vitro study found that sh-GDF11 could promote cell proliferation and inhibit death by blocking the PI3K/Akt/mTOR signaling pathway. In vivo animal experiments further confirmed that sh-GDF11 could suppress hepatocyte apoptosis by inhibiting the PI3K/Akt/mTOR signaling pathway. sh-GDF11 relieved LPS/D-GalN-induced ALF by blocking the PI3K/Akt/mTOR signaling pathway, emphasizing its critical role in LPS/D-GalN-induced ALF treatment.

Cholinergic Anti-inflammatory Pathway Attenuates Acute Liver Failure Through Inhibiting MAdCAM1/α4ß7-mediated Gut-derived Proinflammatory Lymphocytes Accumulation.

BACKGROUND & AIMS: The function of cholinergic anti-inflammatory pathway (CAP) in acute liver failure (ALF) with inflammatory storm remains indefinite. The liver-gut axis has been proved to be crucial for liver homeostasis. Investigation about CAP regulation on liver-gut axis would enrich our understanding over cholinergic anti-inflammatory mechanism. METHODS: Co-injection of lipopolysaccharide and D-galactosamine was used to establish the model of ALF. PNU-282987 was used to activate the CAP. Histological staining, real-time polymerase chain reaction, Western blotting, RNA sequencing, and flow cytometry were conducted. Liver biopsy specimens and patients' serum from patients with liver failure were also analyzed. RESULTS: We confirmed that activating the CAP alleviated hepatocyte destruction, accompanied by a significant decrease in hepatocyte apoptosis, pro-inflammatory cytokines, and NLRP3 inflammasome activation. Moreover, hepatic MAdCAM1 and serum MAdCAM1 levels were induced in ALF, and MAdCAM1 levels were positively correlated with the extent of liver damage and the expression of pro-inflammatory markers. Furthermore, activating the CAP mainly downregulated ectopic expression of MAdCAM1 on endothelial cells, and inhibition of NF-κB p65 nuclear translocation was partly attributed to the decreased MAdCAM1. Notably, in ALF, the aberrant hepatic expression of MAdCAM1 subsequently recruited gut-derived α4ß7+ CD4+T cells to the liver, which exhibited an augmented IFN-γ-secreting and IL-17-producing phenotype. Finally, we revealed that the levels of serum and hepatic MAdCAM1 were elevated in patients with liver failure and closely correlated with clinical course. Increasing hepatic infiltration of ß7+ cells were also confirmed in patients. CONCLUSIONS: Activating the CAP attenuated liver injury by inhibiting MAdCAM1/α4ß7 -mediated gut-derived proinflammatory lymphocytes infiltration, which provides a potential therapeutic target for ALF.

miRNA-ome plasma analysis unveils changes in blood-brain barrier integrity associated with acute liver failure in rats.

BACKGROUND: Hepatic encephalopathy (HE) symptoms associated with liver insufficiency are linked to the neurotoxic effects of ammonia and other toxic metabolites reaching the brain via the blood-brain barrier (BBB), further aggravated by the inflammatory response. Cumulative evidence documents that the non-coding single-stranded RNAs, micro RNAs (miRs) control the BBB functioning. However, miRs' involvement in BBB breakdown in HE is still underexplored. Here, we hypothesized that in rats with acute liver failure (ALF) or rats subjected to hyperammonemia, altered circulating miRs affect BBB composing proteins. METHODS: Transmission electron microscopy was employed to delineate structural alterations of the BBB in rats with ALF (thioacetamide (TAA) intraperitoneal (ip.) administration) or hyperammonemia (ammonium acetate (OA) ip. administration). The BBB permeability was determined with Evans blue dye and sodium fluorescein assay. Plasma MiRs were profiled by Next Generation Sequencing (NGS), followed by in silico analysis. Selected miRs, verified by qRT-PCR, were examined in cultured rat brain endothelial cells. Targeted protein alterations were elucidated with immunofluorescence, western blotting, and, after selected miR mimics transfection, through an in vitro resistance measurement. RESULTS: Changes in BBB structure and increased permeability were observed in the prefrontal cortex of TAA rats but not in the brains of OA rats. The NGS results revealed divergently changed miRNA-ome in the plasma of both rat models. The in silico analysis led to the selection of miR-122-5p and miR-183-5p with their target genes occludin and integrin ß1, respectively, as potential contributors to BBB alterations. Both proteins were reduced in isolated brain vessels and cortical homogenates in TAA rats. We documented in cultured primary brain endothelial cells that ammonia alone and, in combination with TNFα increases the relative expression of NGS-selected miRs with a less pronounced effect of TNFα when added alone. The in vitro study also confirmed miR-122-5p-dependent decrease in occludin and miR-183-5p-related reduction in integrin ß1 expression. CONCLUSION: This work identified, to our knowledge for the first time, potential functional links between alterations in miRs residing in brain endothelium and BBB dysfunction in ALF.

RNF115/BCA2 deficiency alleviated acute liver injury in mice by promoting autophagy and inhibiting inflammatory response.

The E3 ubiquitin ligase RING finger protein 115 (RNF115), also known as breast cancer-associated gene 2 (BCA2), has been linked with the growth of some cancers and immune regulation, which is negatively correlated with prognosis. Here, it is demonstrated that the RNF115 deletion can protect mice from acute liver injury (ALI) induced by the treatment of lipopolysaccharide (LPS)/D-galactosamine (D-GalN), as evidenced by decreased levels of alanine aminotransaminase, aspartate transaminase, inflammatory cytokines (e.g., tumor necrosis factor α and interleukin-6), chemokines (e.g., MCP1/CCL2) and inflammatory cell (e.g., monocytes and neutrophils) infiltration. Moreover, it was found that the autophagy activity in Rnf115-/- livers was increased, which resulted in the removal of damaged mitochondria and hepatocyte apoptosis. However, the administration of adeno-associated virus Rnf115 or autophagy inhibitor 3-MA impaired autophagy and aggravated liver injury in Rnf115-/- mice with ALI. Further experiments proved that RNF115 interacts with LC3B, downregulates LC3B protein levels and cell autophagy. Additionally, Rnf115 deletion inhibited M1 type macrophage activation via NF-κB and Jnk signaling pathways. Elimination of macrophages narrowed the difference in liver damage between Rnf115+/+ and Rnf115-/- mice, indicating that macrophages were linked in the ALI induced by LPS/D-GalN. Collectively, for the first time, we have proved that Rnf115 inactivation ameliorated LPS/D-GalN-induced ALI in mice by promoting autophagy and attenuating inflammatory responses. This study provides new evidence for the involvement of autophagy mechanisms in the protection against acute liver injury.

MicroRNA-181a-5p alleviates acute liver failure in mice by inhibiting HMGB1.

MicroRNAs (miRNAs) control liver diseases, but the role of microRNA-181a-5p in acute liver failure (ALF) is unclear. In this study, the ALF model was generated by injection of D-galactosamine (D-GalN) and lipopolysaccharide (LPS). The levels of miRNAs were assessed by microarray and qRT-PCR. The expression of caspase 3 was detected as the marker of cell apoptosis in ALF by immunohistochemistry and western blot. The targeting of microRNA-181a-5p on the high mobility group box 1 (HMGB1) was verified by dual luciferase assay. The impact of microRNA-181a-5p and HMGB1 was explored by flow cytometry. Results showed that microRNA-181a-5p was significantly down-regulated by D-GalN/LPS in vivo and in vitro, while the level of HMGB1 was up-regulated after the challenge. Furthermore, microRNA-181a-5p overexpression attenuated cell apoptosis in D-GalN/TNF-treated BNLCL2 cells. MicroRNA-181a-5p could directly target HMGB1 mRNA and repress its expressions, in further HMGB1 is involved in microRNA-181a-5p effect on cell apoptosis of ALF. In conclusion, these findings demonstrate that microRNA-181a-5p regulates hepatocyte apoptosis via HMGB1 in the development of ALF, which may provide potential therapeutic targets for ALF. However, the precise underlying mechanism that connects microRNA-181a-5p and HMGB1 remains to be explored.

GAS1 Promotes Ferroptosis of Liver Cells in Acetaminophen-Induced Acute Liver Failure.

Purpose: Acute liver failure (ALF) is a clinically fatal disease that leads to the rapid loss of normal liver function. Acetaminophen (APAP) is a leading cause of drug-induced ALF. Ferroptosis, defined as iron-dependent cell death associated with lipid peroxide accumulation, has been shown to be strongly associated with APAP-induced liver injury. Growth arrest-specific 1 (GAS1) is a growth arrest-specific gene, which is closely related to the inhibition of cell growth and promotion of apoptosis. However, the functional role and underlying mechanism of GAS1 in APAP-induced ferroptosis remain unknown. Methods: We established liver-specific overexpression of GAS1 (GAS1AAV8-OE) mice and the control (GAS1AAV8-vector) mice by tail vein injection of male mice with adeno-associated virus. APAP at 500 mg/kg was intraperitoneally injected into these two groups of mice to induce acute liver failure. The shRNA packaged by the lentivirus inhibits GAS1 gene expression in human hepatoma cell line HepaRG (HepaRG-shNC and HepaRG-shGAS1-2) and primary hepatocytes of mice with liver-specific overexpression of GAS1 were isolated and induced by APAP in vitro to further investigate the regulatory role of GAS1 in APAP-induced acute liver failure. Results: APAP-induced upregulation of ferroptosis, levels of lipid peroxides and reactive oxygen species, and depletion of glutathione were effectively alleviated by the ferroptosis inhibitor, ferrostatin-1, and downregulation of GAS1 expression. GAS1 overexpression promoted ferroptosis-induced lipid peroxide accumulation via p53, inhibiting its downstream target, solute carrier family 7 member 11. Conclusion: Collectively, our findings suggest that GAS1 overexpression plays a key role in aggravating APAP-induced acute liver injury by promoting ferroptosis-induced accumulation of lipid peroxides.

Primary human hepatocytes-laden scaffolds for the treatment of acute liver failure.

Cell-based liver therapies based on retrieving and steadying failed metabolic function(s) for acute and chronic diseases could be a valuable substitute for liver transplants, even though they are limited by the low engraftment capability and reduced functional quality of primary human hepatocytes (PHH). In this paper we propose the use of gelatin-hyaluronic acid (Gel-HA) scaffolds seeded with PHH for the treatment of liver failure. We first optimized the composition using Gel-HA hydrogels, looking for the mechanical properties closer to the human liver and determining HepG2 cells functionality. Gel-HA scaffolds with interconnected porosity (pore size 102 µm) were prepared and used for PHH culture and evaluation of key hepatic functions. PHH cultured in Gel-HA scaffolds exhibited increased albumin and urea secretion and metabolic capacity (CYP and UGT activity levels) compared to standard monolayer cultures. The transplant of the scaffold containing PHH led to an improvement in liver function (transaminase levels, necrosis) and ameliorated damage in a mouse model of acetaminophen (APAP)-induced liver failure. The study provided a mechanistic understanding of APAP-induced liver injury and the impact of transplantation by analyzing cytokine production and oxidative stress induction to find suitable biomarkers of cell therapy effectiveness.

Calycosin inhibits hepatocyte apoptosis in acute liver failure by suppressing the TLR4/NF-κB pathway: An in vitro study.

BACKGROUND: Acute liver failure (ALF) is a serious liver disease that is difficult to treat owing to its unclear pathogenesis. This study aimed to investigate the roles and molecular mechanisms of calycosin (CA) in ALF. METHODS: In this study, the roles and mechanism of CA in ALF were explored using an in vitro lipopolysaccharide (LPS)-induced ALF cell model. Additionally, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay was used to assess the effect of CA on the activity of LPS-induced L02 human liver epithelial cells, and flow cytometry was used to detect apoptosis in L02 cells. Expression levels of apoptosis-related genes, Bax and Bcl-2, were measured using reverse transcription-quantitative polymerase chain reaction and Western blot analysis. Expression levels of inflammatory factors in LPS-induced L02 cells were measured using an enzyme-linked immunosorbent assay. Additionally, the effect of CA on ALF was inhibited via transfection of a toll-like receptor 4 (TLR4)-plasmid to elucidate the relationship between CA and TLR4/nuclear factor (NF)-κB signaling pathway in ALF. RESULTS: CA had no toxic effects on L02 cells, but enhanced the activity of LPS-induced L02 cells in a dose-dependent manner. Apoptosis and inflammatory factor release was increased in ALF, activating the TLR4/NF-κB signaling pathway. However, CA treatment inhibited the apoptosis and release of inflammatory factors. Further mechanistic studies revealed that the upregulation of TLR4 expression reversed the alleviating effects of CA on inflammation and apoptosis in LPS-induced L02 cells. CONCLUSION: CA alleviates inflammatory damage in LPS-induced L02 cells by inhibiting the TLR4/NF-κB pathway and may be a promising therapeutic agent for ALF treatment.

Label-Free Imaging Techniques to Evaluate Metabolic Changes Caused by Toxic Liver Injury in PCLS.

Abuse with hepatotoxic agents is a major cause of acute liver failure. The search for new criteria indicating the acute or chronic pathological processes is still a challenging issue that requires the selection of effective tools and research models. Multiphoton microscopy with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM) are modern label-free methods of optical biomedical imaging for assessing the metabolic state of hepatocytes, therefore reflecting the functional state of the liver tissue. The aim of this work was to identify characteristic changes in the metabolic state of hepatocytes in precision-cut liver slices (PCLSs) under toxic damage by some of the most common toxins: ethanol, carbon tetrachloride (CCl4) and acetaminophen (APAP), commonly known as paracetamol. We have determined characteristic optical criteria for toxic liver damage, and these turn out to be specific for each toxic agent, reflecting the underlying pathological mechanisms of toxicity. The results obtained are consistent with standard methods of molecular and morphological analysis. Thus, our approach, based on optical biomedical imaging, is effective for intravital monitoring of the state of liver tissue in the case of toxic damage or even in cases of acute liver injury.

A Biomimetic Nanoparticle Exerting Protection against Acute Liver Failure by Suppressing CYP2E1 Activity and Scavenging Excessive ROS.

Acute liver failure (ALF) is a severe liver disease caused by many reasons. One of them is the overdosed acetaminophen (APAP), which is metabolized into N-acetyl-p-benzoquinone imine (NAPQI), an excessive toxic metabolite, by CYP2E1, resulting in excessive reactive oxygen species (ROS), exhausted glutathione (GSH), and thereafter hepatocyte necrosis. N-acetylcysteine is the Food and Drug Administration-approved drug for detoxification of APAP, but it has limited clinical application due to the short therapeutic time window and concentration-related adverse effects. In this study, a carrier-free and bilirubin dotted nanoparticle (B/BG@N) is developed, which is formed using bilirubin and 18ß-Glycyrrhetinic acid, and bovine serum albumin (BSA) is then adsorbed to mimic the in vivo behavior of the conjugated bilirubin for hitchhiking. The results demonstrate that B/BG@N can effectively reduce the production of NAPQI as well as exhibit antioxidant effects against intracellular oxidative stress via regulating the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signal axis and reducing the production of inflammatory factors. In vivo study shows that B/BG@N can effectively improve the clinical symptom of the mice model. This study suggests that B/BG@N own increases circulation half-life, improves accumulation in the liver, and dual detoxification, providing a promising strategy for clinical ALF treatment.

P2rx1 deficiency alleviates acetaminophen-induced acute liver failure by regulating the STING signaling pathway.

AIMS: Purinergic signaling-mediated mitochondria dysfunction and innate immune-mediated inflammation act as triggers during acetaminophen (APAP)-induced liver injury (AILI). However, the underlying mechanisms by which purinoceptor regulates mitochondria function and inflammation response in the progression of AILI remains unclear. METHODS: First, the hepatic level of purinergic receptor P2X 1 (P2RX1) was identified in the DILI patients and APAP-induced WT mice. P2rx1 knockout (KO) mice (P2rx1-/-) with 300 mg/kg APAP challenge were used for the analysis of the potential role of P2RX1 in the progression of AILI. Administration of DMX, the activator of stimulator of interferon genes (STING), was performed to investigate the effects of the STING-related pathway on APAP-treated P2rx1-/- mice. RESULTS: The elevated hepatic P2RX1 levels were found in DILI patients and the AILI mice. P2rx1 depletion offered protection against the initial stages of AILI, mainly by inhibiting cell death and promoting inflammation resolution, which was associated with alleviating mitochondria dysfunction. Mechanistically, P2rx1 depletion could inhibit STING-TANK-binding kinase 1 (TBK1)-P65 signaling pathways in vivo. We then showed that DMX-mediated STING activation could greatly aggravate the liver injury of P2rx1-/- mice treated with APAP. CONCLUSION: Our data confirmed that P2RX1 was inducted during AILI, identified P2RX1 as a novel regulator in mitochondria dysfunction and STING pathways, and suggested a promising therapeutic approach for AILI involving the blockade of P2RX1. 1. It first demonstrated the protective effects of P2rx1 deficiency on acetaminophen-induced liver injury (AILI). 2. P2rx1 knockout alleviates mitochondria function and promotes inflammation resolution after APAP treatment. 3. It first reported the regulation of P2RX1 on the STING signaling pathway in the progress of AILI. 4. P2RX1 blockade is a promising therapeutic strategy for AILI.

Upregulated microRNA-450b-5p represses the development of acute liver failure via modulation of liver function, inflammatory response, and hepatocyte apoptosis.

OBJECTIVE: It has been evidenced that microRNAs (miRs) exert crucial effects on acute liver failure (ALF), while the detailed function of miR-450b-5p in ALF progression remained obscure. The purpose of this research was to unravel the regulatory mechanism of miR-450b-5p in ALF via modulating Mouse Double Minute 2 protein (MDM2). METHODS: ALF was induced in mice by intraperitoneal injection of d-galactosamine ( d-GalN) and lipopolysaccharide (LPS). Adenoviruses containing overexpressed miR-450b-5p, MDM2 shRNA, and overexpressed MDM2 sequences were utilized to manipulate miR-450b-5p and MDM2 expression in the liver before the mice were treated with d-GalN/LPS-induced ALF. Subsequently, miR-450b-5p and MDM2 expression levels in liver tissues of ALF mice were examined. Serum biochemical parameters of liver function were tested, serum inflammatory factors were assessed, and the histopathological changes and hepatocyte apoptosis in liver tissues were observed. The relation between miR-450b-5p and MDM2 was verified. RESULTS: In ALF mice, miR-450b-5p was low-expressed while MDM2 was high-expressed. The upregulation of miR-450b-5p or downregulation of MDM2 could alleviate liver function, mitigate the serum inflammatory response and pathological changes in liver tissues, as well as inhibit the apoptosis of hepatocytes. MiR-450b-5p targeted MDM2. MDM2 overexpression reversed the repressive effects of elevated miR-450b-5p on ALF. CONCLUSION: The upregulated miR-450b-5p blocks the progression of ALF via targeting MDM2. This study contributes to affording novel therapeutic targets for ALF treatment.

Geraniol ameliorates acute liver failure induced by lipopolysaccharide/D-galactosamine via regulating macrophage polarization and NLRP3 inflammasome activation by PPAR-γ methylation Geraniol alleviates acute liver failure.

Geraniol (Ger), a natural acyclic monoterpene alcohol, has been reported to exert protective effects through anti-inflammation in Acute liver failure (ALF). However, its specific roles and precise mechanisms underlying anti-inflammatory effects in ALF have not yet fully explored. We aimed to investigated the hepatoprotective effects and mechanisms of Ger against ALF induced by lipopolysaccharide (LPS)/D-galactosamine (GaIN). In this study, the liver tissue and serum of LPS/D-GaIN-induced mice were collected. The degree of liver tissue injury was evaluated by HE and TUNEL staining. Serum levels of liver injury markers (ALT and AST) and inflammatory factors were measured by ELISA assays. PCR and western blotting were conducted to determine the expression of inflammatory cytokines, NLRP3 inflammasome-related proteins, PPAR-γ pathway-related proteins, DNA Methyltransferases and M1/M2 polarization cytokines. Immunofluorescence staining was used to assess the localization and expression of macrophage markers (F4/80 and CD86), NLRP3 and PPAR-γ. In vitro experiments were performed in macrophages stimulated with LPS with or without IFN-γ. Purification of macrophages and cell apoptosis was analyzed using flow cytometry. We found that Ger effectively alleviated ALF in mice, specified by the attenuation of liver tissue pathological damage, inhibition of ALT, AST and inflammatory factor levels, and inactivation of NLRP3 inflammasome. Meanwhile, downregulation M1 macrophage polarization may involve in the protective effects of Ger. In vitro, Ger reduced the activation of NLRP3 inflammasome and apoptosis through regulating PPAR-γ methylation by inhibiting M1 macrophage polarization. In conclusion, Ger protects against ALF through suppressing NLRP3 inflammasome-mediated inflammation and LPS-induced macrophage M1 polarization via modulating PPAR-γ methylation.

The Efficiency of Stem Cells (SCs) Differentiation into Functional Hepatocytes for Treating Liver Disorders: A Systematic Review.

Stem cells provided new opportunity to treat various diseases, including liver disorders. Stem cells are unspecialized cells, stimulating influential research interest be indebted to their multipotent self-renewal capacity and differentiation characteristics into several specialized cell types. Many factors contribute to their differentiation into different cell types such as insulin producing cells, osteoblast, and hepatocytes. Accordingly, wide range methods and materials have been used to transform stem cells into hepatocytes, but effectiveness of differentiation is different and depends on several factors such as cell-to-cell adhesion, cell-to-cell contact, and cell biological change. Search was done in PubMed, Scopus, and WOS to evaluate results of studies about stem cells differentiation for higher efficacy. Among more than 28000 papers, 51 studies were considered eligible for more evaluations. Results indicated that most studies were performed on mesenchymal stem cells compared with other types. Acute liver failure was the most investigated liver disorder, and tissue engineering was the most investigated differentiation methods. Also, functional parameters were the most evaluated parameters in assessing differentiation efficacy. We summarize recent advances in increasing efficiency of stem cells differentiation using varied materials, since promising results of this review, further studies are needed to assess efficiency and safety of these cells transplantation in some liver disease treatment.