Protective Effect Of Resveratrol On H₂O₂-induced IPEC-J2 Cells Apoptosis

Oxidative stress has been one of the important factors of restricting the development of breeding industry.When the organism is exposed under harmful stimulation,reactive oxygen species（ROS）concentration increased that far more than cell anti-oxidation ability,the organism will be in a state of oxidative stress which lead to cell protein abnormal expression,lipids and DNA damage,cell dysfunction and a variety of diseases.The excessive ROS-induced oxidative stress can trigger inflammatory cascade and a series of tissue damage,hydrogen peroxide（H₂O₂）is contributing to a major molecules of ROS regulating oxidative stress.The abnormal intestinal epithelial cells metabolism result in H₂O₂ over production,H₂O₂ as toxic by-products can destroy the intestinal mucosa,induce oxidative stress damage and affect the gastrointestinal barrier function.In recent years there have been numerous studies showed that H₂O₂ mediated oxidative stress is one of the main ways to cause a variety of cell apoptosis.Apoptosis also known as programmed cell death is autonomic death regulated by a series of genes,which is necessary to maintain the organism homeostasis balance by the interaction of pro-apoptosis and anti-apoptosis genes,for example: apoptosis protein molecules the Bcl-2 family have 25 family members,which is divided into anti-apoptotic effect the Bcl-2 subfamily（Mcl-1,Bcl-2,Bcl-w and Bcl-xL）and pro-apoptosis subfamily of BAX（BAX and Bak）;Caspase cysteine protease family is also directly involved in the apoptosis process,which caspase-3 and caspase-9 have been researched widely in the process of apoptosis.At the same time,the cell apoptosis induced by oxidative stress can cause cell DNA sequence fragment,cell shrinkage and the nucleus pycnosis,so the development of natural antioxidants for relief and protection of oxidative stress damage is great significance.Resveratrol（Rel）also known as trans（E）-3,5,4 ’-3 hydroxy stilbene,is natural polyphenol compounds,derived from the grapes,peanuts,mulberries and giant knotweed many kinds of plants.As people for natural resources extensive research,Rel belong to at least 21 families,31 genera,72 species,is a kind of phytoalexin.The present study showed that Rel has anti-cancer,anti-tumor effects both in vivo and in vitro.It has been proved in many kinds of cell lines including cattle bovine MECs,Caco–2 and human umbilical vein endothelial cells.Rel can resist the oxidative stress and apoptosis indued by H₂O₂.IPEC-J2 is pig jejunum epithelial cell line,it is widely used in swine intestinal permeability and inflammation research,but little is known about Rel on IPEC-J2 cell apoptosis,and the specific mechanism is not yet clear.In order to define the effect of Rel on H₂O₂-indued apoptosis in IPEC-J2 cell,follow experiment was designed.H₂O₂-indued oxidative stress model was set,a series of experiments were carry out for testing the effect of Rel on the cell apoptosis induced by H₂O₂.Our results showed that 50μM H₂O₂ treatment IPEC-J2 cells for 24 h can induce apoptosis,the apoptosis rate is up to 40%,and while using 5,10,25,50μM Rel pretreatment for 1 h resperctively,25 and 50μM Rel significantly inhibited IPEC-J2 cell apoptosis.We detected the expression changes of Cleaved caspase3,Bcl-2,Bax,BAD and ERK1/2,AKT,p-p38 signaling pathway molecules by protein immunoblot assay.After H₂O₂ treatment the ratio of Bcl-2/Bax was decreased,meanwhile the expression of the actived BAD and Cleaved caspase3 were increased.However,the results were reversed after the pre-treatment by 50μM Rel for 1h.Our results confirmed that 50μM Rel can active ERK1/2 and AKT,and inhibit H₂O₂-induced IPEC-J2 cell apoptosis by p-p38 signaling pathway.