| With the rapid development of animal husbandry industry in China,large scale,high density intensive culture mode,epidemic disease,overuse of antibiotics and other harmful factors outside stimulation,cause oxidative stress problems in livestock breeding process.Oxidative stress is the numerous disease occurrence and important inducing or promoting factors of development.For animal husbandry industry,it is very important to solve the problem of oxidative stress for the healthy livestock farming.In this study,we used Hericium erinaceus as raw material,response surface method was used to optimize water extract-alcohol precipitation method obtained raw sugar,and then used the DEAE-52 purified cellulose to purify raw sugar.Swine intestinal epithelial cells(IPEC-J2 cells)as the research object,applied H2O2 construction model of oxidative stress,concentration of H2O2 and the best safety effect dose of HEPs was determined by MTT assay.We measured the intracellular ROS level of IPEC-J2 cells and cell antioxidant activity,cell membrane permeability,apoptosis index.Observed the effect of HEPs on ZO-1,Claudin-1,Occludin gene and protein related with tight junction expression in IPEC-J2 cells under oxidative stress by using RT-PCR and Western-blot.Results are as follows:(1)To optimize the extraction process of hericium polysaccharide by the response surface method,got the best condition for extracting time was 3.8 h,25 mL/g,ratio of liquid extraction temperature is 81℃,the extraction yield of 3.09%.(2)Determination of effects of different concentration of H2O2/HEPs on the proliferation rate of IPEC-J2 by MTT method,the optimal concentration of H2O2 concentration was 0.4 mM,HEPs was 20～500 ug/mL,selected 50 μg/mL,100 μg/mL,200 μg/mL as the safety concentration of HEPs for low,medium and high dose group.(3)After pretreatment of HEPs on IPEC-J2 cells,the level of intracellular ROS was significantly decreased under oxidative stress,inhibited the lipid peroxidation process and the activity of SOD and CAT was also significantly increased,enhanced antioxidant capacity.At mean time,HEPs protecting the mitochondrial membrane potential has a polarity,inhibited the cell apoptosis;decreased the release rate of LDH,explained the HEPs maintain the integrity of cell membrane effectively,significantly improved permeability of cell membrane under the oxidative stress.(4)Oxidative stress model of IPEC-J2 cells builded by H2O2 significantly down regulation the expression of ZO-1,Claudin-1,and Occludin genes and proteins associated with tight junction.At the gene level,the high dose of HEPs could increased the transcription level of ZO-1,Claudin-1 and Occludin in oxidative stress to some extent.The expression of ZO-1,Claudin-1 and Occludin in the oxidative stress state could be increased at the middle and high doses of HEPs.Therefore,HEPs can improved the expression of ZO-1,Claudin-1 and Occludin genes and proteins associated with tight junction in IPEC-J2 under oxidative stress,protect the barrier function of the cell membrane,increase the survival rate of cells.In conclusion,HEPs can improved the antioxidant capacity of IPEC-J2 cells under oxidative stress,inhibited the apoptosis process.At the molecular level,HEPs significantly increased the expression of ZO-1,Claudin-1 and Occludin gene and protein associated with tight junction,help to maintain the integrity of the intestinal mechanical barrier and normal permeability,reduce the risk of the gut from exogenous antigen... |
