Research About The Effect Of Astragaloside Ⅳ On Antioxidant Function And Correlative Action Mechanism Of Calves

Oxidative stress destroys the integrity and functionality of mammalian intestinal epithelial cells,and causes gastrointestinal disfunction.Astragaloside IV（ASIV）,as an extract of the traditional Chinese medicine astragalus,has a variety of biological activities,including anti-inflammatory and antioxidant effects.In this study the effect of Astragaloside IV on the antioxidant function of calves and its mechanism,we used Astragaloside IV to fed calves and hydrogen peroxide（H₂O₂）to stimulate the calf small intestinal epithelial cells to construct an oxidative stress model in vitro.1 Effect of Astragaloside IV on growth performance and antioxidant function of calvesIn order to investigate the effect of Astragaloside IV on growth performance and antioxidant function of calves,Holstein male calves were selected and randomly divided into four groups and fed of 0,15,30,and 60 mg/（head·d）.Treatment groups were fed different amounts of Astragaloside IV with 6 L/day milk replacer from 7–60 days,then weaned and offered water,starter,and Chinese wildrye ad libitum for the whole trial period.The results showed that as the dose of Astragaloside IV increased,the dry matter intake of each group did not change significantly,but the final weight and average daily weight gain at 120 days increased significantly.During the experiment,there was no significant difference in the body size of calves in each treatment group,but the activities of CAT,GSH-Px and SOD increased significantly with the increase of Astragaloside IV,and the production of MDA decreased significantly with the increase of feed amount.In addition,IL-6,IL-8,and TNF-αwere not affected by Astragaloside IV.2 Effect of Astragaloside IV on antioxidant function of calf small intestinal epithelial cells and molecular mechanism2.1 Establish an oxidative strss model induced by H₂O₂ in calf small intestinal epithelial cellsThe expression of Nrf2,NQO1 and HO-1 and the cell survival rate were detected by stimulating the calf small intestinal epithelial cells with H₂O₂ at different concentrations and times.The results showed that 300-450μmol/L H₂O₂ induced a significant increase the expression of antioxidant genes in cells,and H₂O₂ treatment above 300μmol/L significantly reduced the cell survival rate.When the H₂O₂concentration reached 350μmol/L,the cell survival rate was 59%.Therefore,350μmol/L H₂O₂ was used to treat calf small intestine epithelial cells for 12 h to establish an oxidative stress model.2.2 Protective effect of Astragaloside IV on oxidative stress induced by H₂O₂ in calf small intestine epithelial cellsIn order to study the protective effect of Astragaloside IV on oxidative damage of cells,this experiment pretreated with different concentrations of Astragaloside IV and added 350μmol/L H₂O₂ to treat calf small intestinal epithelial cells for 12h,and measured of cell survival rate,LDH activity,reactive oxygen species（ROS）level,total antioxidant capacity and antioxidant enzyme activity,malondialdehyde（MDA）formation and apoptosis rate.The results showed that compared with control,Astragaloside IV had no adverse effect on cell viability.Compared with H₂O₂treatment,Astragaloside IV pretreatment and then added H₂O₂ significantly increased cell survival rate and antioxidant enzyme activity,while LDH activity,ROS levels,MDA formation,and apoptosis rate were significantly reduced.The results indicate that Astragaloside IV has protective effect on H₂O₂-induced oxidative damage of calf small intestinal epithelial cells.2.3 Molecular mechanism of Astragaloside IV protect against oxidative damage in calf small intestine epithelial cellsIn order to further explore the molecular mechanism of ASIV protect against the oxidative damage in calf small intestine epithelial cells,this experiment was treated with 350μmol/L H₂O₂ for 12 h after pretreatment with different concentrations of ASIV to detect antioxidant genes and proteins Nrf2,NQO1 and HO-1expression changes and distribution situation.The results showed that ASIV pre-activated the expression of antioxidant genes and proteins in cells,and the protein expression was mainly concentrated in the nucleus.Next,the activation of Nrf2-ARE signaling pathway was inhibited by inhibitor pretreatment,and it was found that the activation of antioxidant genes and proteins was inhibited.Therefore,it was showed that ASIV protect the calf small intestine epithelial cells from oxidative damage by activating the Nrf2-ARE signaling pathway.To sum up,Astragaloside IV has protective effect on oxidative damage in calf small intestine epithelial cells.The mechanism is closely related to the activation of Nrf2-ARE signaling pathway.Meanwhile,this study also provides a research basis for the healthy growth of calves.