Model Of Fulminant Hepatic Failure Research Articles

C–C chemokine receptor 5 is essential for conventional NK cell trafficking and liver injury in a murine hepatitis virus-induced fulminant hepatic failure model

BackgroundPrevious studies have demonstrated that natural killer (NK) cells migrated into the liver from peripheral organs and exerted cytotoxic effects on hepatocytes in virus-induced liver failure.AimThis study aimed to investigate the potential therapeutic role of chemokine receptors in the migration of NK cells in a murine hepatitis virus strain 3 (MHV-3)-induced fulminant hepatic failure (MHV-3-FHF) model and its mechanism.ResultsBy gene array analysis, chemokine (C–C motif) receptor 5 (CCR5) was found to have remarkably elevated expression levels in hepatic NK cells after MHV-3 infection. The number of hepatic CCR5+ conventional NK (cNK) cells increased and peaked at 48 h after MHV-3 infection, while the number of hepatic resident NK (rNK) cells steadily declined. Moreover, the expression of CCR5-related chemokines, including macrophage inflammatory protein (MIP)-1α, MIP-1β and regulated on activation, normal T-cell expressed and secreted (RANTES) was significantly upregulated in MHV-3-infected hepatocytes. In an in vitro Transwell migration assay, CCR5-blocked splenic cNK cells showed decreased migration towards MHV-3-infected hepatocytes, and inhibition of MIP-1β or RANTES but not MIP-1α decreased cNK cell migration. Moreover, CCR5 knockout (KO) mice displayed reduced infiltration of hepatic cNK cells after MHV-3 infection, accompanied by attenuated liver injury and improved mouse survival time. Adoptive transfer of cNK cells from wild-type mice into CCR5 KO mice resulted in the abundant accumulation of hepatic cNK cells and aggravated liver injury. Moreover, pharmacological inhibition of CCR5 by maraviroc reduced cNK cell infiltration in the liver and liver injury in the MHV-3-FHF model.ConclusionThe CCR5-MIP-1β/RANTES axis played a critical role in the recruitment of cNK cells to the liver during MHV-3-induced liver injury. Targeted inhibition of CCR5 provides a therapeutic approach to ameliorate liver damage during virus-induced acute liver injury.

Dietary exposure to polystyrene microplastics exacerbates liver damage in fulminant hepatic failure via ROS production and neutrophil extracellular trap formation

With the increasing influx of microplastics (MPs) into the environment, their potential toxicity represents an increasing threat to human health. However, there is a lack of relevant research surrounding the biological toxicity associated with pre-exposure to MPs under pathological conditions. To fill this gap, we established a mouse model of fulminant hepatic failure after 14 days of pre-exposure to polystyrene (PS) MPs and investigated its biological response process under combined stimulation with lipopolysaccharide (LPS)/d-galactosamine (d-GalN) and PS-MPs. The results indicated that the stress response from exposure to PS-MPs exacerbated the death induced by LPS/d-GalN and reinforced the potential of liver damage in mice. The dominant roles of inflammation promotion, reactive oxygen species (ROS), and neutrophil extracellular traps in this process were confirmed by cellular reactive oxygen species assays and experiments on oxidative stress and inflammatory responses in the liver. Transcriptomic analysis revealed that PS-MPs exacerbated the expression levels of neutrophil extracellular traps in mice treated with LPS/d-GalN, and weakened the expression of genes involved in pathways related to peroxisome, taurine, and hypotaurine metabolism, which was further validated by reverse-transcription quantitative polymerase chain reaction. This study addresses the knowledge gap regarding the adverse effects caused by a pathological state upon exposure to MPs and provides a theoretical reference for further assessment of the underlying health risks of MPs.

Inhibition of EZH2 ameliorates bacteria-induced liver injury by repressing RUNX1 in dendritic cells

Fulminant hepatic failure (FHF) is a clinical syndrome characterized by a sudden and severe impairment in liver function. However, the precise mechanism of immune dysregulation that is significant to FHF pathogenesis remains unclear. Enhancer of zeste homolog 2 (EZH2) has been implicated in inflammation as a regulator of immune cell function. In this study, we investigated the role of EZH2 in an animal model of human FHF induced by Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS). We demonstrated that EZH2 depletion in dendritic cells (DCs) and pharmacological inhibition of EZH2 using GSK126 both significantly ameliorated liver injury and improved the survival rates of mice with P. acnes plus LPS-induced FHF, which could be attributed to the decreased infiltration and activation of CD4+ T cells in the liver, inhibition of T helper 1 cells and induction of regulatory T cells. The expression of EZH2 in DCs was increased after P. acnes administration, and EZH2 deficiency in DCs suppressed DC maturation and prevented DCs from efficiently stimulating CD4+ T-cell proliferation. Further mechanistic analyses indicated that EZH2 deficiency directly increased the expression of the transcription factor RUNX1 and thereby suppressed the immune functions of DCs. The functional dependence of EZH2 on RUNX1 was further illustrated in DC-specific Ezh2-deficient mice. Taken together, our findings establish that EZH2 exhibits anti-inflammatory effects through inhibition of RUNX1 to regulate DC functions and that inhibition of EZH2 alleviates P. acnes plus LPS-induced FHF, probably by inhibiting DC-induced adaptive immune responses. These results highlight the effect of EZH2 on DCs, serving as a guide for the development of a promising immunotherapeutic strategy for FHF.

Melatonin modulates mitophagy, innate immunity and circadian clocks in a model of viral-induced fulminant hepatic failure.

The haemorrhagic disease virus (RHDV) is a non‐cultivable virus that promotes in rabbits an acute disease which accomplishes many characteristics of an animal model of fulminant hepatic failure (FHF). Beneficial effects of melatonin have been reported in RHDV‐infected rabbits. This study investigated whether protection against viral‐derived liver injury by melatonin is associated with modulation of mitophagy, innate immunity and clock signalling. Rabbits were experimentally infected with 2 × 104 haemagglutination units of a RHDV isolate and killed at 18, 24 and 30 hours after infection (hpi). Melatonin (20 mg/kg body weight ip) was administered at 0, 12 and 24 hpi. RHDV infection induced mitophagy, with the presence of a high number of mitophagosomes in hepatocytes and increased expression of mitophagy genes. Greater expression of main innate immune intermediaries and inflammasome components was also found in livers with RHDV‐induced FHF. Both mitophagy and innate immunity activation was significantly hindered by melatonin. FHF induction also elicited an early dysregulation in clock signalling, and melatonin was able to prevent such circadian disruption. Our study discloses novel molecular routes contributing to RHDV‐induced damage progression and supports the potential of melatonin as a promising therapeutic option in human FHF.

Up‐regulation of microRNA‐30b/30d cluster represses hepatocyte apoptosis in mice with fulminant hepatic failure by inhibiting CEACAM1

Recently, impacts of microRNAs have been unraveled in human diseases, and we aimed to confirm the role of miR-30b/30d in fulminant hepatic failure (FHF). Expression of miR-30b/30d and CEACAM1 in serum of FHF patients and healthy people was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. Mice FHF models were established by injection of D-Galn and lipopolysaccharide, and were treated with miR-30b/30d mimics. Oxidative stress, liver injury, and inflammatory reaction in mouse liver tissues were measured using oxidative stress-related factor kits, hematoxylin-eosin staining and enzyme-linked immunosorbent assay, respectively. Moreover, cell cycle distribution and apoptosis of hepatocytes of mice were determined by flow cytometry, and the target relation between miR-30b/30d and CEACAM1 was confirmed by bioinformatic method and dual luciferase reporter gene assay. MiR-30b/30d expression was positively, and CEACAM1 expression was negatively related to prognosis of FHF patients. Up-regulation of miR-30b/30d attenuated oxidative stress, liver injury, and inflammatory reaction, and improved survival rate of FHF mice. Furthermore, elevated miR-30b/30d ameliorated apoptosis and cell cycle arrest of hepatocytes of FHF mice. CEACAM1 was a target gene of miR-30b/30d. This study highlights that up-regulated miR-30b/30d attenuates the progression of FHF by targeting CEACAM1, which may be helpful to FHF treatment.

Intraportally delivered stem cell spheroids localize in the liver and protect hepatocytes against GalN/LPS-induced fulminant hepatic toxicity

BackgroundSystemic inflammatory response syndrome (SIRS) is common in severe fulminant hepatic failure (FHF) and has a high mortality rate (20–50%) due to irreversible cerebral edema or sepsis. Stem cell-based treatment has emerged as a promising alternative therapeutic strategy to prolong the survival of patients suffering from FHF via the inhibition of SIRS due to their immunomodulatory effects.Methods3D spheroids of adipose-derived mesenchymal stem cells (3D-ADSC) were prepared by the hanging drop method. The efficacy of the 3D-ADSC to rescue FHF was evaluated in a d-galactosamine/lipopolysaccharide (GalN/LPS)-induced mouse model of FHF via intraportal transplantation of the spheroids.ResultsIntraportally delivered 3D-ADSC better engrafted and localized into the damaged livers compared to 2D-cultured adipose-derived mesenchymal stem cells (2D-ADSC). Transplantation of 3D-ADSC rescued 50% of mice from FHF-induced lethality, whereas only 20% of mice survived when 2D-ADSC were transplanted. The improved transplantation outcomes correlated with the enhanced immunomodulatory effect of 3D-ADSC in the liver microenvironment.ConclusionThe study shows that the transplantation of optimized 3D-ADSC can efficiently ameliorate GalN/LPS-induced FHF due to improved viability, resistance to exogenous ROS, and enhanced immunomodulatory effects of 3D-ADSC.

Lysophosphatidic acid receptor, LPA6, regulates endothelial blood-brain barrier function: Implication for hepatic encephalopathy

Cerebral edema is a life-threatening neurological condition characterized by brain swelling due to the accumulation of excess fluid both intracellularly and extracellularly. Fulminant hepatic failure (FHF) develops cerebral edema by disrupting blood-brain barrier (BBB). However, the mechanisms by which mediator induces brain edema in FHF remain to be elucidated. Here, we assessed a linkage between brain edema and lysophosphatidic acid (LPA) signaling by utilizing an animal model of FHF and in vitro BBB model. Azoxymethane-treated mice developed FHF and hepatic encephalopathy, associated with higher autotaxin (ATX) activities in serum than controls. Using in vitro BBB model, LPA disrupted the structural integrity of tight junction proteins including claudin-5, occludin, and ZO-1. Furthermore, LPA decreased transendothelial electrical resistances in in vitro BBB model, and induced cell contraction in brain endothelial monolayer cultures, both being inhibited by a Rho-associated protein kinase inhibitor, Y-27632. The brain capillary endothelial cells predominantly expressed LPA6 mRNA, whose knockdown blocked the LPA-induced endothelial cell contraction. Taken together, the up-regulation of serum ATX in hepatic encephalopathy may activate the LPA–LPA6–G12/13–Rho pathway in brain capillary endothelial cells, leading to enhancement of BBB permeability and brain edema.

Overexpression of C‑sis inhibits H2O2‑induced Buffalo rat liver cell apoptosis invitro and alleviates liver injury in a rat model of fulminant hepatic failure.

The present study aimed to investigate the role of the C-sis gene in the apoptosis of hepatocytes in vitro and in the liver function of a rat model of fulminant hepatic failure (FHF). Buffalo rat liver (BRL) cells were treated with hydrogen peroxide (H2O2) to induce apoptosis and then transfected with a C-sis overexpression vector. A rat model of FHF was established, and C-sis was overexpressed. The mRNA and protein expression of C-sis were examined using reverse transcription-polymerase chain reaction and western blot analyses, respectively. Cell viability was assessed by CCK8, and a TUNEL assay was used to examine cell apoptosis. Flow cytometry was used for cell cycle detection. Hematoxylin and eosin staining was used for histological examination. The levels of alanine transaminase (ALT) and aspartate transaminase (AST) were also examined in the rats. The results showed that C-sis was successfully overexpressed in the cells and rat model. Compared with H2O2-treated BRL cells, the overexpression of C-sis significantly inhibited cell apoptosis, promoted cell viability, and decreased the expression of cleaved caspase-3. Similar results were observed in the FHF rats treated with the C-sis overexpression plasmid, compared with those treated with empty plasmids. In addition, in the FHF rats overexpressing C-sis, histological examination showed that liver injury was alleviated, the levels of ALT and AST were significantly decreased, and mortality rate was significantly decreased, compared with those observed in the rats treated with empty plasmids. In conclusion, the overexpression of C-sis inhibited the H2O2-induced apoptosis of BRL cells in vitro, and alleviated liver injury, improved liver function, and decreased mortality rates in rat models of FHF.

Alleviation of lipopolysaccharide/d-galactosamine-induced liver injury in leukocyte cell-derived chemotaxin 2 deficient mice

Leukocyte cell-derived chemotaxin 2 (LECT2) is a secreted pleiotropic protein that is mainly produced by the liver. We have previously shown that LECT2 plays an important role in the pathogenesis of inflammatory liver diseases. Lipopolysaccharide/d-galactosamine (LPS/d-GalN)-induced acute liver injury is a known animal model of fulminant hepatic failure. Here we found that this hepatic injury was alleviated in LECT2-deficient mice. The levels of TNF-α and IFN-γ, which mediate this hepatitis, had significantly decreased in these mice, with the decrease in IFN-γ production notably greater than that in TNF-α. We therefore analyzed IFN-γ-producing cells in liver mononuclear cells. Flow cytometric analysis showed significantly reduced IFN-γ production in hepatic NK and NKT cells in LECT2-deficient mice compared with in wild-type mice. We also demonstrated a decrease in IFN-γ production in LECT2-deficient mice after systemic administration of recombinant IL-12, which is known to induce IFN-γ in NK and NKT cells. These results indicate that a decrease of IFN-γ production in NK and NKT cells was involved in the alleviation of LPS/d-GalN-induced liver injury in LECT2-deficient mice.

Expression of IL-1Ra, IL-6, IL-8, IL-18, TNF-α and IFN-γ genes in peripheral blood leukocytes of rabbits infected with RHDV (Rabbit Haemorrhagic Disease Virus)

Rabbit haemorrhagic disease virus (RHDV) induces a highly contagious and extremely lethal disease that fulfils many requirements of an animal model of fulminant hepatic failure (FHF); however, the pathogenesis of RHD has still not been fully elucidated. Cytokines play an important role in regulation of the immune response and pathogenesis of many diseases, including those caused by viral infections. Furthermore, recent studies indicate a role of the immune response, especially peripheral blood leukocytes (PBL), in the pathogenesis of RHD. Thus, in the present study we investigated the expression of IL-1Ra, IL-6, IL-8, IL-18, TNF-α and IFN-γ genes in PBL of RHDV-infected rabbits. We also compared the expression of genes encoding these cytokines in rabbits with different course of RHDV infection (in animals that died 36 h post infection or survived even over 60 h after infection). The study revealed increased expression of genes encoding pro-inflammatory cytokines: IL-6, IL-8, TNF-α, IFN-γ in PBL of RHDV-infected rabbits. Moreover, the level of cytokine gene expression depended on the course of RHD. Hence, the results obtained indicate the potential role of these cytokines in RHDV infection and their influence on the survival time of infected rabbits.

Immunomodulatory Effects of Adipose Tissue-Derived Stem Cells on Concanavalin A-Induced Acute Liver Injury in Mice.

Cell therapy with adipose tissue-derived stem cells (ASCs) is expected to be a candidate for the treatment of fulminant hepatic failure (FHF), which is caused by excessive immune responses. In order to evaluate the therapeutic effects of ASCs on FHF, the in vitro and in vivo immunomodulatory effects of ASCs were examined in detail in the mouse model. The in vitro effects of ASCs were examined by assessing their influence on the proliferation of lymphomononuclear cells (LMCs) stimulated with three kinds of mitogens: phorbol 12-myristate 13-acetate (PMA) plus ionomycin, concanavalin A (ConA), and lipopolysaccharide (LPS). The proliferation of LMCs was efficiently suppressed in a dose-dependent manner by ASCs in the cases of PMA plus ionomycin stimulation and ConA stimulation, but not in the case of LPS stimulation. The in vivo effects of transplanted ASCs were examined in the murine FHF model induced by ConA administration. The ALT levels and histological inflammatory changes in the ConA-administered mice were apparently relieved by the transplantation of ASCs. The analysis of mRNA expression patterns in the livers indicated that the expressions of the cytokines such as Il-6, Il-10, Ifn-γ, and Tnf-α, and the cell surface markers such as Cd3γ, Cd4, Cd8α, Cd11b, and Cd11c were downregulated in the ASC-transplanted mice. The immunomodulatory and therapeutic effects of ASCs were confirmed in the mouse model both in vitro and in vivo. These suggest that the cell therapy with ASCs is beneficial for the treatment of FHF.

Taurine treatment preserves brain and liver mitochondrial function in a rat model of fulminant hepatic failure and hyperammonemia.

Ammonia-induced mitochondrial dysfunction and energy crisis is known as a critical consequence of hepatic encephalopathy (HE). Hence, mitochondria are potential targets of therapy in HE. The current investigation was designed to evaluate the role of taurine treatment on the brain and liver mitochondrial function in a rat model of hepatic encephalopathy and hyperammonemia. The animals received thioacetamide (400mg/kg, i.p, for three consecutive days at 24-h intervals) as a model of acute liver failure and hyperammonemia. Several biochemical parameters were investigated in the serum, while the animals' cognitive function and locomotor activity were monitored. Mitochondria was isolated from the rats' brain and liver and several indices were assessed in isolated mitochondria. Liver failure led to cognitive dysfunction and impairment in locomotor activity in the rats. Plasma and brain ammonia was high and serum markers of liver injury were drastically elevated in the thioacetamide-treated group. An assessment of brain and liver mitochondrial function in the thioacetamide-treated animals revealed an inhibition of succinate dehydrogenase activity (SDA), collapsed mitochondrial membrane potential, mitochondrial swelling, and increased reactive oxygen species (ROS). Furthermore, a significant decrease in mitochondrial ATP was detected in the brain and liver mitochondria isolated from thioacetamide-treated animals. Taurine treatment (250, 500, and 1000mg/kg) decreased mitochondrial swelling, ROS, and LPO. Moreover, the administration of this amino acid restored brain and liver mitochondrial ATP. These data suggest taurine to be a potential protective agent with therapeutic capability against hepatic encephalopathy and hyperammonemia-induced mitochondrial dysfunction and energy crisis.

Plumbagin protects liver against fulminant hepatic failure and chronic liver fibrosis via inhibiting inflammation and collagen production.

Plumbagin is a quinonoid constituent extracted from Plumbago genus, and it exhibits diverse pharmacological effects. This study thoroughly investigated the effects of plumbagin on thioacetamide-induced acute and chronic liver injury. Results shown that plumbagin increased survival rate, reduced liver congestion and inflammation, and decreased macrophages and neutrophils in the fulminant hepatic failure model, and remarkably diminished liver fibrosis and inflammation in the chronic liver injury model. Furthermore, plumbagin significantly suppress the HSCs/myofibroblasts activation by reduced expression of markers α-SMA and COL-1/3, and reduced macrophage in liver. In the in vitro study, plumbagin induced apoptosis and suppressed the proliferation of LX-2 cells (human HSCs). Plumbagin treatment increased AMPK phosphorylation and attenuated NF-κB, STAT3, and Akt/mTOR signals in LX-2 cells, while SMAD2 phosphorylation was not changed. Noticeably, plumbagin promoted AMPK binding to p300 which is a cofactor of SMAD complex, this may further competitively decreases the p300/SMAD complex initiated transcription of COL-1/3 and α-SMA. Additionally, plumbagin hampered inflammation related NF-κB signal in RAW 264.7 cells. In conclusion, these findings indicate that plumbagin may be a powerful drug candidate to protect the liver from acute and chronic damage by inhibiting inflammation and collagen production.

CRISPR/Cas9 Technology Targeting Fas Gene Protects Mice From Concanavalin-A Induced Fulminant Hepatic Failure.

Fulminant hepatic failure is a life-threatening disease which occurs in patients without preexisting liver disease. Nowadays, there is no ideal therapeutic tool in the treatment of fulminant hepatic failure. Recent studies suggested that a novel technology termed CRISPR/Cas9 may be a promising approach for the treatment of fulminant hepatic failure. In this project, we have designed single chimeric guide RNAs specifically targeting the genomic regions of mouse Fas gene. The in vitro and in vivo effects of sgRNAs on the production of Fas protein were examined in cultured mouse cells and in a hydrodynamic injection-based mouse model, respectively. The in vivo delivery of CRISPR/Cas9 could maintain liver homeostasis and protect hepatocytes from Fas-mediated cell apoptosis in the fulminant hepatic failure model. Our study indicates the clinical potential of developing the CRISPR/Cas9 system as a novel therapeutic strategy to rescue Concanavalin-A-induced fulminant hepatic failure in the mouse model. J. Cell. Biochem. 118: 530-536, 2017. © 2016 Wiley Periodicals, Inc.

Mesenchymal stem cells and their secreted molecules predominantly ameliorate fulminant hepatic failure and chronic liver fibrosis in mice respectively.

BackgroundOrthotopic liver transplantation is the only effective treatment for liver failure but limited with shortage of available donor organs. Recent studies show promising results of mesenchymal stem cells (MSCs)-based therapies.MethodsWe systematically investigate the therapeutic effects of MSCs or MSC-conditioned medium (MSC-CM) in ameliorating fulminant hepatic failure (FHF) and chronic liver fibrosis in mice. In addition, extensive flow cytometry analysis of spleens from vehicle and MSC- and MSC-CM-treated mice was applied to reveal the alteration of inflammatory state.ResultsIn FHF model, MSCs treatment reduced remarkably the death incidents; the analysis of gross histopathology showed that control livers were soft and shrunken with extensive extravasated blood, which was gradually reduced at later time points, while MSC–treated livers showed gross pathological changes, even 24 h after MSC infusion, and hematoxylin and eosin staining revealed dramatical hepatocellular death with cytoplasmic vacuolization suppressed by MSCs treatment; flow cytometry analysis of total lymphocytes showed that macrophages (F4/80) infiltrated into control livers more than MSC-treated livers; by contrast, MSC-CM partially ameliorates FHF. In chronic liver injury model, MSC and MSC-CM both suppressed fibrogenesis and necroinflammatory, and the later was better; activation of hepatic stellate cells (α-SMA) was inhibited; glycogen synthesis and storage (indicated by periodic acid-Schiff -staining) was improved; liver regeneration (Ki67) was promoted while liver apoptosis (TUNEL) was reduced. In the in vitro, MSCs promote macrophage line RAW264.7 apoptosis and MSC-CM promotes apoptosis and inhibits proliferation of HSC line LX-2. We also found that MSCs and MSC-CM could improve spleen; MSC-CM increased levels of Th2 and Treg cells, and reduced levels of Th17 cells, whereas levels of Th1 cells were unchanged; comparatively, MSC treatment did not affect Th17 and Treg cells and only slightly alters inflammatory state; MSC and MSC-CM treatment both substantially down-regulated macrophages in the spleens.ConclusionBoth MSCs and MSC-CM exert therapeutic effects by acting on various key cells during the pathogenesis of FHF and chronic fibrosis, stimulating hepatocyte proliferation and suppressing apoptosis, down-regulating infiltrating macrophages, converting CD4+ T lymphocyte system into an anti-inflammatory state, and facilitating hepatic stellate cell death.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0792-1) contains supplementary material, which is available to authorized users.

Interleukin 17A plays a role in lipopolysaccharide/d-galactosamine–induced fulminant hepatic injury in mice

BackgroundLipopolysaccharide/d-galactosamine (LPS/GalN)–induced hepatic injury is an experimental model of fulminant hepatic failure in which tumor necrosis factor alpha (TNF-α) plays a pivotal role. Moreover, it was reported from our laboratory that interleukin (IL) 17A enhanced production of TNF-α by the Kupffer cell. ObjectiveThe purpose of this study was to determine the role of IL-17A in LPS/GalN-induced hepatic injury in mice. MethodsLPS/GalN was injected into three mouse models: wild-type (WT) mice, IL-17A knockout (KO) mice, or IL-17A KO mice treated with recombinant mouse (rm) IL-17A homodimer (KO + rmIL-17A). Survival was assessed for 24 h after LPS/GalN injection, and histopathologic findings were evaluated at various time points after LPS/GalN injection for neutrophil and apoptosis markers. After LPS/GalN injection, expression of the inflammatory mediators TNF-α, IL-6, monocyte chemotactic protein 1, IL-17A, high-mobility group box 1, and soluble intercellular adhesion molecule 1 was assessed in serum by enzyme-linked immunosorbent assay. ResultsSurvival was higher in KO mice compared with WT mice after LPS/GalN injection. However, in KO + rmIL-17A mice, mortality was not significantly different compared to the other groups. Neutrophil infiltration and apoptosis were significantly greater in WT mice than KO mice. Furthermore, serum alanine aminotransferase, serum TNF-α, monocyte chemotactic protein 1, IL-17A, high-mobility group box 1, and soluble intercellular adhesion molecule 1 levels were also significantly greater in WT mice than KO mice. In KO + rmIL-17A mice, these levels were similar to those in WT mice. ConclusionsIL-17A is a key regulator in hepatic injury caused by neutrophil-induced inflammatory responses after LPS/GalN injection.

Intestinal dendritic cells change in number in fulminant hepatic failure.

To investigate the change in intestinal dendritic cell (DC) number in fulminant hepatic failure (FHF). An animal model of FHF was created. Intestinal CD11b/c was detected by immunohistochemistry and Western blot. Quantitative real-time polymerase chain reaction (PCR) was used to detect intestinal integrin-α mRNA expression. Intestinal CD83, CD86, CD74, CD3 and AKT were detected by immunohistochemistry, Western blot and PCR. Phosphorylated-AKT (p-AKT) was detected by immunohistochemistry and Western blot. In the FHF group [D-galactosamine (D-Galn) + lipopolysaccharide (LPS) group], the mice began to die after 6 h; conversely, in the D-Galn and LPS groups, the activity of mice was poor, but there were no deaths. Immunohistochemistry results showed that in FHF, the expression of CD11b/c (7988400 ± 385941 vs 1102400 ± 132273, P < 0.05), CD83 (13875000 ± 467493 vs 9257600 ± 400364, P < 0.05), CD86 (7988400 ± 385941 vs 1102400 ± 13227, P < 0.05) and CD74 (11056000 ± 431427 vs 4633400 ± 267903, P < 0.05) was significantly increased compared with the normal saline (NS) group. Compared with the NS group, the protein expression of CD11b/c (5.4817 ± 0.77 vs 1.4073 ± 0.37, P < 0.05) and CD86 (4.2673 ± 0.69 vs 1.1379 ± 0.42, P < 0.05) was significantly increased. Itg-α (1.1224 ± 0.3 vs 0.4907 ± 0.19, P < 0.05), CD83 (3.6986 ± 0.40 vs 1.0762 ± 0.22, P < 0.05) and CD86 (1.5801 ± 0.32 vs 0.8846 ± 0.10, P < 0.05) mRNA expression was increased significantly in the FHF group. At the protein level, expression of CD74 in the FHF group (2.3513 ± 0.52) was significantly increased compared with the NS group (1.1298 ± 0.33), whereas in the LPS group (2.3891 ± 0.47), the level of CD74 was the highest (P < 0.05). At the gene level, the relative expression of CD74 mRNA in the FHF group (1.5383 ± 0.26) was also significantly increased in comparison to the NS group (0.7648 ± 0.22; P < 0.05). CD3 expression was the highest in the FHF group (P < 0.05). In the FHF, LPS and D-Galn groups, the expression of AKT at the protein and mRNA levels was elevated compared with the NS group, but there was no statistical significance (P > 0.05). The p-AKT protein expression in the FHF (1.54 ± 0.06), LPS (1.56 ± 0.05) and D-Galn (1.29 ± 0.03) groups was higher than that in the NS group (1.07 ± 0.03) (P < 0.05). In FHF, a large number of DCs mature, express CD86, and activate MHC class II molecular pathways to induce a T cell response, and the AKT pathway is activated.

Intraperitoneal Administration of Fetuin-A Attenuates d-Galactosamine/Lipopolysaccharide-Induced Liver Failure in Mouse

BackgroundFulminant hepatic failure (FHF) is a devastating syndrome, which sometimes results in death or liver transplantation, in which inflammation would aggravate the development of fetuin-A which would act as an anti-inflammatory factor and may be an available approach to attenuate FHF.AimsThe purpose of this study was to investigate the effects of fetuin-A on d-galactosamine/lipopolysaccharide (d-GalN/LPS)-induced liver failure in mice.MethodsA mouse model of FHF induced by d-GalN/LPS was established and fetuin-A was injected intraperitoneally prior to d-GalN/LPS treatment. At different time points after d-GalN/LPS intervention, serum TNF-α and IL-6 levels were measured by ELISA. Fetuin-A mRNA and protein expression in liver tissues was assessed by RT-PCR, Western blotting and immunohistochemical staining. Besides, an observation of liver tissue injury, the apoptosis of hepatocytes, was analyzed by TUNEL assay.ResultsExpression of fetuin-A mRNA and protein in liver tissue were significantly and gradually decreased after d-GalN/LPS administration. A pre-intervention of exogenous fetuin-A significantly improved the liver function, decreased TNF-α and IL-6 expression in peripheral blood, and liver tissue inhibited hepatocyte apoptosis responded to d-GalN/LPS induction so as to decrease the mortality rates of FHF mouse. Meanwhile, fetuin-A was negatively correlated with the hepatic pathological score and TNF-α protein staining in FHF mouse.ConclusionsAn intraperitoneal injection of fetuin-A attenuates d-GalN/LPS-induced FHF in mice. Fetuin-A might be a protective agent of liver damage partly through inhibiting liver inflammatory response and hepatocyte apoptosis.

Autophagic response in the Rabbit Hemorrhagic Disease, an animal model of virally-induced fulminant hepatic failure

The Rabbit Hemorrhagic Disease Virus (RHDV) induces a severe disease that fulfils many requirements of an animal model of fulminant hepatic failure. However, a better knowledge of molecular mechanisms contributing to liver damage is required, and it is unknown whether the RHDV induces liver autophagy and how it relates to apoptosis. In this study, we attempted to explore which signalling pathways were involved in the autophagic response induced by the RHDV and to characterize their role in the context of RHDV pathogenesis. Rabbits were infected with 2 × 104 hemmaglutination units of a RHDV isolate. The autophagic response was measured as presence of autophagic vesicles, LC3 staining, conversion of LC3-I to autophagosome-associated LC3-II and changes in expression of beclin-1, UVRAG, Atg5, Atg12, Atg16L1 and p62/SQSTM1. RHDV-triggered autophagy reached a maximum at 24 hours post-infection (hpi) and declined at 30 and 36 hpi. Phosphorylation of mTOR also augmented in early periods of infection and there was an increase in the expression of the endoplasmic reticulum chaperones BiP/GRP78, CHOP and GRP94. Apoptosis, measured as caspase-3 activity and expression of PARP-1, increased significantly at 30 and 36 hpi in parallel to the maximal expression of the RHDV capsid protein VP60. These data indicate that RHDV infection initiates a rapid autophagic response, perhaps in an attempt to protect liver, which associates to ER stress development and is independent from downregulation of the major autophagy suppressor mTOR. As the infection continues and the autophagic response declines, cells begin to exhibit apoptosis.

Advances of Notch signaling regulating fetal liver stem/progenitor cells: implications for the therapy of end-stage liver diseases

Cell therapy has a very promising potential for end-stage liver diseases (ESLD).Fetal liver stem/progenitor cells (FLSPCs) have advantages of safety,high survival and proliferation rates,and a small volume,all which make them ideal for liver disease stem cell therapy.During the early phase of our study,we applied a three-step separation method to enrich FLSPCs and obtained a separation efficiency similar to that of the flow-cell sorting method.Additionally,using a fulminant hepatic failure model in rats,we have demonstrated that FLSPCs can contribute to morphological and functional recovery of the liver.This manuscript will discuss how FLSPCs can be induced to accurately differentiate into hepatocytes and cholangiocytes and how FLSPCs maintain self-renewal.The Notch signaling plays a critical role in regulating the differentiation and self-renewal of many types of stem cells.Our previous findings have shown that the Notch signaling plays an important role in FLSPCs differentiation into hepatocytes.Therefore,the Notch signaling might be involved in the differentiation and self-renewal of FLSPCs.We conducted a study on the regulatory effects and relative molecular mechanisms of the Notch signaling on FLSPCs and found the corresponding interfering target,which might become an index for the clinical application of FLSPCs. Key words: Fetal liver stem/progenitor Cells (FLSPCs); Cell therapy; End-stage liver diseases (ESLDs) ; Notch signaling pathway