Mouse Model Of Acute Liver Failure Research Articles

Mesenchymal Stem Cells Alleviate Acute Liver Failure through Regulating Hepatocyte Apoptosis and Macrophage Polarization.

Acute liver failure (ALF) is a life-threatening clinical problem with limited treatment options. Administration of human umbilical cord mesenchymal stem cells (hUC-MSCs) may be a promising approach for ALF. This study aimed to explore the role of hUC-MSCs in the treatment of ALF and the underlying mechanisms. A mouse model of ALF was induced by lipopolysaccharide and d-galactosamine administration. The therapeutic effects of hUC-MSCs were evaluated by assessing serum enzyme activity, histological appearance, and cell apoptosis in liver tissues. The apoptosis rate was analyzed in AML12 cells. The levels of inflammatory cytokines and the phenotype of RAW264.7 cells co-cultured with hUC-MSCs were detected. The C-Jun N-terminal kinase/nuclear factor-kappa B signaling pathway was studied. The hUC-MSCs treatment decreased the levels of serum alanine aminotransferase and aspartate aminotransferase, reduced pathological damage, alleviated hepatocyte apoptosis, and reduced mortality in vivo. The hUC-MSCs co-culture reduced the apoptosis rate of AML12 cells in vitro. Moreover, lipopolysaccharide-stimulated RAW264.7 cells had higher levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β and showed more CD86-positive cells, whereas the hUC-MSCs co-culture reduced the levels of the three inflammatory cytokines and increased the ratio of CD206-positive cells. The hUC-MSCs treatment inhibited the activation of phosphorylated (p)-C-Jun N-terminal kinase and p-nuclear factor-kappa B not only in liver tissues but also in AML12 and RAW264.7 cells co-cultured with hUC-MSCs. hUC-MSCs could alleviate ALF by regulating hepatocyte apoptosis and macrophage polarization, thus hUC-MSC-based cell therapy may be an alternative option for patients with 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.

Dihydroquercetin improves experimental acute liver failure by targeting ferroptosis and mitochondria-mediated apoptosis through the SIRT1/p53 axis

BackgroundFerroptosis and mitochondria-mediated apoptosis are both involved in the pathogenesis of acute liver failure (ALF). Ferroptosis-produced reactive oxygen species (ROS) trigger the chain oxidation of polyunsaturated phospholipids and promote mitochondrial apoptosis. Dihydroquercetin (DHQ) also plays an important protective role against liver injury. PurposeHere, we aimed to investigate the protective effects of DHQ on ALF. We also explored the underlying mechanism. MethodsWe established a Lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced ALF mouse model and tumor necrosis factor-α (TNF-α)/D-Gal-induced ALF LO2 cell model. 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) and Dihydroethidium (DHE) were used to detect total ROS levels. Lipid ROS was assessed using C11-BODIPY flow cytometry. Lipid peroxidative products levels were detected using MDA ELISA assay and 4-hydroxynonenal (4-HNE) immunohistochemistry. QRT-PCR and western blots were used to test mRNA and protein expression levels, respectively. Cell viability was evaluated with CCK8 assay, and apoptosis was analyzed using flow cytometry. ResultsDHQ treatment improved LPS/D-Gal-induced ALF, as well as TNF-α/D-Gal-induced reductions in LO2 viability and increased sirtuin 1 (SIRT1) expression. DHQ pretreatment also reduced the accumulation of ROS, reduced lipid peroxidation, elevated mitochondrial membrane potentials (ΔΨm), and decreased liver cell apoptosis both in vivo and in vitro. Additionally, the knockdown of SIRT1 and p53 activator (Tenovin-6) treatment reversed DHQ's inhibitory effects on ferroptosis and mitochondria-mediated apoptosis in vitro. DHQ enhanced p53 deacetylation by both up-regulating SIRT1 expression and directly bonding to SIRT1. We also found that Tenovin-6′s stimulatory effects on ferroptosis and mitochondria-mediated apoptosis in the DHQ-treated LO2 ALF cell model were partially attenuated by overexpression of solute carrier family 7member 11 (SLC7A11), as well as by apoptotic protease activating factor 1 (Apaf-1) knockdown. ConclusionOur results suggest that DHQ alleviated ALF by inhibiting both ferroptosis and mitochondria-mediated apoptosis by regulating the SIRT1/p53 axis. Thus, DHQ may serve as a novel therapy for ALF.

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

BackgroundTransplantation 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. MethodsFlow 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). ResultsAfter 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. ConclusionsHypoxic 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.

C-C chemokine receptor type 7 (CCR7) regulates hepatic CD8 + T cell homeostasis and response to acute liver injury.

Acute liver failure (ALF) is a rare but life-threatening condition, and DILI, particularly acetaminophen toxicity, is the leading cause of ALF. Innate immune mechanisms further perpetuate liver injury, while the role of the adaptive immune system in DILI-related ALF is unclear. We analyzed liver tissue from 2 independent patient cohorts with ALF and identified hepatic T cell infiltration as a prominent feature in human ALF. CD8 + T cells were characterized by zonation toward necrotic regions and an activated gene expression signature. In murine acetaminophen-induced liver injury, intravital microscopy revealed zonation of CD8 + but not CD4 + T cells at necrotic areas. Gene expression analysis exposed upregulated C-C chemokine receptor 7 (CCR7) and its ligand CCL21 in the liver as well as a broadly activated phenotype of hepatic CD8 + T cells. In 2 mouse models of ALF, Ccr7-/- mice had significantly aggravated early-phase liver damage. Functionally, CCR7 was not involved in the recruitment of CD8 + T cells, but regulated their activation profile potentially through egress to lymphatics. Ccr7-/- CD8 + T cells were characterized by elevated expression of activation, effector, and exhaustion profiles. Adoptive transfer revealed preferential homing of CCR7-deficient CD8 + T cells to the liver, and depletion of CD8 + T cells attenuated liver damage in mice. Our study demonstrates the involvement of the adaptive immune system in ALF in humans and mice. We identify the CCR7-CCL21 axis as an important regulatory pathway, providing downstream protection against T cell-mediated liver injury.

Microfluidic Fabrication of MicroRNA-Induced Hepatocyte-Like Cells/Human Umbilical Vein Endothelial Cells-Laden Microgels for Acute Liver Failure Treatment.

Acute liver failure (ALF) is a critical life-threatening disease that occurs due to a rapid loss in hepatocyte functions. Hepatocyte transplantation holds great potential for ALF treatment, as it rapidly supports liver biofunctions and enhances liver regeneration. However, hepatocyte transplantation is still limited by renewable and ongoing cell sources. In addition, intravenously injected hepatocytes are primarily trapped in the lungs and have limited efficacy because of the rapid clearance in vivo. Here, we designed a Y-shaped DNA nanostructure to deliver microRNA-122 (Y-miR122), which could induce the hepatic differentiation and maturation of human mesenchymal stem cells. mRNA sequencing analysis revealed that the Y-miR122 promoted important hepatic biofunctions of the induced hepatocyte-like cells including fat and lipid metabolism, drug metabolism, and liver development. To further improve hepatocyte transplantation efficiency and therapeutic effects in ALF treatment, we fabricated protective microgels for the delivery of Y-miR122-induced hepatocyte-like cells based on droplet microfluidic technology. When cocultured with human umbilical vein endothelial cells in microgels, the hepatocyte-like cells exhibited an increase in hepatocyte-associated functions, including albumin secretion and cytochrome P450 activity. Notably, upon transplantation into the ALF mouse model, the multiple cell-laden microgels effectively induced the restoration of liver function and enhanced liver regeneration. Overall, this study presents an efficient approach from the generation of hepatocyte-like cells to hepatocyte transplantation in ALF therapy.

Carfilzomib Mitigates Lipopolysaccharide/D-Galactosamine/Dimethylsulfoxide-Induced Acute Liver Failure in Mice.

Acute liver failure (ALF) is a disease accompanied by severe liver inflammation. No effective therapy is available yet apart from liver transplantation; therefore, developing novel treatments for ALF is urgently required. Inflammatory mediators released by NF-кB activation play an essential role in ALF. Proteasome inhibitors have many medical uses, such as reducing inflammation and NF-кB inhibition, which are believed to account for most of their repurposing effects. This study was undertaken to explore the possible protective effects and the underlying mechanisms of carfilzomib, a proteasome inhibitor, in a mouse model of ALF induced by lipopolysaccharide/D-galactosamine/dimethylsulfoxide (LPS/GalN/DMSO). Carfilzomib dose-dependently protected mice from LPS/GalN/DMSO-induced liver injury, as indicated by the decrease in serum alanine aminotransferase and aspartate aminotransferase levels. LPS/GalN/DMSO increased TNF-α, NF-кB, lipid peroxidation, NO, iNOS, cyclooxygenase-II, myeloperoxidase, and caspase-3 levels. Carfilzomib administration mitigated LPS/GalN/DMSO-induced liver damage by decreasing the elevated levels of TNF-α, NF-кB, lipid peroxidation, nitric oxide, iNOS, cyclooxygenase-II, myeloperoxidase, caspase-3, and histopathological changes. A restored glutathione level was also observed in the carfilzomib-treated LPS/GalN/DMSO mice. Our results demonstrate that carfilzomib protects against LPS/GalN/DMSO-induced ALF by inhibiting NF-кB, decreasing inflammatory mediators, oxidative/nitrosative stress, neutrophil recruitment, and apoptosis, suggesting that carfilzomib may be a potential therapeutic agent for ALF.

Identification of key immune-related genes associated with LPS/D-GalN-induced acute liver failure in mice based on transcriptome sequencing.

The aim of this study was to identify key immune-related genes in acute liver failure (ALF) by constructing an ALF mouse model for transcriptome sequencing. The C57BL/6 mouse with ALF model was induced by lipopolysaccharide (LPS)/ D-galactosamine (D-GalN). After successful modelling, the liver tissues of all mice were obtained for transcriptome sequencing. The key immune-related genes in mice with ALF were identified by differential expression analysis, immune infiltration analysis, weighted gene co-expression network analysis (WGCNA), enrichment analysis, and protein-protein interaction (PPI) analysis. An LPS/D-GalN-induced ALF mouse model was successfully constructed, and transcriptome sequencing was performed. Significant differences in the proportions of monocytes, macrophages M0, macrophages M1 and neutrophils were shown by immune infiltration analysis, and 5255 genes highly associated with these four immune cells were identified by WGCNA. These immune genes were found to be significantly enriched in the TNF signalling pathway by enrichment analysis. Finally, PPI analysis was performed on genes enriched in this pathway and three key genes (CXCL1, CXCL10 and IL1B) were screened out and revealed to be significantly upregulated in ALF. Key immune-related genes in ALF were identified in this study, which may provide not only potential therapeutic targets for treating ALF and improving its prognosis, but also a reliable scientific basis for the immunotherapy of the disease.

Liposome cocktail activator modulates hepatocytes and remodels the microenvironment to mitigate acute liver failure

Acute liver failure (ALF) is a mortal and critical hepatic disease, in which oxidative stress, inflammation storm and hepatocyte death are crucial in the pathogenesis. Hence, in contrast to the control of a single link, a combination therapy targeting multiple pathogenic links of the disease will be a favorable means to control the progression of the disease. In this study, we constructed dimethyl itaconate-loaded liposomes modified with dodecyl gallate as a cocktail activator to investigate its functional role in acetaminophen (APAP)-induced ALF. Our results demonstrated that the cocktail activator acted on hepatocytes and triggered cocktail efficacy, thereby simultaneously attenuating APAP-induced hepatocyte damage and remodeling the damage microenvironment. The cocktail activator could effectively scavenge reactive oxygen species, inhibit excessive inflammatory responses and reduce cell death in impaired hepatocytes for detoxification. More importantly, the cocktail activator could remodel the damage microenvironment, thus further promoting hepatocyte expansion and specifically switching macrophages from the M1 to M2 phenotype for a favorable liver regeneration of ALF. Furthermore, in APAP-induced ALF mouse model, the cocktail activator improved liver function, alleviated histopathological damage and increased survival rate. In summary, these findings indicate that the cocktail activator may provide a promising therapeutic approach for ALF treatment as a nanomedicine.

Inhibition of inflammatory liver injury by the HMGB1-A box through HMGB1/TLR-4/NF-κB signaling in an acute liver failure mouse model.

We aimed to investigate the preventive effect of high mobility group box 1 (HMGB1)-A box and the mechanism by which it alleviates inflammatory injury in acute liver failure (ALF) by inhibiting the extracellular release of HMGB1. BALB/c mice were intraperitoneally (i.p.) administered LPS/D-GalN to establish an ALF mouse model. HMGB1-A box was administered (i.p.) 1 h before establishing the ALF mouse model. The levels of extracellularly released HMGB1, TLR-4/NF-κB signaling molecules, the proinflammatory cytokines TNF-α, IL-1β, and IL-6 and COX-2 were measured in the liver tissue and/or serum by Immunohistochemistry, Western blotting and Enzyme-linked immunosorbent assay (ELISA). The levels of extracellularly released HMGB1, TLR-4/NF-κB signaling molecules and proinflammatory cytokines were measured in Huh7 cells as well as LPS- and/or HMGB1-A box treatment by confocal microscopy, Western blotting and ELISA. In the ALF mouse model, the levels of HMGB1 were significantly increased both in the liver and serum, TLR-4/NF-κB signaling molecules and proinflammatory cytokines also was upregulated. Notably, HMGB1-A box could reverse these changes. HMGB1-A box could also cause these changes in LPS-induced Huh7 cells. HMGB1-A box played a protective role by inhibiting inflammatory liver injury via the regulation of HMGB1/TLR-4/NF-κB signaling in the LPS/D-GaIN-induced ALF mouse model, which may be related to inhibiting the extracellular release of HMGB1.

Dynamics of Acute Liver Injury in Experimental Models of Hepatotoxicity in the Context of Their Implementation in Preclinical Studies on Stem Cell Therapy.

Experimental models using carbon tetrachloride (CCl4) and D-galactosamine (D-GalN) can be used in preclinical assessment of acute liver failure (ALF) therapies. Unfortunately, these models are characterized by different dynamics of liver injury depending on the animal strain, administered hepatotoxin, and its dose. The aim of this study was to compare known rat and mouse models of ALF with a view to their future introduction into preclinical cell therapy experiments. In particular, based on histopathological and molecular changes, we suggested experimental time cut-off points for an effective stem cell therapeutic intervention. ALF was induced by a single intraperitoneal injection of CCl4 in mice (50 μL/100 g b.w.) and rats (200 μL/100 g b.w.) and D-GalN in mice (150 mg/100 g b.w.) and rats (50 mg/100 g b.w.). Blood and liver samples were collected 12 h, 24 h, 48 h and 7 days after intoxication. Blood morphology, liver function blood tests, histopathological changes, proliferation activity, apoptosis, fibrosis, and gene expression were analysed to assess liver damage. At 12 h, 24 h, and 48 h after CCl4 injection, mouse livers showed moderate inflammatory infiltration and massive pericentral necrosis. In rats treated with CCl4, minor lymphocytic infiltration in the liver parenchyma was seen at 12 h, followed by necrosis that appeared around central veins at 24 h and persisted to 48 h. In D-GalN-injected mice, the first histopathological signs of liver injury appeared at 48 h. In the livers of D-GalN-treated rats, moderate pericentral inflammatory infiltration occurred after 12 h, 24 h, and 48 h, accompanied by increased proliferation and apoptosis. All histological changes were accompanied by decreasing expression of certain genes. In most experimental groups of rats and mice, both histological and molecular parameters returned to the baseline values between 48 h and 7 days after intoxication. In mice and rats with CCl4-induced ALF, signs of liver failure can be seen as early as 12 h and develop to 48 h. In the D-GalN-induced model, mice are more resistant to the hepatotoxic effect than rats (after 12 h), and the early hepatitis phase can be observed much later, after 48 h. These cut-off points seem to be optimal for suppressing inflammation and applying effective stem cell therapy for acute liver injury.

Mesenchymal stem cells derived from different perinatal tissues donated by same donors manifest variant performance on the acute liver failure model in mouse

BackgroundMesenchymal stem cells (MSCs) derived from different tissues have variant biological characteristics, which may induce different performances in the treatment of diseases. At present, it is difficult to know which type of MSC is most suitable for acute liver failure (ALF), and there is no parallel study to compare MSCs from different tissues of the same donor.MethodsIn this study, we derived MSCs from three different perinatal tissues of the same donor: cord lining (CL), cord–placenta junction (CPJ) and fetal placenta (FP), respectively, for compared gene expression profiles by transcriptome sequencing, and ability of proliferation and immune regulation in vitro. In addition, the therapeutic effects (e.g., survival rate, histological evaluation, biochemical analysis) of CL-MSCs, FP-MSCs and CPJ-MSCs on ALF mouse model were compared.ResultsThe transcriptome analysis showed that FP-MSCs have significantly high expression of chemokines compared to CPJ-MSCs and CL-MSCs, similar to the q-PCR result. Of note, we found that CPJ-MSCs and FP-MSCs could improve the survival rate of mice with ALF induced by carbon tetrachloride, but CL-MSCs had no difference with Sham group. Moreover, we also found that biomarkers of ALF (e.g., MDA, SOD and GSH-px) significantly improved post-CPJ-MSCs and FP-MSCs treatment, but not CL-MSCs and Sham group. However, CL-MSCs treatment leads to inflammatory reaction in the early stage (day 3) of ALF treatment but not found with other groups.ConclusionsIt is important to select the MSCs derived from different tissues with variant performance for therapeutic purpose, and the CPJ-MSCs and FP-MSCs cells can significantly improve the syndrome of ALF which is highly recommended for a potential therapeutic options for ALF.

Noncanonical Wnt5a/JNK Signaling Contributes to the Development of D-Gal/LPS-Induced Acute Liver Failure.

Acute liver failure (ALF) is a deadly clinical disorder with few effective treatments and unclear pathogenesis. In our previous study, we demonstrated that aberrant Wnt5a expression was involved in acute-on-chronic liver failure. However, the role of Wnt5a in ALF is unknown. We investigated the expression of Wnt5a and its downstream c-Jun N-terminal kinase (JNK) signaling in a mouse model of ALF established by coinjection of D-galactosamine (D-Gal) and lipopolysaccharide (LPS) in C57BL/6 mice. We also investigated the role of Box5, a Wnt5a antagonist, in vivo. Moreover, the effect of Wnt5a/JNK signaling on downstream inflammatory cytokine expression, phagocytosis, and migration in THP-1 macrophages was studied in vitro. Aberrant Wnt5a expression and JNK activation were detected in D-Gal/LPS-induced ALF mice. Box5 pretreatment reversed JNK activation and eventually decreased the mortality rate of D-Gal/LPS-treated mice, with reduced hepatic necrosis and apoptosis, serum ALT and AST levels, and liver inflammatory cytokine expression, although the latter was not significant. We further demonstrated that recombinant Wnt5a (rWnt5a)-induced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA expression and increased THP-1 macrophage phagocytosis in a JNK-dependent manner, which could be restored by Box5. In addition, rWnt5a-induced migration of THP-1 macrophages was also reversed by Box5. Our findings suggested that Wnt5a/JNK signaling plays an important role in the development of ALF and that Box5 could have particular hepatoprotective effects in ALF.

Naringenin affords protection against lipopolysaccharide/D-galactosamine-induced acute liver failure: Role of autophagy

Acute liver failure (ALF) is considered a fatal clinical disorder and novel therapeutic interventions are mandatory. Naringenin is a flavonoid with anti-inflammatory, antioxidant and antiapoptotic effects that have displayed beneficial effects in different animal models of ALF. The current study aimed at investigating the hepatoprotective effect and the possible underlying molecular mechanisms of naringenin in lipopolysaccharide (LPS)/D-galactosamine (D-Gal) mouse model of ALF. Interestingly, naringenin pretreatment substantially alleviated LPS/D-Gal-induced liver injury, enhanced survival, improved liver function and ameliorated histopathological liver changes. Importantly, naringenin potently activated autophagy as evidenced by the increased Beclin-1 expression and LC3 II/LC3 I ratio. Furthermore, results demonstrated that naringenin alleviated oxidative stress by inducing nuclear factor-erythroid 2-related factor 2 (Nrf2) and increasing hepatic SOD activity and GSH level as well as ameliorated endoplasmic reticulum (ER) stress. Likewise, naringenin mitigated LPS/D-Gal-triggered inflammation by suppressing NF-κB and NLRP3 pathways. Accordingly, apoptotic cell death provoked by LPS/D-Gal challenge was markedly attenuated as depicted by the decrease in caspase-3 and p53 in naringenin-treated mice. To investigate the contribution of autophagy to naringenin-conferred hepatoprotection, autophagy was inhibited using 3-methyladenine (3 MA). Strikingly, 3 MA co-treatment abolished the hepatoprotective effect of naringenin, a finding that strongly suggests that naringenin-afforded protection is, at least in part, attributed to autophagy. Taken together, the present study revealed that naringenin exerted a prominent hepatoprotective effect by promoting autophagy with consequent attenuation of inflammatory responses, oxidative stress, ER stress and apoptosis. Our results provide evidence that naringenin use holds a promise as a potential therapeutic agent for ALF management.

Oyster-Derived Tyr-Ala (YA) Peptide Prevents Lipopolysaccharide/D-Galactosamine-Induced Acute Liver Failure by Suppressing Inflammatory, Apoptotic, Ferroptotic, and Pyroptotic Signals.

Models created by the intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (D-GalN) have been widely used to study the pathogenesis of human acute liver failure (ALF) and drug development. Our previous study reported that oyster (Crassostrea gigas) hydrolysate (OH) had a hepatoprotective effect in LPS/D-GalN-injected mice. This study was performed to identify the hepatoprotective effect of the tyrosine-alanine (YA) peptide, the main component of OH, in a LPS/D-GalN-injected ALF mice model. We analyzed the effect of YA on previously known mechanisms of hepatocellular injury in the model. LPS/D-GalN-injected mice showed inflammatory, apoptotic, ferroptotic, and pyroptotic liver injury. The pre-administration of YA (10 mg/kg or 50 mg/kg) significantly reduced the liver damage factors. The hepatoprotective effect of YA was higher in the 50 mg/kg YA pre-administered group than in the 10 mg/kg YA pre-administered group. These results showed that YA had a hepatoprotective effect by reducing inflammation, apoptosis, ferroptosis, and pyroptosis in the LPS/D-GalN-injected ALF mouse model. We suggest that YA can be used as a functional peptide for the prevention of acute liver injury.

Distinct effects of different doses of kaempferol onD‑GalN/LPS‑induced ALF depend on the autophagy pathway.

Kaempferol, a flavonoid compound, has various biological functions, such as anti-inflammatory and antitumor activities. Acute liver failure (ALF) is a lethal clinical syndrome that occurs due to severe damage of the liver function. In the present study, the mechanisms underlying the therapeutic effects of kaempferol in ALF were evaluated. An ALF mouse model was established using D-galactosamine (D-GalN; 700 mg/kg)/lipopolysaccharide (LPS; 10 µg/kg). A total of 2 h before the administration of D-GalN/LPS, mice were pretreated with different doses of kaempferol (2.5, 5, 10, 20 and 40 mg/kg), and 6 h after injection of D-GalN/LPS, mice were euthanized. The survival rate, liver function and levels of inflammatory cytokines were assessed. The results demonstrated that kaempferol pretreatment protected hepatocytes from ALF induced by D-GalN/LPS via regulation of the autophagy pathway, both in vivo and in vitro. Pretreatment with a high dose of kaempferol significantly decreased the survival rates and increased severe liver damage; however, pretreatment with a low dose of kaempferol had the opposite effect. Furthermore, pretreatment with a high dose of kaempferol enhanced the levels of proinflammatory cytokines [TNF-α, IL-6, IL-12p40, IL-1β, C-X-C motif chemokine ligand (CXCL)-2, CXCL-10] and markers of the MAPK signaling pathway [phosphorylated (p)-JNK, p-ERK, p-p38], whereas pretreatment with a low dose of kaempferol had the opposite effect. Pretreatment with a high dose of kaempferol decreased autophagy, whereas pretreatment with a low dose of kaempferol increased autophagy in vivo and in vitro. It was also shown that pretreatment with 3-methyadenine or autophagy related 7 small interfering RNA, to inhibit autophagy, partially abrogated the hepatoprotective effects of pretreatment with 5 mg/kg kaempferol in the ALF mouse model. These results demonstrate that the effects of different doses of kaempferol on D-GalN/LPS-induced ALF varies based on the dose, and that kaempferol exerted its effects via regulation of the autophagy pathway.

Exosomes derived from human umbilical cord mesenchymal stem cells alleviate acetaminophen-induced acute liver failure through activating ERK and IGF-1R/PI3K/AKT signaling pathway

This study aimed to investigate the therapeutic potential of human umbilical cord mesenchymal stem cells derived exosomes (hUCMSC-Exo) in acute liver failure (ALF) in mice as well as its underlying mechanism. We found that a single tail vein administration of hucMSC-Exo effectively enhanced the survival rate, inhibited apoptosis in hepatocytes, and improved liver function in APAP-induced mouse model of ALF. Furthermore, the deletion of glutathione (GSH) and superoxide dismutase (SOD), generation of malondialdehyde (MDA), and the over production of cytochrome P450 E1 (CYP2E1) and 4-hydroxynonenal (4-HNE) caused by APAP were also inhibited by hucMSC-Exo, indicating that hucMSC-Exo inhibited APAP-induced apoptosis of hepatocytes by reducing oxidative stress. Moreover, hucMSC-Exo significantly down-regulated the levels of inflammatory cytokines IL-6, IL-1β, and TNF-α in APAP-treated livers. Western blot showed that hucMSC-Exo significantly promoted the activation of ERK1/2 and IGF-1R/PI3K/AKT signaling pathways in APAP-injured LO2 cells, resulting in the inhibition of apoptosis of LO2 cells. Importantly, PI3K inhibitor LY294002 and ERK1/2 inhibitor PD98059 could reverse the function of hucMSC-Exo on APAP-injured LO2 cells in some extent. Our results suggest that hucMSC-Exo offer antioxidant hepatoprotection against APAP in vitro and in vivo by inhibitiing oxidative stress-induced apoptosis via upregulation of ERK1/2 and PI3K/AKT signaling pathways.

Verapamil inhibits early acute liver failure through suppressing the NLRP3 inflammasome pathway.

Acute liver failure (ALF) is a rare disease characterized by the sudden onset of serious hepatic injury, as manifested by a profound liver dysfunction and hepatic encephalopathy in patients without prior liver disease. In this paper, we aim to investigate whether verapamil, an antagonist of TXNIP, inhibits early ALF through suppressing the NLRP3 inflammasome pathway. Firstly, an ALF mouse model was induced by lipopolysaccharide (LPS)/D‐galactosamine (GalN) treatment. The optimal concentration of verapamil in treating early ALF mice was determined followed by investigation on its mechanism in LPS/GalN‐induced liver injury. Western blot analysis and co‐immunoprecipitation were performed to determine the activation of the TXNIP/NLRP3 inflammasome pathway. Subsequently, overexpression of NLRP3 in mouse liver was induced by transfection with AAV‐NRLP3 in vivo and in vitro to identity whether verapamil inhibited early ALF through suppressing the activation of NLRP3 inflammasome. We found that ALF was induced by LPS/GalN in mice but was alleviated by verapamil through a mechanism that correlated with suppression of the NLRP3 inflammasome pathway. Oxidative stress and inflammatory response were induced by intraperitoneal injection of LPS/GalN, but alleviated with injection of verapamil. Overexpression of NLRP3 via AAV in mouse liver in vivo and in vitro reduced the therapeutic effect of verapamil on LPS/GalN‐induced ALF. Taken together, the TXNIP antagonist verapamil could inhibit activation of the NLRP3 inflammasome, inflammatory responses and oxidative stress to alleviate LPS/GalN‐induced ALF.

Silencing long noncoding RNA NEAT1 alleviates acute liver failure via the EZH2-mediated microRNA-139/PUMA axis.

This study aimed to investigate the role of long noncoding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) in the development of ALF. We collected blood samples from patients with acute liver failure (ALF) and established an ALF mouse model induced by D-galactosamine/Lipopolysaccharide (D-GalN/LPS) for in vivo studies. Peripheral blood mononuclear cells (PMBCs) induced with LPS were isolated for in vitro experiments. Survival tests, histological analysis, and biochemical indicator assays were conducted. Luciferase assay was performed to determine the binding affinity between microRNA-139 (miR-139) and p53-upregulated modulator of apoptosis (PUMA). Expression of lncRNA NEAT1, enhancer of zeste homolog 2 (EZH2), and PUMA was upregulated, while the expression of miR-139 was downregulated in clinical samples and D-GalN/LPS induced ALF mouse model. LncRNA NEAT1 promoted the enrichment of H3K27me3 on the promoter region of miR-139 via EZH2, which led to suppression of miR-139. The inhibition of miR-139 resulted in the upregulation of its downstream target PUMA. The NEAT1/miR-139/PUMA pathway upregulated the production of pro-inflammatory cytokines, tumor necrosis factor alpha, interleukin (IL)-6, and IL-1β, thereby mediating the progression of ALF. In conclusion, silencing lncRNA NEAT1 upregulated the expression of miR-139 through EZH2, leading to the downregulation of PUMA, which alleviated the development of ALF.

Mahuang Decoction Antagonizes Acute Liver Failure via Modulating Tricarboxylic Acid Cycle and Amino Acids Metabolism.

Acute liver failure (ALF) is a serious clinical disorder with high fatality rates. Mahuang decoction (MHD), a well-known traditional Chinese medicine, has multiple pharmacological effects, such as anti-inflammation, anti-allergy, anti-asthma, and anti-hyperglycemia. In this study, we investigated the protective effect of MHD against ALF. In the lipopolysaccharide and D-galactosamine (LPS/D-GalN)-induced ALF mouse model, the elevated activities of the serum alanine and aspartate transaminases as well as the liver pathological damage were markedly alleviated by MHD. Subsequently, a metabolomics study based on the ultrahigh performance liquid chromatograph coupled with Q Exactive Orbitrap mass spectrometry was carried to clarify the therapeutic mechanisms of MHD against ALF. A total of 36 metabolites contributing to LPS/D-GalN-induced ALF were identified in the serum samples, among which the abnormalities of 27 metabolites were ameliorated by MHD. The analysis of metabolic pathways revealed that the therapeutic effects of MHD are likely due to the modulation of the metabolic disorders of tricarboxylic acid (TCA) cycle, retinol metabolism, tryptophan metabolism, arginine and proline metabolism, nicotinate and nicotinamide metabolism, phenylalanine metabolism, phenylalanine, tyrosine and tryptophan synthesis, as well as cysteine and methionine metabolism. This study demonstrated for the first time that MHD exerted an obvious protective effect against ALF mainly through the regulation of TCA cycle and amino acid metabolism, highlighting the importance of metabolomics to investigate the drug-targeted metabolic pathways.