刺五加苷E对脂多糖介导的仔...细胞屏障功能基因表达的影响_胡枭偲.pdf

吉林农业大学学报 2023，45（2）：213-220http:/Email:jlndxb Journal of Jilin Agricultural University刺五加苷E对脂多糖介导的仔猪小肠上皮细胞屏障功能基因表达的影响*胡枭偲，赵宝，范越蠡，车东升*吉林农业大学动物科学技术学院，动物生产及产品质量安全教育部重点实验室，吉林省动物营养与饲料科学重点实验室，吉林省生猪产业技术科技创新中心，长春130118摘 要：通过脂多糖（Lipopolysaccharide，LPS）诱导仔猪小肠组织上皮细胞（IPEC-J2）建立损伤模型，探讨刺五加苷E（Eleutheroside E，EE）对IPEC-J2屏障功能的保护作用。采用单因素试验方法，分为对照组C组（等量的完全培养基），E组（完全培养基+0.1 g/L EE），L组（完全培养基+10 mg/L LPS），EL组（完全培养基添加0.1 g/L EE预处理6 h后再添加10 mg/L的 LPS进行培养），采用RTCA及q-PCR方法对IPEC-J2细胞的增殖活性、炎症因子以及紧密连接蛋白mRNA表达量变化进行检测。EE对LPS介导的IPEC-J2的增殖活性试验结果显示：与C组相比，EE对IPEC-J2细胞增殖活性显著提高，LPS对IPEC-J2细胞有显著的抑制作用（P0.05），且EL组IPEC-J2增殖活性与L组相比明显提高（P0.05）。EE对LPS介导的IPEC-J2的免疫细胞因子影响的结果显示：与对照组相比，EE显著降低了炎性细胞因子IL-6、TNF-、INF-的mRNA相对表达量（P0.01），抗炎性细胞因子IL-10、TGF-显著升高（P0.01）；L组的炎性细胞因子mRNA相对表达量显著升高（P0.01），抗炎性细胞因子mRNA相对表达量显著降低（P0.01）。EE预处理组相比于LPS损伤组，IL-6、TNF-、INF-mRNA相对表达量显著降低（P0.01），IL-10 mRNA相对表达量显著升高（P0.05）。EE对LPS介导的IPEC-J2细胞紧密连接蛋白mRNA相对表达量的影响试验结果显示：与C组比，E组Occludin（P0.05）、ZO-1和 Claudin-3（P0.01）的mRNA相对表达量显著升高，LPS显著下调Occludin、Claudin-3和ZO-1的mRNA相对表达量（P0.01）。与LPS损伤组相比，EE预处理组显著上调了Occludin（P0.05）、Claudin-3和ZO-1（P0.01）的mRNA相对表达量。结果表明：添加EE能够缓解LPS诱导的IPEC-J2细胞增殖活性的降低，改变细胞因子和紧密连接蛋白的基因表达，进而起到保护肠道机械屏障和免疫屏障功能的作用。关键词：刺五加苷E；脂多糖；仔猪小肠上皮细胞；紧密连接蛋白；细胞因子中图分类号：S828 文献标志码：A 文章编号：1000-5684（2023）02-0213-08DOI：10.13327/j.jjlau.2021.1227引用格式：胡枭偲，赵宝，范越蠡，等.刺五加苷E对脂多糖介导的仔猪小肠上皮细胞屏障功能基因表达的影响J.吉林农业大学学报，2023，45（2）：213-220.Effects of Eleutheroside E on Barrier Function Gene Expression Mediated by Lipopolysaccharide in Piglet Intestinal Epithelial Cells*HU Xiaocai，ZHAO Bao，FAN Yueli，CHE Dongsheng*Key Laboratory of Animal Production and Product Quality and Security of the Ministry of Education，Jilin Provincial Key Laboratory of Animal Nutrition and Feed Science，Jilin Provincial Swine Industry Technical Innovation Center，College of Animal Science and Technology，Jilin Agricultural University，Changchun 130118，ChinaAbstract：The purpose of this study was to establish an injury model of piglet intestinal epithelial*基金项目：国家重点研发计划项目（2021YFD1300201-3），吉林省科技发展计划项目（20210202045NC，20190301039NY）作者简介：胡枭偲，男，在读硕士，研究方向：单胃动物营养。收稿日期：2021-04-22*通信作者：车东升，E-mail：吉林农业大学学报 2023 年 4 月Journal of Jilin Agricultural University 2023，Aprilcell(IPEC-J2)induced by lipopolysaccharide(LPS),and to explore the protective effect of eleutheroside E(EE)on the barrier function of IPEC-J2.Experimental design:Single factor test method was used,treatments were divided into control group C(the same amount of complete medium),E group(complete medium+0.1 g/L EE),L group(complete medium+10 mg/L LPS),EL group(complete medium pretreated with 0.1 g/L EE for 6 h and then cultured with 10 mg/L LPS),and proliferation activity,inflammatory factors and tight junction proteins mRNA of IPEC-J2 cells were analyzed by RTCA and q-PCR methods.Changes in expression levels were detected.The results of the proliferation activity test of EE on IPEC-J2 mediated by LPS showed that compared with group C,EE significantly increased the proliferation activity of IPEC-J2 cells,and LPS had a significant inhibitory effect on IPEC-J2 cells(P0.05).Compared with group L,the proliferation activity of IPEC-J2 in the EL group was significantly increased(P0.05).The results of EE on immune cytokines of LPS-mediated IPEC-J2 showed that compared with the control group,EE significantly decreased the relative mRNA expressions of inflammatory cytokines IL-6,TNF-and INF-(P 0.01),and the anti-inflammatory cytokines IL-10 and TGF-were significantly increased(P0.01);the relative expression of inflammatory cytokine mRNA in group L was significantly increased(P0.01),and the anti-inflammatory cytokine mRNA relative expression level was significantly decreased(P0.01).Compared with the LPS injury group,the relative expressions of IL-6,TNF-and INF-mRNA in the EE pretreatment group were significantly decreased(P0.01),and the relative expression of IL-10 mRNA was significantly increased(P0.05).The effect of EE on the relative expression of tight junction proteins mRNA in IPEC-J2 cells mediated by LPS showed that compared with group C,the mRNA of Occludin(P0.05),ZO-1 and Claudin-3(P0.01)relative mRNA expression levels were significantly increased in group E,and LPS significantly down-regulated the relative mRNA expressions of Occludin,Claudin-3 and ZO-1(P0.01).Compared with the LPS injury group,the EE pretreatment group significantly up-regulated the relative mRNA expressions of Occludin(P0.05),Claudin-3 and ZO-1(P0.01).In conclusion,the addition of EE could alleviate the LPS-induced decrease in the proliferation activity of IPEC-J2 cell,alter the gene expression of cytokines and tight junction proteins,and then play a role in protecting the intestinal mechanical barrier and immune barrier functions.Key words：eleutheroside E；lipopolysaccharide；piglet intestinal epithelial cell；tight junction protein；cytokine近年来，抗生素通常被用于预防和治疗牲畜疾病，然而抗生素的过量使用会造成环境污染和病原体耐药性等问题。因此，开发有效的免疫调节剂是生产健康动物的必要条件1。一些植物提取物已被证明是有效的免疫调节剂2，并且是治疗炎症性疾病的药物3。刺五加（Acanthopanax senticosus）又被称为西伯利亚人参，是主要分布在中国东北地区、俄罗斯、日本和朝鲜的一种传统中草药4-5，具有活血化瘀、滋补脾肾等功效，