| ObjectiveTo observe the protective and antioxidative effects of astragaloside Ⅳand Puerarin compatibility on gastric mucosal injury induced by ethanol and indomethacin in rats,analyze whether the compatibility of two component(Astragaloside Ⅳ and Puerarin)has a certain synergistic effect in protecting gastric mucosa,and study its mechanism.Methods1.HPLC was used to detect the content of astragaloside Ⅳ and puerarin in Huangqi-Gegen Water decoction.Huangqi-Gegen Water decoction was prepared according to the following method:Huangqi blending Gegen by a ratio of 3:1 which is the optimum compatibility ratiodipped 10 times volumn of water and boiled for 30min,repeated 3 times.Then the content of Astragalus Ⅳ and Puerarin in water decoction were determinated by HPLC.2.The protective effect of Astragaloside Ⅳ and Puerarin compatibility on the gastric mucosal injury induced by ethanol in rats.SD rats were divided into 14 groups:1)normal group,2)model group,3)positive group(Cimetidine),4)Astragaloside(AS-Ⅳ)high dose group,5)AS-Ⅳ medium dose group,6)AS-Ⅳ low dose group,7)puerarin(Pur)high dose group,8)Pur medium dose group,9)Pur low-dose group,10)Astragaloside medium dose plus puerarin medium dose(AS + P medium),11)Astragaloside middle dose plus puerarin low dose(AS medium + P low),12)Astragaloside low dose plus puerarin middle dose(AS low + P medium),13)Astragaloside low dose +Pueraria medium dose(AS low+ P medium),14)Huangqi-Gegen pairs group.Rats in all groups were given medicine continuously for 4 days,except Huangqi-Gegen pairs group,other groups were injected by intraperitoneally(ip);Huangqi-Gegen pairs group was administrated by gavage(ig).75mins after the last administration,acute gastric mucosal injury was induced by ethanol 5ml/Kg orally.Then 75minslater,stomachs of rats were obtained.Gastric mucosal injury scores were measured and calculated,and gastric pathology were observed under microscope.Superoxide dismutase enzyme(SOD),malondialdehyde(MDA),and reactive oxygen species(ROS)levels were detected in gastric tissue.3.Effect of Huangqi-Gegen pairs on gastric mucosal injury in rats induced by indomethacinSD rats were divided into six groups:1)normal group,2)model group,3)positive group,4)Huangqi-Gegen high dose group,5)Huangqi-Gegen medium dose group,6)Huangqi-Gegen low dose group.All groups were administrated by gavage for 4 days.75min after the last administration,all groups except the normal group were injected with indomethacin suspension solution at the dose of 35mg/Kg,6hrs later,all animals were killed,and gastric mucosal injury scores were calculated,pathological changes of gastric mucosa were observed,and the levels of MDA,SOD,ROS in gastric mucosa were detected.4.Effects of Astragaloside Ⅳ-Puerarin compatibility on gastric mucosal injury in rats induced by indomethacinSD rats were divided into 13 groups:1)normal group,2)model group,3)positive group,4)AS-IV high-dose group,5)AS-IV medium dose group,6)AS-IV low dose group,7)Pur high dose group,8)Pur medium dose group,9)Pur low dose group,10)AS medium + P medium dose group,11)AS medium + P low dose group,12)AS low + P medium dose group,13)AS low + P low dose group.The method of administration was the same as 2.75min after the last administration,except the normal group,each group was injected with indomethacin suspension at 45mg/Kg.6hrs later,all rats were killed,gastric mucosal injury scores were calculated,pathological changes of gastric mucosa were observed,and the levels of MDA,SOD,ROS in gastric mucosa were detected.5.Effects of Huangqi-Gegen pairs,Astragaloside Ⅳ,Puerarin and Astragaloside Ⅳ-puerarin compatibility on gene expression and significant protein in gastric Kear1-Nrf2-ARE signaling pathway..Western-blot and Real time PCR were respectively used to detect the protein and gene expression of nuclear transcription factor(Nrf2),heme oxygenase(HO-1),NDPH quinone dehydrogenase(NQ01),glutathione peroxidase 2(GPx2),glutathione S transferase(GST)in gastric mucosa which obtained from the groups rat in experiment 2 and 4.Results1.The contents of Astragaloside and puerarin in Huangqi-Gegen Water decoction(3:1)were 1.02mg/ml,0.058mg/ml by HPLC Detection,RSD%was less than 3%.2.Effect of Astragaloside Ⅳ-Puerarin compatibility,Huangqi-Gegen pairs on the gastric mucosal injury induced by ethanol in rats.1)Morphology:The injury of gastric mucosa in rats induced by ethanol was serious,the appearance of mucous membrane was dark red,and the cord-like,linear and spotted hemorrhages were common.Compared with the normal group,the pathological changes showed that the gastric mucosa was damaged,the lamina propria of the epithelial cells were separated,the gastric mucosa was necrotic,the submucosal epithelium was damaged,the hemorrhage was lessened,Edema,glandular arrangement irregularity,nuclear pyknosis and even dissolved,telangiectasia,compression,red blood cell extravasation,visible inflaimmatory cell infiltration;the extent and hemorrhage of gastric mucosal injury in the administration groups were significantly lower than the model group,especially AS-IV high and medium dose group,Pur high-dose group,AS medium + P medium compatibility group.2)Gastric mucosal injury score:The administration groups could reduce ethanol-induced gastric mucosal injury score in different degrees.It was shown that a certain dose-effect relationship in the three dose groups of AS-IV and Pur.The score of gastric mucosal injury in the AS medium + P medium group was lower than that in the same dose of AS-Ⅳ and Pur.3)SOD,MDA,ROS:Compared with the normal group,the activity of SOD decreased and the content of MDA and ROS increased in model group..The content of MDA in gastric mucosal of the administration groups was reduced while the activity of SOD was increased;the content of MDA in AS low + P low group was lower significantly than AS-Ⅳ,Pur group at the same dose;Compared with the same dose of AS-Ⅳ group,the level of ROS in AS low + P low group was decreased significantly too.3.Effect of Huangqi-Gegen pairs on indomethacin-induced gastric mucosal injury in ratsIndomethacin intraperitoneal injection can lead to gastric mucosal injury,which showed linear,punctate hemorrhage and erosion-based,While the contents of ROS and MDA in the gastric mucosa increased and the SOD activity decreased.The high,middle and low dosage groups of Huangqi-Gegen pairs could improve the appearance and pathological changes,reduce the injury score and the levels of MDA and ROS of the gastric mucosa induced by indomethacin.4.Effect of Astragaloside IV-Puerarin compatibility on gastric mucosal injury induced by indomethacin in rats1)Morphology:45mg/g indomethacin intraperitoneally injected could cause significant damage of gastric mucosal in the rats.It was showed that the structure of gastric pits was damaged obviously in the model group,epithelial cell degenerated,the gastric mucosal edema,hemorrhage,gastric glandular arranged disorder,atrophy,and inflammatory cells infiltrated.Mucosal appearance and pathological changes could be significantly improved in the positive group,AS-IV groups,AS medium dose+P low dose group,and AS low dose+P low dose group.2)Gastric mucosal injury score:Compared with the model group,the scores of positive group,AS-Ⅳ high dose,medium dose groups,AS medium dose + P low dose group,and AS low dose + P low dose group were obviously decreased.Gastric mucosal injury scores in AS low+P low dose group was significantly lower than that in Pur low dose group.However,compared with model group,there was no significant difference of gastric mucosal injury scores in the Pur groups.3)MDA,SOD,ROS:The content of ROS,MDA increased in indomethacin-induced gastric mucosal injury in rats,while the SOD activity decreased;Compared with the model group,the content of MDA decreased in AS-IV high,medium dose group,AS medium+P medium group,AS low+ P medium group,AS low+ P low group;The SOD activity in gastric mucosal significantly increased in AS-IV high and medium dose groups,Pur high and low dose groups,AS medium dose+ P medium dose group,AS low+ P low dose group(compared with the model group);Except AS low+P medium dose group,the level of ROS in gastric mucosal significantly decreased in administration groups(compared with the model group).5.Molecular mechanism of Astragaloside Ⅳ-Puerarin compatibility,Huangqi-Gegen pairs on the protection of gastric mucosal injury.1)Compared with the normal group,the expression of Nrf2,HO-1,NQ01,GPX2,GST protein and gene in gastric mucosa significantly increased in ethanol-induced gastric mucosal injury model rats;The administration groups except positive group could further up-ragulated the expression of the protein and gene mentioned aboved.2)The expression of HO-1,NQ01,GPX2,GST protein and gene in gastric mucosal were significantly increased in indomethacin-induced gastric mucosal injury model rats;AS-IV medium group can further up-regulate the expression of these genes.Conclusion1.Astragaloside IV,Puerarin and its compatibility,Huangqi-Gegen pairs have certain protective effect on gastric mucosa injury induced by ethanol,and resist the oxidative stress of gastric mucosa.2.Huangqi-Gegen pairs and Astragaloside IV have a certain protective effect of indomethacin-induced gastric mucosal injury,resist the oxidative stress of gastric mucosa.3.Compatibility of Astragaloside IV and Puerarin had certain synergistic effect on gastric mucosal injury in rats.4.The protecting effect of Astragaloside,Puerarin and their combination,Huangqi-Gegen pairs on gastric mucosal injury may be related to activating the Nrf2_ARE pathway,promotion of Nrf2 nuclear translocation,up-regulation of the expression of HO-1,NQO1,GPx2,GST genes and protein;... |
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