| 地锦草水提物在IPEC-J2细胞中的抗炎与抗氧化作用 |
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| 引用本文： | 陈瑾,谢景昊,韩莹倩,杨彦宾,王月影,李和平.地锦草水提物在IPEC-J2细胞中的抗炎与抗氧化作用[J].中国畜牧兽医,2023,50(2):704-712. |
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| 作者姓名： | 陈瑾 谢景昊 韩莹倩 杨彦宾 王月影 李和平 |
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| 作者单位： | 1. 河南农业大学, 农业农村部动物生化与营养重点实验室, 郑州 450046;2. 河南省动物生长发育调控重点实验室, 郑州 450046 |
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| 基金项目： | 河南省科技攻关计划(212102110356、222102110402) |
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| 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、200 μg/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5 μg/mL LPS处理,ELP组细胞用5 μg/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0 μg/mL EH组相比,5、50 μg/mL EH组IPEC-J2细胞活力显著增加(P＜0.05);10 μg/mL EH组IPEC-J2细胞活力极显著增加(P＜0.01),125 μg/mL EH组IPEC-J2细胞活力下降,但差异不显著(P＞0.05),200 μg/mL EH组IPEC-J2细胞活力极显著下降(P＜0.01)。因此,后续选用10 μg/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),Keap1、Nrf2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),Keap1、Nrf2、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10 μg/mL EH水提物作用效果最佳。
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| 关 键 词： | 地锦草水提物 IPEC-J2细胞 LPS 氧化应激 Keap1/Nrf2信号 |
| 收稿时间： | 2022-07-13 |
Anti-inflammatory and Antioxidant Effects of Euphorbiae humifusae Aqueous Extract in IPEC-J2 Cells |
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| CHEN Jin,XIE Jinghao,HAN Yingqian,YANG Yanbin,WANG Yueying,LI Heping.Anti-inflammatory and Antioxidant Effects of Euphorbiae humifusae Aqueous Extract in IPEC-J2 Cells[J].China Animal Husbandry & Veterinary Medicine,2023,50(2):704-712. |
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| Authors: | CHEN Jin XIE Jinghao HAN Yingqian YANG Yanbin WANG Yueying LI Heping |
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| Institution: | 1. Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs, Henan Agricultural University, Zhengzhou 450046, China;2. Key Laboratory of Animal Growth and Development of Henan Province, Zhengzhou 450046, China |
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| Abstract: | 【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 (0,5,10,50,125,200 μg/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 5 μg/mL LPS.The cells in ELP group were co-cultured with 5 μg/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 factor α (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,5,50 μg/mL EH increased significantly the viability of IPEC-J2 cells (P＜0.05).EH concentration was at 10 μg/mL,the increased cell viability was very significant (P＜0.01).When EH concentration was at 125 μg/mL,IPEC-J2 cell viability decreased,there was no statistical significance (P＞0.05).EH concentration is at 200 μg/mL,the decreased cell viability was extremely significant (P＜0.01).The concentration of EH treatment was 10 μg/mL in subsequent experiment.Compared with the CT group,in LPS group,the IL-6 and TNF-α genes 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 Keap1 ,Nrf2 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-α gene 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 Keap1 ,Nrf2 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 10 μg/mL EH water extract had the best effect. |
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| Keywords: | Euphorbiae humifusae aqueous extract IPEC-J2 cells LPS oxidative stress Keap1/Nrf2 signaling |
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