摘要
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]
作者
罗波文
邹田德
陈丽玲
曾永娣
郭晓波
游金明
LUO Bowen;ZOU Tiande;CHEN Lilin;ZENG Yongdi;GUO Xiaobo;YOU Jinming(Jiangxi Province Key Laboratory of Animal Nutrition,Engineering Research Center of Feed Development,Jiangxi Agricultural University,Nanchang 330045,China;Jiangxi University of Traditional Chinese Medicine,Nanchang 330004,China)
出处
《动物营养学报》
CAS
CSCD
北大核心
2020年第5期2108-2115,共8页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
国家自然科学基金项目(31760688)
江西省生猪产业技术体系(JXARS 03-营养与饲料岗)。