Mice ; Animals ; Tandem Mass Spectrometry ; Alpha-Amanitin ; Chromatography, Liquid ; Metabolomics ; Chromatography, High Pressure Liquid/methods*

PURPOSE: Glehnia littoralis has been reported to have several pharmacological properties but no in vivo reports describing the protective effects of this plant on α-amanitin-induced hepatotoxicity have been published. α-Amanitin is a peptide found in several mushroom species that accounts for the majority of severe mushroom poisonings leading to severe hepatonecrosis. In our previous in vitro study, we found that α-amanitin induced oxidative stress, which may contribute to its severe hepatotoxicity. The aim of this study was to investigate whether Glehnia littoralis acetate extract (GLEA) has protective antioxidant effects on α-amanitin-induced hepatotoxicity in a murine model. METHODS: Swiss mice (n=40 in all groups) were divided into four groups (n=10/group). Three hours after giving α-amanitin (0.6 mg/kg, i.p.) to the mice, they were administered silibinin (50 mg/kg/d, i.p.) or Glehnia littoralis ethyl acetate extract (100 mg/kg/d, oral) therapies once a day for 3 days. After 72 hours of treatment, each subject was killed, cardiac blood was aspirated for hepatic aminotransferase measurement, and liver specimens were harvested to evaluate the extent of hepatonecrosis. The degree of hepatonecrosis was assessed by a pathologist blinded to the treatment group and divided into 4 categories according to the grade of hepatonecrosis. RESULTS: GLEA significantly improved the beneficial functional parameters in α-amanitin-induced hepatotoxicity. In the histopathological evaluation, the toxicity that was generated with α-amanitin was significantly reduced by GLEA, showing a possible hepatoprotective effect. CONCLUSION: In this murine model, Glehnia littoralis was effective in limiting hepatic injury after α-amanitin poisoning. Increases of aminotransferases and degrees of hepatonecrosis were attenuated by this antidotal therapy.
Agaricales ; Alpha-Amanitin* ; Animals ; Antidotes ; Antioxidants ; Apiaceae* ; In Vitro Techniques ; Liver ; Mice ; Models, Animal ; Mushroom Poisoning ; Oxidative Stress ; Plants ; Poisoning ; Transaminases

PURPOSE: Glehnia littoralis has been used to treat ischemic stroke, phlegm, cough, systemic paralysis, antipyretics and neuralgia. The pharmacological mechanisms of Glehnia littoralis include calcium channel block, coumarin derivatives, anticoagulation, anti-convulsive effect, as well as anti-oxidant and anti-inflammatory effects. Alpha-amanitin (α-amanitin) is a major toxin from extremely poisonous Amanita fungi. Oxidative stress, which may contribute to severe hepatotoxicity was induced by α-amanitin. The aim of this study was to investigate whether Glehnia littoralis ethyl acetate extract (GLEA) has the protective antioxidant effects on α-amanitin -induced hepatotoxicity. METHODS: Human hepatoma cell line HepG2 cells were pretreated in the presence or absence of GLEA (50, 100 and 200µg/ml) for 4 hours, then exposed to 60µmol/L of α-amanitin for an additional 4 hours. Cell viability was evaluated using the MTT method. AST, ALT, and LDH production in a culture medium and intracellular MDA, GSH, and SOD levels were determined. RESULTS: GLEA (50, 100 and 200µg/ml) significantly increased the relative cell viability by 7.11, 9.87, and 14.39%, respectively, and reduced the level of ALT by 10.39%, 34.27%, and 52.14%, AST by 9.89%, 15.16%, and 32.84%, as well as LDH by 15.86%, 22.98%, and 24.32% in culture medium, respectively. GLEA could also remarkably decrease the level of MDA and increase the content of GSH and SOD in the HepG2 cells. CONCLUSION: In the in vitro model, Glehnia littoralis was effective in limiting hepatic injury after α-amanitin poisoning. Its antioxidant effect is attenuated by antidotal therapy.
Alpha-Amanitin* ; Amanita ; Antioxidants ; Antipyretics ; Apiaceae* ; Calcium Channels ; Carcinoma, Hepatocellular ; Cell Line ; Cell Survival ; Cough ; Coumarins ; Fungi ; Hep G2 Cells ; Humans ; In Vitro Techniques* ; Methods ; Neuralgia ; Oxidative Stress ; Paralysis ; Poisoning ; Stroke

OBJECTIVETo investigate the effects of transcriptional inhibitors 5, 6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) and alpha-Amanitin on the localization of Nrf2 in the nucleus.

METHODSA549 cells were treated with DRB (50 mg/L) or alpha-Amanitin (2.5 mg/L)for 1 h and 6 h in serum-free medium, respectively. The expressions of Nrf2, HO-1, NQO1 and AKR1C were detected by Western blotting analysis. The localization of Nrf2 was determined by laser scanning confocal microscopy after cells were treated with either DRB or agr:-Amanitin for 1 h.

RESULTSThe expressions of Nrf2 and Nrf2-ARE gene batteries HO-1, AKR1C and NQO1 were decreased after 6 h treated with either DRB or alpha-Amanitin. The expression of SC35 was up-regulated but RNA Pol II was down-regulated; Y12 and NPC did not significantly change. The localization of Nrf2 in the cell nucleus did not change significantly.

CONCLUSIONDRB and alpha-Amanitin can down-regulate the expression of Nrf2 and its targeting proteins HO-1, AKR1C and NQO1, but may have no effect on the localization of Nrf2.

20-Hydroxysteroid Dehydrogenases ; genetics ; metabolism ; Alpha-Amanitin ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Dichlororibofuranosylbenzimidazole ; pharmacology ; Heme Oxygenase-1 ; genetics ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Nucleic Acid Synthesis Inhibitors ; pharmacology

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.
Alpha-Amanitin/pharmacology ; Animals ; Antibodies, Monoclonal/analysis/metabolism ; Antibodies, Protozoan/analysis/metabolism ; Dactinomycin/pharmacology ; Fluorescent Antibody Technique, Direct ; Gene Expression/*physiology ; Green Fluorescent Proteins/genetics ; Hela Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Nucleic Acid Synthesis Inhibitors/pharmacology ; Nucleolus Organizer Region/drug effects/*metabolism ; Pol1 Transcription Initiation Complex Proteins/metabolism ; Protein Sorting Signals/physiology ; Protozoan Proteins/*biosynthesis/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Toxoplasma/*physiology ; Transfection