Effect of ultrasonic modification on the protective activity of Flammulina velutipes polysaccharide to prevent ethanol-induced injury on GES-1 cells

Flammulina velutipes(F.velutipes)polysaccharides were modified by ultrasound at the rated power of 150 W and 900 W.The monosaccharide composition,ultraviolet-visible,and Fourier transform infrared spectral characteristics of F.velutipes polysaccharides(FVP)and their ultrasonic modification products(U-FVPs)were determined.The protective effects of FVP and U-FVPs on human gastric mucosal cells GES-1 were confi rmed for the first time.The mole ratios of glucose and galactose were decreased and the mole ratio of mannose was increased after ultrasonic modification.Compared with the original FVP and the FVP modifi ed by ultrasound of 150 W(U-FVP1),the FVP modifi ed by ultrasound of 900 W(U-FVP2)could better prevent ethanol-induced damage to GES-1 cells.With increasing ultrasound intensity,the protective effect of FVPs on GES-1 cells was significantly enhanced by more effective prevention of intracellular reactive oxygen species(ROS)production and more promotion of expression of triglyceride factor 2(TFF2),prostaglandin E2(PGE2),epidermal growth factor(EGF),and transforming growth factorβ1(TGF-β1)mRNA.The ultrasonic modifi cation might be an effective way to develop novel F.velutipes polysaccharides that could effectively resist the gastric injury caused by excessive alcohol consumption.

Tamarix chinensis Lour inhibits chronic ethanol-induced liver injury in mice

BACKGROUND Tamarix chinensis Lour(TCL)is a shrub that usually grows in arid or semiarid desert areas and saline-alkali fields.It is a traditional Chinese herbal medicine with hepatoprotective,antioxidant,antibacterial,and antitumor activities.AIM To investigate the possible protective effects of TCL against liver injury induced by chronic ethanol intake.METHODS C57BL/6J male mice were fed a Lieber-DeCarli lipid diet containing alcohol and received(by gavage)a water-alcohol extract(80%)of TCL(100 and 200 mg/kg BW)or distilled water for 4 wk.After euthanasia,liver tissues were observed histologically with hematoxylin and eosin staining and Oil red O staining,and the levels of alanine aminotransferase,aspartate transaminase,hepatic lipids,reactive oxygen species,malondialdehyde,and superoxide dismutase were measured.In addition,expression of the NOD-like receptor family,pyrin domain-containing 3(NLRP3)inflammasome and downstream proinflammatory cytokines were determined.RESULTS Compared with the ethanol group,mice in the TCL-treated group(200 mg/kg)had significantly lower serum levels of alanine aminotransferase(mean,34.1 IU/L vs 45.3 IU/L,P<0.01)and aspartate transaminase(mean,89.6 IU/L vs 115.7 IU/L,P<0.01),as well as marked reduction of hepatic tissue reactive oxygen species(decreased by 27.5%,P<0.01)and malondialdehyde(decreased by 76.6%,P<0.01)levels,with a significant increase of superoxide dismutase(Increased by 73.2%,P<0.01).Expression of the NLRP3 inflammasome and its downstream cytokines[interleukin(IL)-1β,tumor necrosis factor-α,and IL-6],and recruitment of natural killer T cells to the liver,were reduced in the TCLtreated incubation with a Lieber-DeCaril ethanol lipid diet group.CONCLUSION These findings suggest that a TCL extract(200 mg/kg)protects against chronic ethanol-induced liver injury,probably by inhibiting the NLRP3-caspase-1-IL-1βsignaling pathway and suppressing oxidative stress.

On the Protective Effects of Aplysin on Ethanol-induced Oxidative Damage in Rat Primary Hepatocytes

[Objective] To investigate the protective effects of Aplysin on ethanol-induced oxidative damage in rat primary hepatocytes.[Methods]Rat primary hepatocytes were obtained via the portal vein collagenase IV in situ perfusion technique followed by a Percoll density gradient centrifuge; MTT test was used to determine the optimum dose of Aplysin and ethanol,and detect the cell vitality in primary hepatocytes; supernatants of primary hepatocytes were harvested to measure AST and LDH level,and the SOD,GSH-PX activities and MDA content in primary hepatocytes were observed; flow cytometry was used to detect the cell apoptosis rate; DNA damage in primary hepatocytes were detected by single-cell gel electrophoresis assay; The level of mitochondrial membrane potential in primary hepatocytes was tested by fluorogenic probe JC-1; the CYP2E1 activity in primary hepatocytes were detected by colorimetry; the proteins of CYP2E1 were detected by Western blotting. [Results]300 mmol/L dose of ethanol and 30 mg/L dose of Aplysin were the optimal dosages and were used in the subsequent experiments. Hepatocyte vitality was significantly increased in the Aplysin group relative to that in the ethanol group,and Aplysin inhibited the release of AST and LDH( P < 0. 05). For the Aplysin treatment group,the activities of hepatocyte SOD and GSH were significantly increased and MDA was markedly lowered as compared with those in the ethanol group( P < 0. 05). And Aplysin can alleviate hepatocyte apoptosis significantly,and hepatocyte DNA damage rate of II-III level and IV level were significantly lowered in the Aplysin treatment group as compared with those in the ethanol group,and Aplysin has evidently improved on alcohol induced mitochondria damage of hepatocyte. Primary hepatocytes activities and protein expression of CYP2E1 were markedly lowered in the Aplysin treatment group as compared with those in the ethanol group( P < 0. 05). [Conclusion] Aplysin has protective effects on liver oxidative damage induced by alcohol of primary cultured rat hepatocytes by blocking CYP2E1 activation,relieving oxidative stress,and sharpening the oxidation resistance ability.

Potential effects of L-NAME on alcohol-induced oxidative stress

AIM: Nitric oxide (NO) is a highly reactive oxidant synthesized from L-arginine by nitric oxide synthase (NOS). NO may cause injury through the generation of potent radicals. Nw-nitro-L-arginine methyl ester (L-NAME) is a non-selective inhibitor of NOS. We aimed to evaluate whether L-NAME treatment had protective effects against oxidative stress in rats intragastrically fed with ethanol during a 4 wk-period. METHODS: Thirty-six male Wistar rats were divided into 3 equal groups: group 1 (control group-isocaloric dextrose was given), group 2 (6 g/kg·d ethanol-induced group) and group 3 (both ethanol 6 g/kg·d and L-NAME 500 mg/L in drinking water-given group). Animals were sacrificed at the end of 4 wk-experimental period, and intracardiac blood and liver tissues were obtained. Biochemical measurements were performed both in plasma and in homogenized liver tissues. Alanine amino transferase (ALT), aspartate amino transferase (AST), malondialdehyde (MDA), NO, superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) levels were measured by spectrophotometry. RESULTS: ALT and AST in group 2 (62 U/L and 128 U/L, respectively) were higher than those in group 1 (24 U/L and 38 U/L) and group 3 (37 U/L and 81 U/L) (P<0.001 for both). Plasma and tissue levels of MDA in group 2 (4.66 μmol/L and 0.55 μmol/mg protein) were higher than in group 1 (2.65 μmol/L and 0.34 nmol/mg protein) and group 3 (3.43 μmol/L and 0.36 nmol/mg protein) (P<0.001 for both). Plasma and liver tissue levels of NO in group 2 (54.67 μmol/L and 586.50 nmol/mg protein) were higher than in group 1 (34.67 μmol/L and 435.33 nmol/mg protein) and group 3 (27.50 μmool/L and 412.75 nmol/mg protein ) (P<0.001 for both). Plasma and liver tissue SOD activities in group 2 (15.25 U/mL and 5.38 U/ mg protein, respectively) were lower than in group 1 (20.00 U/mL and 8.13 U/ mg protein) and group 3 (19.00 U/mL and 6.93 U/ mg protein) (P<0.001 for both). Plasma and liver tissue CAT activities in group 2 (145 U/mL and 37 U/ mg protein, respectively) were lower than in group 1 (176 U/mL and 73 U/mg protein) and group 3 (167 U/mL and 61 U/mg protein) (P<0.001 for both). Meanwhile, erythrocytes and liver tissue levels of GSH in group 2 (4.12 mg/g Hb and 5.38 nmol/mg protein, respectively) were lower than in group 1 (5.52 mg/g Hb and 4.49 nmol/mg protein) and group 3 (5.64 mg/g Hb and 4.18 nmol/mg protein) (P<0.001 for both). CONCLUSION: Our findings show that L-NAME may produce a restorative effect on ethanol-induced liver damage via decreasing oxidative stress and increasing antioxidant status.

S2A-4 Epigenetic Mechanisms Underlying Fetal Alcohol Spectrum Disorders

Maternal alcohol consumption is the leading known non-genetic cause of mental retardation.Prenatal alcohol exposure can cause a range of structural and functional birth defects,which are defined as Fetal Alcohol Spectrum Disorders(FASD).Growing evidence suggests that excessive cell death in selected cell populations is a major component of the pathogenesis of FASD.This suggests that a strategy for protecting against ethanol’s teratogenesis by epigenetically regulating the genes involved in the apoptotic pathway is promising for effective intervention and prevention of FASD.We have recently found that treatment with ethanol resulted in a significant decrease in miR-125b expression in neural crest cells(NCCs)and mouse embryos.We also validated that Bcl-2 antagonist killer 1(Bak1)and p53-upregulated modulator of apoptosis(PUMA)are the direct targets of miR-125b in NCCs.In addition,overexpression of miR-125b significantly reduced the ethanol-induced increase in Bak1 and PUMA protein expression,caspase-3 activation,and apoptosis in NCCs,indicating that miR-125b can modulate ethanol-induced apoptosis by the regulation of Bcl-2 and p53 pathways.Furthermore,microinjection of miR-125b mimic resulted in a significant increase in miR-125b expression and a decrease in the protein expression of Bak1 and PUMA in ethanol-exposed mouse embryos.Up-regulation of miR-125b also significantly reduced ethanol-induced caspase-3 activation and diminished ethanol-induced growth retardation in mouse embryos.Our studies have also shown that exposure to ethanol resulted in a significant increase in the activities of histone deacetylase(HDAC)and DNA methyltransferase(DNMT),and increased the methylation of Bcl-2 promoter in NCCs.In addition,ChIP-qPCR assay revealed that ethanol exposure significantly decreased acetyl-histone H3 binding to the Bcl-2 promoter and the expression of Bcl-2.Supplementing with sulforaphane(SFN),an isothiocyanate derived from cruciferous vegetables and a dual epigenetic regulator which can inhibit both DNMTs and HDACs,reversed the ethanol-induced hypermethylation of Bcl-2 promoter and reduction in acetyl-histone H3 binding to the Bcl-2 promoter.Treatment with SFN also restored the expression of Bcl-2 in ethanol-exposed NCCs.Furthermore,supplementing with SFN diminished ethanol-induced apoptosis in NCCs and in mouse embryos exposed to ethanol in vivo.These results demonstrate that SFN can epigenetically restore the expression of Bcl-2 and attenuate ethanol-induced apoptosis by decreasing methylation and increasing histone acetylation at the Bcl-2 promoter.These findings support the potential of dietary consumption of SFN or SFN-rich broccoli sprouts to attenuate ethanol-induced apoptosis and confer in vivo protection against FASD through epigenetic regulation of the expression of anti-apoptotic genes.

Voacanga grandifolia(Miq.)Rolfe protects against alcohol-induced liver toxicity in rats

Objective:To evaluate the ethanol extract of Voacanga grandifolia for hepatoprotective and antioxidant potential against ethanol-induced liver toxicity in rats.Methods:Sprague-Dawley rats were administered ethanol(7 g/kg)and then treated with 100 and 200 mg/kg of Voacanga grandifolia extract.The phytochemical constituents and antioxidant potential of Voacanga grandifolia extract were evaluated by GC-MS and in vitro antioxidant assays.Biochemical indicators for liver damage and pro-apoptotic and antiapoptotic gene expression were determined using biochemical kits,ELISA,and qRT-PCR,respectively.Additionally,histopathological study of the liver was performed.Results:GC-MS identified propanoic acid,meso-erythritol,D-pinitol,myo-inositol,and hexadecanoic acid in Voacanga grandifolia extract.Voacanga grandifolia extract(100 and 200 mg/kg)increased the concentration of enzymatic antioxidants while diminishing the levels of inflammatory cytokines and biochemical indicators.qRT-PCR assay showed that Voacanga grandifolia extracts upregulated antiapoptotic gene expression while downregulating pro-apoptotic gene expression.Furthermore,the plant extract improved the hepatic architecture of ethanol-intoxicated rats.Conclusions:Voacanga grandifolia extract demonstrates hepatoprotective activity against alcohol-induced liver injury in rats and could be a potential hepatoprotective agent.