Antioxidant defense of Nrf2 vs pro-inflammatory system of NF-κB during the amoebic liver infection in hamster.

Entamoeba histolytica is the causative agent of amoebic liver abscess (ALA), which course with an uncontrolled inflammation and nitro-oxidative stresses, although it is well known that amoeba has an effective defence mechanisms against this toxic environment, the underlying molecular factors responsible for progression of tissue damage remain largely unknown. The purpose of the present study was to determine during the acute stage of ALA in hamsters, the involvement of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and nuclear factor-kappa B (NF-κB), which are activated in response to oxidative stress. From 12 h post-infection the ALA was visible, haematoxylin-eosin and Masson's trichrome stains were consistent with these observations, and alanine aminotransferase, alkaline phosphatase and γ-glutamyl transpeptidase serum activities were increased too. At 48 h after infection, liver glycogen content was significantly reduced. Western blot analyses showed that 4-Hydroxy-2-nonenal peaked at 12 h, while glycogen synthase kinase-3ß, cleaved caspase-3, pNF-κB, interleukin-1ß and tumour necrosis factor-α were overexpressed from 12 to 48 h post-infection. Otherwise, Nrf2 and superoxide dismutase-1, decreased at 48 h and catalase declined at 36 and 48 h. Furthermore, heme oxygenase-1 was increased at 12 and 24 h and decreased to normal levels at 36 and 48 h. These findings suggest for the first time that the host antioxidant system of Nrf2 is influenced during ALA.

Modulation of innate immune response by the vagus nerve in experimental hepatic amebiasis in rats.

The parasympathetic nervous system has a crucial role in immunomodulation of the vagus nerve, its structure provides a pathogen detection system, and a negative feedback to the immune system after the pathogenic agent has been eliminated. Amebiasis is a disease caused by the protozoan parasite Entamoeba histolytica, considered the third leading cause of death in the world. The rats are used as a natural resistance model to amoebic liver infection. The aim of this study is to analyze the interaction of Entamoeba histolytica with neutrophils, macrophages, and NK cells in livers of intact and vagotomized rats. Six groups were studied (n = 4): Intact (I), Intact + amoeba (IA), Sham (S), Sham + amoeba (SA), Vagotomized (V) and Vagotomized + amoeba (VA). Animals were sacrificed at 8 h post-inoculation of E. histolytica. Then, livers were obtained and fixed in 4% paraformaldehyde. Tissue liver slides were stained with H-E, PAS and Masson. The best development time for E. histolytica infection was at 8 h. Amoeba was identified with a monoclonal anti-220 kDa E. histolytica lectin. Neutrophils (N) were identified with rabbit anti-human neutrophil myeloperoxidase, macrophages (Mɸ) with anti-CD68 antibody and NK cells (NK) with anti-NK. Stomachs weight and liver glycogen were higher in V. Collagen increased in VA, whereas vascular and neutrophilic areas were decreased. There were fewer N, Mɸ, NK around the amoeba in the following order IA > SA > VA (p < 0.05 between IA and VA). In conclusion, these results suggest that the absence of parasympathetic innervation affects the participation of neutrophils, macrophages and NK cells in the innate immune response, apparently by parasympathetic inhibition on the cellular functions and probably for participation in sympathetic activity.

α-1 Adrenergic receptor antagonist doxazosin reverses hepatic stellate cells activation via induction of senescence.

BACKGROUND: Activated hepatic stellate cells (aHSCs) are the main effector cells during liver fibrogenesis. α-1 adrenergic antagonist doxazosin (DX) was shown to be anti-fibrotic in an in vivo model of liver fibrosis (LF), but the mechanism remains to be elucidated. Recent studies suggest that reversion of LF can be achieved by inducing cellular senescence characterized by irreversible cell-cycle arrest and acquisition of the senescence-associated secretory phenotype (SASP). AIM: To elucidate the mechanism of the anti-fibrotic effect of DX and determine whether it induces senescence. METHODS: Primary culture-activated rat HSCs were used. mRNA and protein expression were measured by qPCR and Western blot, respectively. Cell proliferation was assessed by BrdU incorporation and xCelligence analysis. TGF-ß was used for maximal HSC activation. Norepinephrine (NE), PMA and m-3M3FBS were used to activate alpha-1 adrenergic signaling. RESULTS: Expression of Col1α1 was significantly decreased by DX (10 µmol/L) at mRNA (-30 %) and protein level (-50 %) in TGF-ß treated aHSCs. DX significantly reduced aHSCs proliferation and increased expression of senescence and SASP markers. PMA and m-3M3FBS reversed the effect of DX on senescence markers. CONCLUSION: Doxazosin reverses the fibrogenic phenotype of aHSCs and induces the senescence phenotype.

Evaluation of the PEΔIII-LC3-KDEL3 Chimeric Protein of Entamoeba histolytica-Lectin as a Vaccine Candidate against Amebic Liver Abscess.

Entamoeba histolytica is an intestinal parasite that causes dysentery and amebic liver abscess. E. histolytica has the capability to invade host tissue by union of virulence factor Gal/GalNAc lectin; this molecule induces an adherence-inhibitory antibody response as well as to protect against amebic liver abscess (ALA). The present work showed the effect of the immunization with PEΔIII-LC3-KDEL3 recombinant protein. In vitro, this candidate vaccine inhibited adherence of E. histolytica trophozoites to HepG2 cell monolayer, avoiding the cytolysis, and in a hamster model, we observed a vaccine-induced protection against the damage to tissue liver and the inhibition of uncontrolled inflammation. PEΔIII-LC3-KDEL3 reduced the expression of TNF-α, IL-1ß, and NF-κB in all immunized groups at 4- and 7-day postinfection. The levels of IL-10, FOXP3, and IFN-γ were elevated at 7 days. The immunohistochemistry assay confirmed this result, revealing an elevated quantity of +IFN-γ cells in the liver tissue. ALA formation in hamsters immunized was minimal, and few trophozoites were identified. Hence, immunization with PEΔIII-LC3-KDEL3 herein prevented invasive amebiasis, avoided an acute proinflammatory response, and activated a protective response within a short time. Finally, this recombinant protein induced an increase of serum IgG.

Growth hormone ameliorates high glucose-induced steatosis on in vitro cultured human HepG2 hepatocytes by inhibiting de novo lipogenesis via ChREBP and FAS suppression.

OBJECTIVE: Growth hormone (GH) deficiency has been associated with increased steatosis but the molecular mechanism has not been fully elucidated. We investigated the effect of GH on lipid accumulation of HepG2 cells cultured on an in vitro steatosis model and examined the potential involvement of insulin-like growth factor 1 (IGF-1) as well as lipogenic and lipolytic molecules. METHODS: Control and steatosis conditions were induced by culturing HepG2 cells with 5.5 or 25 mmol/l glucose for 24 h, respectively. Afterward, cells were exposed to 0, 5, 10 or 20 ng/ml GH for another 24 h. Lipid content was quantified as well as mRNA and protein levels of IGF-1, carbohydrate responsive element-binding protein (ChREBP), sterol regulatory element-binding protein 1c (SREBP1c), fatty acid synthase (FAS), carnitine palmitoyltransferase 1A (CPT1A), and peroxisome proliferator-activated receptor alpha (PPAR-alpha) by qPCR and western blot, respectively. Data were analyzed by one-way ANOVA and the Games-Howell post-hoc test. RESULTS: In the steatosis model, HepG2 hepatocytes showed a significant 2-fold increase in lipid amount as compared to control cells. IGF-1 mRNA and protein levels were significantly increased in control cells exposed to 10 ng/ml GH, whereas high glucose abolished this effect. High glucose also significantly increased both mRNA and protein of ChREBP and FAS without having effect on SREBP1c, CPT1A and PPAR-alpha. However, GH inhibited ChREBP and FAS production, even in HepG2 hepatocytes cultured under steatosis conditions. CONCLUSIONS: Growth hormone ameliorates high glucose-induced steatosis in HepG2 cells by suppressing de novo lipogenesis via ChREBP and FAS down-regulation.