Study On The Effects And Mechanisms Of Lactobacillus Acidophilus On Oxidative Stress Induced By Oxidative Stress In Porcine Intestinal Epithelial Cells

The purpose of this experiment was to oxidative stress model cell lines,Western blot（Western Blot）,real time fluorescence quantitative PCR（RT-PCR）and other technical means to explore the intrinsic relationship between Lactobacillus acidophilus and oxidative stress in piglets intestinal epithelial cells and elucidate the NF-κB signaling pathway in the oxidative stress of piglet intestinal epithelial cells mediated role in the process of from the cellular and molecular level.The research results provide a theoretical basis for the mechanism of action of Lactobacillus acidophilus will reveal oxidative stress regulation of epithelial cells in small intestine of piglets,and provide a theoretical basis for application of Lactobacillus acidophilus in piglet feed.The results are as follows:（1）the survival rate of cells decreased with the increase of H₂O₂ concentration,H₂O₂ concentration in 1.2¹.8 m M,although the survival rate of cells decreased but the difference was not significant（P>0.05）,the survival rate is about 60%.The supernatant was observed in SOD,GSH-Px,there are significant differences in CAT activity,and MDA content was not significant the difference.Among them,with the concentration of H₂O₂ increased,SOD,GSH-Px,CAT activity was significantly decreased（P<0.05）.There were significant differences in each group,the content of MDA in cells and SOD,GSH-Px,CAT activity had no significant difference.The content of MDA decreased with the concentration of H₂O₂ increased.These results suggested that oxidative stress of 1.2 m M H₂O₂ can induce IPEC-J2 cells,can be used as the final concentration of construct the model of oxidative stress in cells in vitro.（2）with the increase of Lactobacillus acidophilus concentration,cell viability increased with the increase of Lactobacillus acidophilus.concentration,the cell viability increased gradually,when the concentration is greater than 1×108 CFU-m L-1,cell viability than the control group;when Lactobacillus acidophilus concentration greater than 1×109 CFU-m L-1.Cell viability was not significantly different（P>0.05）.The supernatant was observed in SOD,there are significant differences in GSH-Px activity,CAT activity and no significant difference.There are significant differences in the content of MDA in cells.Among them,along with the increase of Lactobacillus acidophilus,the concentration of SOD,the activity of GSH-Px increased significantly（P<0.05）,decreased the content of MDA（P<0.05）.Add the Lactobacillus acidophilus group,cytoplasmic ZO-1,Occludin,Claudin protein expression was lower than control group and hydrogen peroxide group（P<0.05）.Hydrogen peroxide group was higher than control group,but the difference was not significant.（3）hydrogen peroxide can increase the intracellular phosphorylation of I kappa B alpha.Lactobacillus acidophilus after pretreatment of P-I kappa B alpha protein significantly decreased.Bay inhibitors can reverse this decline.Lactobacillus acidophilus can significantly increase the expression of intracellular phosphorylation of NF-kappa B protein（P<0.05）Lactobacillus acidophilus bacteria.Increased expression of TLR2 protein in cells（P<0.05）.The expression of the three inhibitors can significantly inhibit TLR2 protein within the cell（P<0.05）.Lactobacillus acidophilus can significantly increase the expression of IL-1 beta protein within the cell（P<0.05）.PDTC,Bay,parthenolide pretreatment could inhibit the expression of（P<0.05）compared with the control group,hydrogen peroxide and Lactobacillus acidophilus can increase the intracellular expression of Myd88 m RNA（P<0.05）.Compared with Lactobacillus acidophilus group,the expression of Bay after pretreatment with inhibitors of m RNA can downregulate Myd88,PDTC pretreatment group but significantly enhanced the expression of the gene Parthenolide,the difference was not significant.Compared with the control group,the other groups TLR2,TLR3 m RNA expression was significantly decreased,the difference was not significant.Compared with the control group,hydrogen peroxide group and Lactobacillus acidophilus group,the expression of PDTC and parthenolide group were significantly decreased TLR4 m RNA.Compared with the control.Hydrogen peroxide group group,the expression of bay inhibitor group and parthenolide group were significantly decreased p65 m RNA,and Lactobacillus acidophilus and Lactobacillus acidophilus pretrearment group significantly increased the expression of p65 m RNA（P<0.05）.Compared with the control group,hydrogen peroxide group,Lactobacillus acidophilus group and bay pretreatment group were significantly increased the relative expression of IKK beta m RNA（P<0.05）.The other groups compared with the control group,I kappa B alpha m RNA expression was significantly decreased in other groups.The difference was not significant.Hydrogen peroxide can significantly increase the expression of tumor necrosis factor,Lactobacillus acidophilus pretreatment High expression can significantly decrease tumor necrosis factor（P<0.05）.Lactobacillus acidophilus significantly increased IL-1 beta,relative expression of IL-8 m RNA（P<0.05）.Bay and parthenolide inhibitors can significantly mitigate the high expression（P<0.05）.Therefore,we can conclude that: oxidative stress 1.2 m M H₂O₂ can induce IPEC-J2 cells,can be used as the final concentration of the test construct the model of oxidative stress in cells in vitro.Lactobacillus acidophilus can promote cell growth,enhance cellular antioxidant levels,reduce the expression of tight junction proteins in the cytoplasm.The high expression of eosinophils Lactobacillus pretreatment could significantly reduce TNFalpha and other cytokines,reduce expression of IKK induced by H₂O₂,inhibition of NF-κB signaling pathway occurs in a certain extent.