Novel insights into autophagy in gastrointestinal pathologies, mechanisms in metabolic dysfunction-associated fatty liver disease and acute liver failure.

In this editorial, we comment on three articles published in a recent issue of World Journal of Gastroenterology. There is a pressing need for new research on autophagy's role in gastrointestinal (GI) disorders, and also novel insights into some liver conditions, such as metabolic dysfunction-associated fatty liver disease (MAFLD) and acute liver failure (ALF). Despite advancements, understanding autophagy's intricate mechanisms and implications in these diseases remains incomplete. Moreover, MAFLD's pathogenesis, encompassing hepatic steatosis and metabolic dysregulation, require further elucidation. Similarly, the mechanisms underlying ALF, a severe hepatic dysfunction, are poorly understood. Innovative studies exploring the interplay between autophagy and GI disorders, as well as defined mechanisms of MAFLD and ALF, are crucial for identifying therapeutic targets and enhancing diagnostic and treatment strategies to mitigate the global burden of these diseases.

Understanding the molecular crossroads in acute liver failure: A pathway to new therapies.

In this editorial we comment on the article published in a recent issue of the World Journal of Gastroenterology. Acute liver failure (ALF) is a critical condition characterized by rapid hepatocellular injury and organ dysfunction, and it often necessitates liver transplant to ensure patient survival. Recent research has elucidated the involvement of distinct cell death pathways, namely ferroptosis and pyroptosis, in the pathogenesis of ALF. Ferroptosis is driven by iron-dependent lipid peroxidation, whereas pyroptosis is an inflammatory form of cell death; both pathways contribute to hepatocyte death and exacerbate tissue damage. This comprehensive review explores the interplay between ferroptosis and pyroptosis in ALF, highlighting the role of key regulators such as silent information regulator sirtuin 1. Insights from clinical and preclinical studies provide valuable perspectives on the dysregulation of cell death pathways in ALF and the therapeutic potential of targeting these pathways. Collaboration across multiple disciplines is essential for translating the experimental insights into effective treatments for this life-threatening condition.

The SIRT2-AMPK axis regulates autophagy induced by acute liver failure.

This study explores the role of SIRT2 in regulating autophagy and its interaction with AMPK in the context of acute liver failure (ALF). This study investigated the effects of SIRT2 and AMPK on autophagy in ALF mice and TAA-induced AML12 cells. The results revealed that the liver tissue in ALF model group had a lot of inflammatory cell infiltration and hepatocytes necrosis, which were reduced by SIRT2 inhibitor AGK2. In comparison to normal group, the level of SIRT2, P62, MDA, TOS in TAA group were significantly increased, which were decreased in AGK2 treatment. Compared with normal group, the expression of P-PRKAA1, Becilin1 and LC3B-II was decreased in TAA group. However, AGK2 enhanced the expression of P-PRKAA1, Becilin1 and LC3B-II in model group. Overexpression of SIRT2 in AML12 cell resulted in decreased P-PRKAA1, Becilin1 and LC3B-II level, enhanced the level of SIRT2, P62, MDA, TOS. Overexpression of PRKAA1 in AML12 cell resulted in decreased SIRT2, TOS and MDA level and triggered more autophagy. In conclusion, the data suggested the link between AMPK and SIRT2, and reveals the important role of AMPK and SIRT2 in autophagy on acute liver failure.

Application of TSPO-Specific Positron Emission Tomography Radiotracer as an Early Indicator of Acute Liver Failure Induced by Propacetamol, a Prodrug of Paracetamol.

Acetaminophen overdose is a leading cause of acute liver failure (ALF), and effective treatment depends on early prediction of disease progression. ALF diagnosis currently requires blood collection 24-72 h after APAP ingestion, necessitating repeated tests and hospitalization. Here, we assessed earlier ALF diagnosis using positron emission tomography (PET) imaging of translocator proteins (TSPOs), which are involved in molecular transport, oxidative stress, apoptosis, and energy metabolism, with the radiotracer [18F]GE180. We intraperitoneally administered propacetamol hydrochloride to male C57BL/6 mice to induce ALF. We performed in vivo PET/CT imaging 3 h later using the TSPO-specific radiotracer [18F]GE180 and quantitatively analyzed the PET images by determining the averaged standardized uptake value (SUVav) in the liver parenchyma. We assessed liver TSPO expression levels via real-time polymerase chain reaction, Western blotting, and immunohistochemistry. [18F]GE180 PET imaging 3 h after propacetamol administration (1500 mg/kg) significantly increased liver SUVav compared to controls (p = 0.001). Analyses showed a 10-fold and 4-fold increase in TSPO gene and protein expression, respectively, in the liver, 3 h after propacetamol induction compared to controls. [18F]GE180 PET visualized and quantified propacetamol-induced ALF through TSPO overexpression. These findings highlight TSPO PET's potential as a non-invasive imaging biomarker for early-stage ALF.

Mefunidone ameliorates acute liver failure in mice by inhibiting MKK4-JNK pathway.

Acute liver failure (ALF) is a critical condition that can lead to substantial liver dysfunction. It is characterized by complex clinical manifestations and rapid progression, presenting significant challenges in diagnosis and treatment. We investigated the protective effect of mefunidone (MFD), a novel antifibrosis pyridone agent, on ALF in mice, and explored its potential mechanism of action. MFD pretreatment can alleviate lipopolysaccharide (LPS) and d-galactosamine (D-GalN)-induced ALF, reduce hepatocyte apoptosis, and reduce inflammation and oxidative stress. Additionally, MFD alleviated LPS/D-GalN-stimulated reactive oxygen species (ROS) production and cell death in AML12 cells. RNA sequencing enrichment analysis showed that MFD significantly affected the Mitogen-Activated Protein Kinase (MAPK) pathway. In vivo and in vitro experiments showed that MFD inhibited MKK4 and JNK phosphorylation. JNK activation caused by MKK4 and JNK activators could eliminate the therapeutic effect of MFD on AML12. In addition, MFD pretreatment alleviated ConA-induced ALF, reduced inflammation and oxidative stress in mice, and reduced mouse mortality. These results suggest that MFD can potentially protect against ALF, partially by inhibiting the MKK4-JNK pathway, and is a promising new therapeutic drug for ALF.

Selecting serum-free hepatocyte cryopreservation stage and storage temperature for the application of an "off-the-shelf" bioartificial liver system.

The bioartificial liver (BAL) system can potentially rescue acute liver failure (ALF) patients by providing partial liver function until a suitable donor liver can be found or the native liver has self-regenerated. In this study, we established a suitable cryopreservation process for the development of an off-the-shelf BAL system. The viability of hepatocyte spheroids cryopreserved in liquid nitrogen was comparable to that of fresh primary hepatocyte spheroids. When hepatocyte spheroids were subjected to cryopreservation in a deep freezer, no statistically significant differences were observed in ammonia removal rate or urea secretion rate based on the cryopreservation period. However, the functional activity of the liver post-cryopreservation in a deep freezer was significantly lower than that observed following liquid nitrogen cryopreservation. Moreover, cryopreserving spheroid hydrogel beads in a deep freezer resulted in a significant decrease (approximately 30%) in both ammonia removal and urea secretion rates compared to the group cryopreserved in liquid nitrogen. The viabilities of spheroid hydrogel beads filled into the bioreactor of a BAL system were similar across all four groups. However, upon operating the BAL system for 24 h, the liver function activity was significantly higher in the group comprising hydrogel beads generated after thawing hepatocyte spheroids cryopreserved in liquid nitrogen. Consequently, the manufacturing of beads after the cryopreservation of hepatocyte spheroids is deemed the most suitable method, considering efficiency, economic feasibility, and liver function activity, for producing a BAL system.

Hepatic Sirt6 activation abrogates acute liver failure.

Acute liver failure (ALF) is a deadly illness due to insufficient detoxification in liver induced by drugs, toxins, and other etiologies, and the effective treatment for ALF is very limited. Among the drug-induced ALF, acetaminophen (APAP) overdose is the most common cause. However, the molecular mechanisms underlying APAP hepatoxicity remain incompletely understood. Sirtuin 6 (Sirt6) is a stress responsive protein deacetylase and plays an important role in regulation of DNA repair, genomic stability, oxidative stress, and inflammation. Here, we report that genetic and pharmacological activation of Sirt6 protects against ALF in mice. We first observed that Sirt6 expression was significantly reduced in the liver tissues of human patients with ALF and mice treated with an overdose of APAP. Then we developed an inducible Sirt6 transgenic mice for Cre-mediated overexpression of the human Sirt6 gene in systemic (Sirt6-Tg) and hepatic-specific (Sirt6-HepTg) manners. Both Sirt6-Tg mice and Sirt6-HepTg mice exhibited the significant protection against APAP hepatoxicity. In contrast, hepatic-specific Sirt6 knockout mice exaggerated APAP-induced liver damages. Mechanistically, Sirt6 attenuated APAP-induced hepatocyte necrosis and apoptosis through downregulation of oxidative stress, inflammation, the stress-activated kinase JNK activation, and apoptotic caspase activation. Moreover, Sirt6 negatively modulated the level and activity of poly (ADP-ribose) polymerase 1 (PARP1) in APAP-treated mouse liver tissues. Importantly, the specific Sirt6 activator MDL-800 exhibited better therapeutic potential for APAP hepatoxicity than the current drug acetylcysteine. Furthermore, in the model of bile duct ligation induced ALF, hepatic Sirt6-KO exacerbated, but Sirt6-HepTg mitigated liver damage. Collectively, our results demonstrate that Sirt6 protects against ALF and suggest that targeting Sirt6 activation could be a new therapeutic strategy to alleviate ALF.

Endotoxin tolerance ameliorates lipopolysaccharide/D-galactosamine-induced acute liver failure by negative regulation of the NF-κB/NLRP3 and activation of Nrf2/HO-1 via Sitr1.

Acute liver failure (ALF) is a potentially fatal disorder characterized by extensive hepatocyte necrosis and rapid decline in liver function. Numerous factors, including oxidative stress, cell death, and inflammatory responses, are associated with its pathogenesis. Endotoxin tolerance (ET) refers to the phenomenon in which the body or cells exhibit low or no response to high-dose lipopolysaccharide (LPS) stimulation after pre-stimulation with low-dose LPS. However, the specific mechanism through which ET regulates LPS/D-galactosamine (D-GalN)-induced ALF remains unclear. An ALF mouse model was established by intraperitoneal injection of D-GalN (400 mg/kg) and LPS (10 mg/kg). A low dose of LPS (0.1 mg/kg/d) was continuously administered to mice for 5 d before modeling to assess the protective effect of ET. The data from this study showed that ET alleviated the inflammatory response in mice with LPS/D-GalN-induced ALF. ET inhibited LPS-induced oxidative damage and pyroptosis in macrophages in vitro. RNA sequencing analysis showed that the NF-κB/NLRP3 pathway was linked to the anti-inflammatory and antioxidative effects of ET. Furthermore, using western blot, RT-qPCR, and immunofluorescence, we verified that ET inhibited the NF-κB/NLRP3 pathway and triggered the Nrf2/HO-1 signaling pathway to attenuate oxidative stress and cell pyroptosis. Sirt1 knockdown reversed this protective effect. In summary, our research elucidates that ET prevents ALF advancement by upregulating Sirt1 levels, triggering the Nrf2/HO-1 signaling axis, and suppressing the NF-κB/NLRP3 signaling cascade to inhibit oxidative stress and cell pyroptosis. Our results provide a mechanistic explanation for the protective effect of ET against ALF.

Hepatocyte-specific METTL3 ablation by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), resulted in acute liver failure (ALF) and postnatal lethality.

AIM: In 2019, to examine the functions of METTL3 in liver and underlying mechanisms, we generated mice with hepatocyte-specific METTL3 homozygous knockout (METTL3Δhep) by simultaneously crossing METTL3fl/fl mice with Alb-iCre mice (GPT) or Alb-Cre mice (JAX), respectively. In this study, we explored the potential reasons why hepatocyte-specific METTL3 homozygous disruption by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), resulted in acute liver failure (ALF) and then postnatal lethality. MAIN METHODS: Mice with hepatocyte-specific METTL3 knockout were generated by simultaneously crossing METTL3fl/fl mice with Alb-iCre mice (GPT; Strain No. T003814) purchased from the GemPharmatech Co., Ltd., (Nanjing, China) or with Alb-Cre mice (JAX; Strain No. 003574) obtained from The Jackson Laboratory, followed by combined-phenotype analysis. The publicly available RNA-sequencing data deposited in the NCBI Gene Expression Omnibus (GEO) database under the accession No.: GSE198512 (postnatal lethality), GSE197800 (postnatal survival) and GSE176113 (postnatal survival) were mined to explore the potential reasons why hepatocyte-specific METTL3 homozygous deletion by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), leads to ALF and then postnatal lethality. KEY FINDINGS: Firstly, we observed that hepatocyte-specific METTL3 homozygous deficiency by Alb-iCre mice (GPT) or by Alb-Cre mice (JAX) caused liver injury, abnormal lipid accumulation and apoptosis. Secondly, we are surprised to find that hepatocyte-specific METTL3 homozygous deletion by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), led to ALF and then postnatal lethality. Our findings clearly demonstrated that METTL3Δhep mice (GPT), which are about to die, exhibited the severe destruction of liver histological structure, suggesting that METTL3Δhep mice (GPT) nearly lose normal liver function, which subsequently contributes to ALF, followed by postnatal lethality. Finally, we unexpectedly found that as the compensatory growth responses of hepatocytes to liver injury induced by METTL3Δhep (GPT), the proliferation of METTL3Δhep hepatocytes (GPT), unlike METTL3Δhep hepatocytes (JAX), was not evidenced by the significant increase of Ki67-positive hepatocytes, not accompanied by upregulation of cell-cycle-related genes. Moreover, GO analysis revealed that upregulated genes in METTL3Δhep livers (GPT), unlike METTL3Δhep livers (JAX), are not functionally enriched in terms associated with cell cycle, cell division, mitosis, microtubule cytoskeleton organization, spindle organization, chromatin segregation and organization, and nuclear division, consistent with the loss of compensatory proliferation of METTL3Δhep hepatocytes (GPT) observed in vivo. Thus, obviously, the loss of the compensatory growth capacity of METTL3Δhep hepatocytes (GPT) in response to liver injury might contribute to, at least partially, ALF and subsequently postnatal lethality of METTL3Δhep mice (GPT). SIGNIFICANCE: These findings from this study and other labs provide strong evidence that these phenotypes (i.e., ALF and postnatal lethality) of METTL3Δhep mice (GPT) might be not the real functions of METTL3, and closely related with Alb-iCre mice (GPT), suggesting that we should remind researchers to use Alb-iCre mice (GPT) with caution to knockout gene in hepatocytes in vivo.

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.

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.

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.

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.

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.

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.

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.

Treatment with ß-Adrenoceptor Agonist Isoproterenol Reduces Non-parenchymal Cell Responses in LPS/D-GalN-Induced Liver Injury.

There is an increasing evidence indicating the involvement of the sympathetic nervous system (SNS) in liver disease development. To achieve an extensive comprehension of the obscure process by which the SNS alleviates inflammatory damage in non-parenchymal liver cells (NPCs) during acute liver failure (ALF), we employ isoproterenol (ISO), a beta-adrenoceptor agonist, to mimic SNS signaling. ISO was administered to C57BL/6J mice to establish an acute liver failure (ALF) model using LPS/D-GalN, which was defined as ISO + ALF. Non-parenchymal cells (NPCs) were isolated from liver tissues and digested for tandem mass tag (TMT) labeled proteomics to identify differentially expressed proteins (DEPs). The administration of ISO resulted in a decreased serum levels of pro-inflammatory cytokines, e.g., TNF-α, IL-1ß, and IL-6 in ALF mice, which alleviated liver damage. By using TMT analysis, it was possible to identify 1587 differentially expressed proteins (DEPs) in isolated NPCs. Notably, over 60% of the DEPs in the ISO + ALF vs. ALF comparison were shared in the Con vs. ALF comparison. According to enrichment analysis, the DEPs influenced by ISO in ALF mice were linked to biological functions of heme and fatty acid metabolism, interferon gamma response, TNFA signaling pathway, and mitochondrial oxidation function. Protein-protein interaction network analysis indicated Mapk14 and Caspase3 may serve as potentially valuable indicators of ISO intervention. In addition, the markers on activated macrophages, such as Mapk14, Casp1, Casp8, and Mrc1, were identified downregulated after ISO initiation. ISO treatment increased the abundance of anti-inflammatory markers in mouse macrophages, as evidenced by the immunohistochemistry (IHC) slides showing an increase in Arg + staining and a reduction in iNOS + staining. Furthermore, pretreatment with ISO also resulted in a reduction of LPS-stimulated inflammation signaling markers, Mapk14 and NF-κB, in human THP-1 cells. Prior treatment with ISO may have the potential to modify the biological functions of NPCs and could serve as an innovative pharmacotherapy for delaying the pathogenesis and progression of 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.