嗜酸乳杆菌对氧化应激仔猪小肠上皮细胞抗氧化能力和紧密连接蛋白表达的影响

Effects of Lactobacillus acidophilus on Antioxidative Ability and Expression of Tight Junction Protein in Intestinal Epithelial Cells of Piglets under Oxidative Stress

本研究旨在探讨嗜酸乳杆菌对氧化应激仔猪小肠上皮细胞(IPEC J2)抗氧化能力及紧密连接蛋白-1(ZO 1)、闭锁蛋白(occludin)和闭合蛋白(claudin)蛋白表达的影响。首先将IPEC J2分为9组,分别加入0(对照)、0.6、0.8、1.0、1.2、1.4、1.6、1.8和2.0 mmol/L的过氧化氢(H2 O2),每组6个复孔,根据细胞活力确定最佳H2 O2浓度。然后根据结果选择1.2 mmol/L的H2 O2建立氧化应激IPEC J2模型,将细胞分为7组,分别加入为0(对照)、1.0×10^5、1.0×10^6、1.0×10^7、1.0×10^8、1.0×10^9和1.0×10^10 CFU/mL嗜酸乳杆菌,每组6个复孔,处理时间为24 h,分析嗜酸乳杆菌对氧化应激IPEC J2抗氧化能力。最后选择1.2 mmol/L的H2O2建立氧化应激IPEC J2模型,加入1.0×10^9 CFU/mL嗜酸乳杆菌继续培养24 h,将细胞分为4组,分别是对照组(添加等量的磷酸盐缓冲液)、H2O2氧化应激组、嗜酸乳杆菌组、嗜酸乳杆菌组+H2O2组(先加入H2O2后加入嗜酸乳杆菌),每组6个重复,测定ZO 1、occludin和claudin蛋白的表达量。结果显示:1)与对照组相比,当H2 O2浓度高于0.6 mmol/L时,细胞活力显著降低(P<0.05)。与对照组和0.6~1.0 mmol/L H2O2组相比,当H2 O2浓度处于1.2~1.8 mmol/L时,细胞活力显著下降(P<0.05)。2)与对照组相比,当嗜酸乳杆菌浓度高于1.0×108 CFU/mL时,细胞活力显著提高(P<0.05)。3)在氧化应激条件下,随着嗜酸乳杆菌浓度的增加,细胞活力逐渐上升。与对照组相比,当嗜酸乳杆菌浓度低于1.0×10^7 CFU/mL时,细胞活力显著降低(P<0.05),当嗜酸乳杆菌浓度高于1.0×10^9 CFU/mL时,细胞活力显著提高(P<0.05)。4)与对照组相比,当嗜酸乳杆菌浓度高于1.0×106 CFU/mL时,细胞中丙二醛(MDA)含量显著降低(P<0.05),嗜酸乳杆菌浓度高于1.0×10^7 CFU/mL时,细胞中超氧化物歧化酶(SOD)活性显著上升(P<0.05),其他各组中谷胱甘肽过氧化物酶(GSH Px)活性较对照组显著升高(P<0.05)。5)与H2O2氧化应激组相比,嗜酸乳杆菌组和嗜酸乳杆菌+H2 O2组中细胞浆内ZO 1和occludin蛋白表达量显著降低(P<0.05);与对照组和H2O2氧化应激组相比,嗜酸乳杆菌组和嗜酸乳杆菌+H2O2组中clau din蛋白表达量显著降低(P<0.05)。由此可知,嗜酸乳杆菌能够促进细胞的生长,增强细胞的抗氧化能力,降低胞浆内紧密连接蛋白的表达量。

The purpose of this study was to investigate the effects of Lactobacillus acidophilus on antioxidative ability and expression of tight junction protein(occludin,ZO 1 and claudin 1)in intestinal epithelial cells of piglets(IPEC J2)under oxidative stress.Firstly,the IPEC J2 were treated with different H2O2 concentrations,including 0(control),0.6,0.8,1.0,1.2,1.4,1.6,1.8 and 2.0 mmol/L,with 6 replications in each group.The optimum concentration of H2O2 was determined according to cell activity.And then,the 1.2 mmol/L H2O2 were used to induce oxidative stress.The cells were divided into 7 groups,and added 0(control),1.0×105,1.0×10^6,1.0×10^7,1.0×10^8,1.0×10^9 and 1.0×10^10 CFU/mL Lactobacillus acidophilus,respectively,with 6 replications in each group,and treated for 24 h,analyzing the antioxidant ability of Lactobacillus aci dophilus in IPEC J2 under oxidative stress.At last,1.2 mmol/L H2O2 was selected to establish the IPEC J2 oxidative stress model,and then 1.0×10^9 CFU/mL Lactobacillus acidophilus was added to culture cells for 24 h,and then cells was divided into four groups:the control group(adding equivalent phosphate buffer),the H2O2 oxidative stress group,the Lactobacillus acidophilus group,and the Lactobacillus acidophilus+H2O2 group(adding H2O2 first and then Lactobacillus acidophilus),with 6 replications in each group.The expres sions of epithelial tight junction proteins(occludin,ZO 1 and claudin 1)were measured.The results showed as follows:1)compared with the control group,at the concentration of H2O2≥0.6 mmom/L,the cell activity was significantly reduced(P<0.05).Compared with control group and 0.6 to 1.0 mmol/L H2O2 groups,the activities of cells were decreased significantly at the concentration between 1.2 and 1.8 mmol/L H2O2(P<0.05).2)Compared with control group,at the concentration of Lactobacillus acidophilus≥1.0×10^8 CFU/mL,the cell activity was significantly increased(P<0.05).3)Under oxidative stress,with the in crease of Lactobacillus acidophilus concentration,cell activity was increaseed gradually.Compared with control group,at the concentration of Lactobacillus acidophilus≤1.0×10^7 CFU/mL,the cell activity was significantly decreased(P<0.05),at the concentration of Lactobacillus acidophilus≥1.0×10^9 CFU/mL,cell activity was increased significantly(P<0.05).4)Compared with control group,at the concentration of Lactobacillus aci dophilus≥1.0×10^6 CFU/mL,the content of malonaldehyde(MDA)in cells was significantly reduced(P<0.05).Compared with control group,at the concentration of Lactobacillus acidophilus≥1.0×10^7 CFU/mL,superoxide dismutase(SOD)activity was significantly increased(P<0.05),and glutathion peroxidase(GSH Px)activity in cells in other groups was significantly increased compared with control group(P<0.05).5)Compared with the H2O2 oxidative stress group,the protein expressions of ZO 1,occludin and claudin proteins in Lactobacillus acidophilus group and Lactobacillus acidophilus+H2O2 group were significantly decreased(P<0.05).Compared with the control group and H2O2 oxidative stress group,claudin protein expression in Lacto bacillus acidophilus group and Lactobacillus acidophilus+H2O2 group was significantly decreased(P<0.05).In conclusion,Lactobacillus acidophilus can promote the growth of cells,enhance the antioxidant ability of cells,and reduce the expression of intracytoplasmic tight junction protein.[Chinese Journal of Animal Nutri tion,2020,32(5):2108 2115]