Protective Effects And Mechanism Of Astragal Oside IV On Gastric Mucosal Injury Induced By Ethanol In Rat

BackgroudWith the rising of people’s living standard,consumption of alcohol has been heightened.The incidence of disease of digestive tract of drinkers is higher than those who do not drink.Acute gastric ulcer and gastric mucosal injury are becoming more common in drink crowd.As one of the most important physiologic barrier,damage of gastric mucosa could be lead to a mount of diseases.There is a long history and rich clinical experience of Traditional Chinese medicine in treatment of gastric mucosal injury diseases.To study a effective method of prevention and treatment of gastric mucosal injury induced by alcohol in TCM is necessary which could be a solution of clinical problems.As a classical qi-invigorating herb,astragalus membranaceus has a long history in treatment of digestive system disease,that is the principal drug in formula of Huangqi Jianzhong Decoction and Buzhong Yiqi Decoction which are used to treat abdominal pain,diarrhea and epigastric fullness.Astragaloside IV(AS-Ⅳ)is the main content of astragalus membranaceus which exhibits anti-inflammatory,anti-oxidant,anti-asthma,anti-diabetes,cardioprotectiv-effect and immunoenhanced effects.Our previous researchs indicated that astrageloside was good to pretect gastric mucosal injury,the astragalosides and AS-IV could promote repairing of GES-1 cell which demaged by alcohol.Therefore,On the basis of the previous research,the present study was designed to investigated the protective effects and mechanism of AS-Ⅳon gastric mucosal injury induced by ethanol in a rat model.ObjectiveTo investigated the protective effect of AS-Ⅳ on gastric mucosal injury induced by ethanol in rat model.To research the mechanism of anti-inflammation,anti-oxidative stress of mitochondria and anti-apoptosis of AS-Ⅳ in protecting gastric mucosal injury.Methods1.The research of protective effect of AS-Ⅳ on gastric mucosal injury induced by ethanol in rat model.Adult SD rats were placed individually in metabolic cages with raised floors of wide mesh in order to avoid coprophagy that interferes with the induction of gastric injury.After 1 week for acclimatization,animals were randomized block according to weight divided into seven experimental groups with 8 rats in each group.The control and model groups received daily i.p.injections of saline at lml/100g for 4 days,The animals in the AS-Ⅳ 4,AS-IV 2,AS-IV 1 groups respectively received daily i.p.injections of AS-IV at 4mg/kg,2mg/kg,lmg/kg for 4 days.The rats in solvent group were injected 10%solvent blended anhydrous ethanol and propylene glycol in ratio of 2:1 at lml/100g for 4days.The OME group received omeprazole injection at 40mg/kg for 4 days.On the third day of the experiment,all the animals were fasted for 24 h but had free access to water.On the fourth day,the rats were administered water deprivation after received intraperitoneal injection,75mins later,absolute ethanol was given orally to animals except control group at 5ml/kg,the control group received intraperitoneal administration of saline at same volum.The animals were euthanized 1 hour after ethanol administration and their stomachs were immediately removed and opened along the greater curvature.The stomachs were rinsed with cold saline to remove the gastric contents and blood clots in order to evaluate the extent of gastric mucosal injury.The extent of gastric damage was quantified via measuring by Guth and microscope.2.The research of anti-inflammatory effect of AS-Ⅳ in protecting gastric mucosal injury induced by ethanol.10%gastric mucosal homogenate supernatant was used for measurement of levels of MPO,TNF-α,IL-1β and IL-10 in tissue samples and colorimetric method and ELISA assay kits were used.Western-blot and RT-PCR were used to measure the NF-κ B p65 protein and mRNA.3.The research of anti-oxidative stress of mitochondria effect of AS-IV in protecting gastric mucosal injury induced by ethanol.The mitochondria of gastric cells were isolated,the indexs of oxidative stress such as SOD,MDA and GSH were measured at the level of mitochondria.The mitochondrial transmerabrane potential of control group,model group,AS-Ⅳ 4 group and OME group were detected by fluorescence method.4.The research of anti-apoptosis effect of AS-Ⅳ in protecting gastric mucosal injury induced by ethanol.TUNEL staining technique was used to study the gastric cell apoptosis rate.Caspase 3 and Caspase 9 activity were evaluated by colorimetric method.Western-blot and RT-PCR were used to measure the proteins and genes of apoptosis relate factors such as Cyt-c,BID,AIF.Bax and Bcl-2 proteins and genes were detected at the level of mitochondria.Results1.Intragastric administration of absolute ethanol triggered several linear hemorrhagic damage and multifocal erosions compared to the vehicle-treated control group,and the gastric mucosal surface is in dark red.AS-Ⅳ at dose of 4mg/kg and 2mg/kg significantly reduced gastric injury scores(P<0.05)compared to model and solvent groups.The solvent group was no different with the model group in macroscopic damage and injury scores.From the view of histologic observation,gastric sections from control group displayed an intact architecture of the gastric wall with few leukocytes in the mucosa.In contrast,administration of ethanol triggered a severe gastric injury with high scores of microscopic damage characterized by hemorrhagic injury,submucosal edema,inflammatory cell infiltration and epithelial cell loss.The solvent group was no different with the model group.Pretreatment with AS-IV 4mg/kg and 2mg/kg attenuated ethanol-induced pathological changes(P<0.05).2.The levels of MPO,TNF-α,IL-1β and NF-κ B p65 were raised by administration of absolute ethanol in rat gastric mucosa tissue,while the IL-10 was reduced.The solvent group was no different with the model group.Pretreatment of AS-Ⅳ 4mg/kg,AS-Ⅳ 2mg/kg and AS-Ⅳ lma/kg could reduce the level of MPO(P<0.05).Pretreat with AS-Ⅳ 4mg/kg and AS-Ⅳ 2mg/kg reduced the degree of TNF-α,IL-1 β(P<0.05).Concentration of IL-10 in AS-Ⅳ 4 group was higher than model group(P<0.05).3.The levels of SOD and GSH in model group were lower and the MDA was higher than contral group(P<0.05),The solvent group was no different with the model group.pretreat with AS-Ⅳ 4mg/kg increased the degree of GSH(P<0.05),MDA concentration of AS-Ⅳ 4 and AS-Ⅳ 1 were lower than model group.Pretreatment of AS-Ⅳ could not raise the level of SOD reduced by administration of ethanol.4.The result of TUNEL indicated that gastric cell apoptosis rate caused by ethanol was higher than control,the solvent group was no different with the model group.AS-IV at dose of 4mg/kg and 2mg/kg significantly reduced the cell apoptosis rate(P<0.05).Caspase 3 and Caspase 9 activity was raised by ethanol,the solvent group was no different with the model group,AS-Ⅳ 4,AS-Ⅳ 2 and AS-Ⅳ 1 groups were more lower than model group(P<0.05).The mitochondrial membrane potential of model group was lower than control group(P<0.05),the AS-IV 4 group and OME group were more higher than model group(P<0.05).5.The result of Western Blot showed that the the expression of Bid protein in the gastric tissue of rats with gastric mucosal injury induced by ethanol was significantly decreased(P<0.01),the expression of Bid protein in AS-IV 4 group was significantly higher than that in model group;there was no difference between the solvent group and the model group;the AS-Ⅳ 2 and AS-IV 1 groups were no difference with the model group as well.As the expression of Cyt-C protein,the model group was significantly higher than the normal group(P<0.01);model group and solvent group was no difference;AS-IV 4,AS-IV 2 and AS-Ⅳ 1 groups could reduce the expression of Cyt-C in different degrees(P<0.05).The expression of AIF protein in model group was significantly higher than that in normal group(P<0.01);there was no difference between the solvent group and the model group;AS-Ⅳ 4 and AS-IV 2 groups could decrease the expression of AIF as well(P<0.05),while the AS-IV 1 group was no difference with model group.The expression of Bax was significantly higher in the model group(P<0.01);There was no significant difference between the solvent group and the model group;the expression of Bax was decreased in AS-IV 4,AS-Ⅳ 2 and AS-Ⅳ 1 groups,(P<0.05).The expression of Bcl-2 protein in model group was significantly lower than that in model group(P<0.01);There was no significant difference between the solvent group and the model group;similarly AS-Ⅳ 2 group and AS-Ⅳ 1 group were no difference with model group.6.The expression of Bid mRNA was significantly increased in ethanol-induced gastric mucosal injury in rats(P<0.01);There was no significant difference between the solvent group and the model group;compared with the model group,the expression of Bid mRNA was significantly decreased in AS-IV 4 group;meanwhile,there were no difference among AS-IV 2 group,AS-Ⅳ 1 group and model group.The expression of Cyt-C mRNA in the model group was significantly higher than that in the normal group(P<0.01);there was no significant difference between the solvent group and the model group;the expression of Cyt-C mRNA in AS-IV 4 group was significantly lower than that in model group(P<0.01);it was not different among AS-Ⅳ 2 group,AS-Ⅳ 1 group and model group.The expression of AIF mRNA in model group was significantly higher than that in normal group(P<0.01).AS-Ⅳ 4 group can significantly down-regulate the expression of AIF mRNA(P<0.01);there was no significant difference between the solvent group and the model group;it was not different among AS-Ⅳ 2 group,AS-Ⅳ 1 group and model group.After stimulation with ethanol,the expression of Bax mRNA in gastric tissue was significantly increased(P<0.01);There was no significant difference between the solvent group and the model group.The expression of Bax mRNA in the AS-IV 4 group was significantly lower than that in the model group(P<O.05);there were no difference among AS-Ⅳ 2 group,AS-Ⅳ 1 group and model group.Bcl-2 mRNA expression in the model group was significantly lower than that in the normal group(P<0.01);AS-Ⅳ 4 group significantly up-regulated the expression of Bcl-2 mRNA(P<0.01);it was not different among AS-Ⅳ 2 group,AS-Ⅳ 1 group,solvent group and model groupConclusion1.In the course of ethanol-induced gastric mucosal injury,mitochondrial oxidative stress and mitochondrial pathway apoptosis were observed in rat gastric tissue.Astragaloside could reduce mitochondrial oxidative stress by reducing mitochondrial lipid peroxidation and increasing mitochondrial GSH concentration.2.In the process of ethanol-induced gastric mucosal injury,the gastric tissue of rats had inflammatory response which characterised as neutrophil aggregation,high expression of pro-inflammatory cytokines TNF-α and IL-1β,low expression of suppression of inflammatory cytokines IL-10 and high expression of inflammatory protein NF-Kb p65.Astragaloside could alleviate the inflammatory response via reducing the activity of MPO,decreasing the expression of TNF-α,IL-1β and NF-Kb p65 and increasing the expression of IL-10.3.Astragaloside intraperitoneal injection has an effect of protecting ethanol-induced gastric mucosal injury in rats,which may be related to its resistance to inflammation and mitochondrial oxidative stress-induced apoptosis in rat gastric tissue.