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

Effects of Eleutheroside E on Barrier Function Gene Expression Mediated by Lipopolysaccharide in Piglet Intestinal Epithelial Cells

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(P<0.05),且EL组IPEC-J2增殖活性与L组相比明显提高(P<0.05)。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-α、INF-γ的mRNA相对表达量(P<0.01),抗炎性细胞因子IL-10、TGF-β显著升高(P<0.01);L组的炎性细胞因子mRNA相对表达量显著升高(P<0.01),抗炎性细胞因子mRNA相对表达量显著降低(P<0.01)。EE预处理组相比于LPS损伤组,IL-6、TNF-α、INF-γmRNA相对表达量显著降低(P<0.01),IL-10 mRNA相对表达量显著升高(P<0.01),TGF-β的mRNA相对表达量上调,但差异不显著(P>0.05)。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(P<0.05)、ZO-1和Claudin-3(P<0.01)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(P<0.01)。与LPS损伤组相比,EE预处理组显著上调了Occludin(P<0.05)、Claudin-3和ZO-1(P<0.01)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。

The purpose of this study was to establish an injury model of piglet intestinal epithelial cell(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(P<0.05).Compared with group L,the proliferation activity of IPEC-J2 in the EL group was significantly increased(P<0.05).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-αand INF-γ(P<0.01),and the anti-inflammatory cytokines IL-10 and TGF-βwere significantly increased(P<0.01);the relative expression of inflammatory cytokine mRNA in group L was significantly increased(P<0.01),and the anti-inflammatory cytokine mRNA relative expression level was significantly decreased(P<0.01).Compared with the LPS injury group,the relative expressions of IL-6,TNF-αand INF-γmRNA in the EE pretreatment group were significantly decreased(P<0.01),and the relative expression of IL-10 mRNA was significantly increased(P<0.01).The relative expression of TGF-βmRNA was up-regulated,but the difference was not significant(P>0.05).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(P<0.05),ZO-1 and Claudin-3(P<0.01)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(P<0.01).Compared with the LPS injury group,the EE pretreatment group significantly up-regulated the relative mRNA expressions of Occludin(P<0.05),Claudin-3 and ZO-1(P<0.01).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.