摘要
【目的】为了揭示地锦草(EH)在猪小肠上皮细胞(IPEC-J2)中的抗炎和抗氧化作用。【方法】利用不同浓度EH水提物(0、5、10、50、125、200μg/mL)处理IPEC-J2细胞12 h,通过CCK-8法检测IPEC-J2细胞活力,确定EH处理细胞的最佳浓度。将IPEC-J2细胞随机分为对照组(CT)、脂多糖(LPS)组(LPS)、EH+LPS组(ELP),每组3个重复。CT组细胞正常培养不做任何处理,LPS组细胞用5μg/mL LPS处理,ELP组细胞用5μg/mL LPS和最佳浓度EH共处理,各组细胞均处理12 h后,收集细胞和上清。利用实时荧光定量PCR方法检测细胞中白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)、kelch样环氧氯丙烷相关蛋白1(Keap1)、核因子E2相关因子(Nrf2)、血红素加氧酶1(HO-1)mRNA表达;ELISA法检测上清液中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的含量,化学荧光法检测上清液中活性氧(ROS)水平。【结果】与0μg/mL EH组相比,5、50μg/mL EH组IPEC-J2细胞活力显著增加(P<0.05);10μg/mL EH组IPEC-J2细胞活力极显著增加(P<0.01),125μg/mL EH组IPEC-J2细胞活力下降,但差异不显著(P>0.05),200μg/mL EH组IPEC-J2细胞活力极显著下降(P<0.01)。因此,后续选用10μg/mL EH处理IPEC-J2细胞。与CT组相比,LPS组IPEC-J2细胞中IL-6、TNF-α基因mRNA的表达极显著增加(P<0.01),上清液中MDA含量和细胞ROS的荧光强度极显著增加(P<0.01),Keap 1、Nrf 2和HO-1基因mRNA表达极显著下降(P<0.01),抗氧化酶SOD、GSH-Px的含量极显著下降(P<0.01)。与LPS组相比,ELP组IPEC-J2细胞中IL-6基因mRNA表达极显著下降(P<0.01),TNF-α基因mRNA的表达显著下降(P<0.05),细胞上清液中MDA含量和细胞ROS的荧光强度均极显著下降(P<0.01),Keap 1、Nrf 2、HO-1基因mRNA表达极显著增加(P<0.01),GSH-Px含量极显著升高(P<0.01),SOD含量显著升高(P<0.05)。【结论】EH水提物在猪小肠上皮细胞中通过激活Keap1/Nrf2信号,提高抗氧化酶SOD和GSH-Px含量,启动下游靶基因HO-1表达,发挥抗炎和抗氧化的作用,且10μg/mL EH水提物作用效果最佳。
【Objective】In order to reveal the anti-inflammatory and antioxidation effects of Euphorbiae humifusae(EH)in swine intestinal epithelial cells(IPEC-J2).【Method】Different concentrations of EH aqueous extract(0,5,10,50,125,200μg/mL)were used to treat IPEC-J2 cells for 12 hours.The viability of IPEC-J2 cells was detected by CCK-8 method,determining the optimal concentration of EH treatment.IPEC-J2 cells were randomly divided into control group(CT),lipopolysaccharide group(LPS),EH+LPS group(ELP),with three replicates in each group.The cells in CT group were cultured in DMEM medium containing 10%fetal bovine serum.The cells in LPS group were treated with 5μg/mL LPS.The cells in ELP group were co-cultured with 5μg/mL LPS and EH,cells in each group was treated for 12 hours,collecting the cells and supernatant.The expression of interleukin-6(IL-6),tumor necrosis factorα(TNF-α),kelch like epichlorohydrin related protein 1(Keap1),nuclear factor E2 related factor 2(Nrf2),heme oxygenase-1(HO-1)mRNA were detected by real time fluorescent quantitative PCR,and the content of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in the supernatant were detected by ELISA,and the reactive oxygen species(ROS)level in the cells was detected by chemical fluorescence method.【Result】Compared with EH untreated cells,5,50μg/mL EH increased significantly the viability of IPEC-J2 cells(P<0.05).EH concentration was at 10μg/mL,the increased cell viability was very significant(P<0.01).When EH concentration was at 125μg/mL,IPEC-J2 cell viability decreased,there was no statistical significance(P>0.05).EH concentration is at 200μg/mL,the decreased cell viability was extremely significant(P<0.01).The concentration of EH treatment was 10μg/mL in subsequent experiment.Compared with the CT group,in LPS group,the IL-6 and TNF-αgenes mRNA expression in IPEC-J2 cells were extremely significantly increased(P<0.01),and the content of MDA in the supernatant and the fluorescence intensity of cells ROS were extremely significantly increased(P<0.01).The expression of Keap 1,Nrf 2 and HO-1 genes mRNA were extremely significantly reduced(P<0.01),the content of antioxidant enzymes SOD and GSH-Px were extremely significantly decreased(P<0.01).Compared with LPS group,in ELP group,the mRNA expression of IL-6 gene was extremely significantly reduced(P<0.01),the mRNA expression of TNF-αgene was significantly decreased(P<0.05).The content of MDA in the supernatant and the fluorescence intensity of cells ROS were extremely significant decreased(P<0.01).The mRNA expression of Keap 1,Nrf 2 and HO-1 genes were significantly up-regulated(P<0.01),the content of GSH-Px was increased extremely significant(P<0.01).The content of SOD was also significantly increased(P<0.05).【Conclusion】EH aqueous extract played an anti-inflammatory and antioxidant role in IPEC-J2 cells by activating the Keap1/Nrf2 signal,increasing the contents of antioxidant enzymes SOD and GSH-Px,activating the expression of downstream target genes HO-1,and 10μg/mL EH water extract had the best effect.
作者
陈瑾
谢景昊
韩莹倩
杨彦宾
王月影
李和平
CHEN Jin;XIE Jinghao;HAN Yingqian;YANG Yanbin;WANG Yueying;LI Heping(Key Laboratory of Animal Biochemistry and Nutrition,Ministry of Agriculture and Rural Affairs,Henan Agricultural University,Zhengzhou 450046,China;Key Laboratory of Animal Growth and Development of Henan Province,Zhengzhou 450046,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2023年第2期704-712,共9页
China Animal Husbandry & Veterinary Medicine
基金
河南省科技攻关计划(212102110356、222102110402)。
作者简介
陈瑾,联系方式:E-mail:1455763159@qq.com。;通信作者:李和平,E-mail:liheping1972@126.com。
