白藜芦醇对脂多糖诱导的猪小肠上皮细胞炎性损伤和屏障功能障碍的缓解作用

Mitigating Effects of Resveratrol on Lipopolysaccharide-Induced Inflammatory Damage and Barrier Dysfunction in Porcine Small Intestinal Epithelial Cells

本试验旨在研究白藜芦醇对脂多糖(LPS)诱导的猪小肠上皮细胞(IPEC-J2细胞)炎性损伤与屏障功能障碍的缓解作用。将IPEC-J2细胞在含有不同浓度的LPS和白藜芦醇的培养基中分别培养18 h后,MTT法检测细胞活性确定LPS造模浓度(10.0 mg/L)和白藜芦醇预处理浓度(5.0、20.0、35.0μmol/L)。分别用上述确定的预处理浓度(5.0、20.0、35.0μmol/L)的白藜芦醇预处理IPEC-J2细胞后进行LPS(10.0 mg/L)诱导,同时设置对照组(不添加LPS与白藜芦醇)与LPS单独刺激组(添加10.0 mg/L LPS),测定细胞活性、细胞迁移率、炎症因子分泌量、Toll样受体4(TLR4)/核转录因子-κB(NF-κB)信号通路及紧密连接相关基因mRNA相对表达量。结果显示:相较于LPS单独刺激组,20.0μmol/L白藜芦醇预处理可以显著缓解由LPS刺激引起的IPEC-J2细胞活性的降低(P<0.05);5.0和20.0μmol/L白藜芦醇预处理组IPEC-J2细胞迁移率较LPS单独刺激组显著提高(P<0.05);5.0、20.0和35.0μmol/L白藜芦醇预处理组细胞上清液中白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)含量显著低于LPS单独刺激组(P<0.05),白细胞介素-10(IL-10)含量显著高于LPS单独刺激组(P<0.05);5.0和20.0μmol/L白藜芦醇预处理显著缓解了LPS刺激引起的细胞中IL-8、TNF-α、白细胞介素-1β(IL-1β)、TLR4、髓样分化因子88(MyD88)的mRNA相对表达量的升高(P<0.05)以及IL-10与NF-κB抑制蛋白α(IκBα)mRNA相对表达量的降低(P<0.05);5.0和20.0μmol/L白藜芦醇预处理后细胞中封闭蛋白-1(claudin-1)、闭锁蛋白(occludin)和闭锁小带蛋白-1(ZO-1)mRNA相对表达量显著提高(P<0.05),且35.0μmol/L白藜芦醇预处理后细胞中ZO-1 mRNA相对表达量显著提高(P<0.05)。综上所述,白藜芦醇通过提高细胞迁移率、调节炎症细胞因子分泌、抑制TLR4/NF-κB信号通路过度激活,提高紧密连接相关基因表达,缓解LPS诱导的IPEC-J2细胞的炎性损伤和屏障功能障碍。

The aim of this experiment was to investigate the alleviating effect of resveratrol(RES)on lipopolysaccharide(LPS)-induced inflammatory damage and barrier dysfunction in porcine small intestinal epithelial cells(IPEC-J2 cells).IPEC-J2 cells were cultured for 18 h in medium supplemented with different concentrations of LPS and RES,respectively,and the cell activity was determined by MTT assay to determine the LPS modeling concentration(10.0 mg/L)and RES pretreatment concentrations(5.0,20.0 and 35.0μmol/L).The cells were pretreated with the above concentrations(5.0,20.0 and 35.0μmol/L)of RES and then subjected to LPS(10.0 mg/L)induction.A control group(no addition of LPS and RES)and an LPS alone stimulation group(addition of 10.0 mg/L LPS alone)were set up to determine cell activity,cell migration rate,inflammatory cytokine secretion,and the mRNA relative expression levels of TLR4/NF-κB signaling pathway and tight junction related genes.The results showed that,compared with LPS alone stimulation group,20.0μmol/L RES representment significantly alleviated the decrease of IPEC-J2 cell activity induced by LPS stimulation(P<0.05);the migration rate of IPEC-J2 cells was significantly increased in the 5.0 and 20.0μmol/L RES pretreatment groups compared with the LPS alone stimulation group(P<0.05).The interleukin-8(IL-8)and tumor necrosis factor-α(TNF-α)contents were significantly lower and the interleukin-10(IL-10)content was significantly higher in the supernatant of cells in the 5.0,20.0 and 35.0μmol/L RES pretreatment groups than those in the LPS alone stimulation group(P<0.05).Compared with LPS alone stimulation group,5.0 and 20.0μmol/L RSE pretreatment significantly alleviated the increase of the mRNA relative expression levels of IL-8,TNF-α,interleukin-1β(IL-1β),TLR4 and myeloid differentiation factor 88(MyD88)and the decrease of the mRNA relative expression levels of IL-10 and NF-κB inhibitor proteinα(IκBα)in cells induced by LPS stimulation(P<0.05);the mRNA relative expression levels of claudin-1,ocludin and zona occludens-1(ZO-1)were significantly increased after 5.0 and 20.0μmol/L RES pretreatment(P<0.05),and the mRNA relative expression level of ZO-1 significantly increased after 35.0μmol/L RES pretreatment(P<0.05).In conclusion,RES alleviated LPS-induced inflammatory damage and barrier dysfunction in IPEC-J2 cells by increasing cell migration rate,regulating inflammatory factor secretion,curbing excessive activation of TLR4/NF-κB signaling pathway and increasing expression of tight junction-related genes.