地锦草水提物在IPEC-J2细胞中的抗炎与抗氧化作用

doi:10.16431/j.cnki.1671-7236.2023.02.028

摘要/Abstract

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。

关键词: 地锦草水提物; IPEC-J2细胞; LPS; 氧化应激; Keap1/Nrf2信号

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.

Key words: Euphorbiae humifusae aqueous extract; IPEC-J2 cells; LPS; oxidative stress; Keap1/Nrf2 signaling

中图分类号:

S852.2

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.

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 and Veterinary Medicine, 2023, 50(2): 704-712.

参考文献

[1] ZHU L H,ZHAO K L,CHEN X L,et al.Impact of weaning and an antioxidant blend on intestinal barrier function and antioxidant status in pigs[J]. Journal of Animal Science,2012,90(8):2581-2589.
[2] 华朱鸣.寡糖和小肽抗消化道氧自由基作用的研究[D].杭州:浙江工商大学,2012. HUA Z M.Effects of oligosaccharides and small peptides on scavenging oxygen free radicals in digestive system[D].Hangzhou:Zhejiang Gongshang University,2012.(in Chinese)
[3] 侯静,黎理,蔡毅.地锦草组药用植物的研究进展[J].华西药学杂志,2020,35(2):218-224. HOU J,LI L,CAI Y.Research advances on medicinal plants of Sect Chamaesyce[J]. West China Journal of Pharmaceutical Sciences, 2020,35(2):218-224.(in Chinese)
[4] 陈桂华,宋群,向佐湘.不同处理方法对地锦草发芽率的影响[J].草业科学,2010,27(7):10-12. CHEN G H,SONG Q,XIANG Z X.Effects of different treatments on the germination rate of Euphorbia humifusa[J]. Pratacultural Science,2010,27(7):10-12.(in Chinese)
[5] MUSTAFA I,FAISAL M N,HUSSAIN G,et al.Efficacy of Euphorbia helioscopia in context to a possible connection between antioxidant and antidiabetic activities:A comparative study of different extracts[J]. BMC Complementary Medicine and Therapies, 2021,21(1):62.
[6] 郑文贺,闫超.地锦草总黄酮对肠源性脓毒症大鼠的肠道保护作用[J].福建中医药,2022,53(1):35-39. ZHENG W H,YAN C.Protective effect of total flavonoids of Euphorbia humifusaon intestinal tract in rats with enterogenous sepsis[J]. Fujian Journal of Traditional Chinese Medicine,2022,53(1):35-39.(in Chinese)
[7] JIAO Y,WANG S,JIANG L,et al.2-undecanone protects against fine particles-induced heart inflammation via modulating Nrf2/HO-1 and NF-κB pathways[J]. Environmental Toxicology,2022,37(7):1642-1652.
[8] 胡流芳,王迎,任汝静,等.Keap1-Nrf2/ARE信号通路的抗氧化应激作用及其调控机制[J].国际药学研究杂志,2016,43(1):146-152. HU L F,WANG Y,REN R J,et al.Anti-oxidative stress actions and regulation mechanisms of Keap1-Nrf2/ARE signal pathway[J]. Global Advances in Pharmaceutical Research,2016,43(1):146-152.(in Chinese)
[9] 罗淑娟,陈小芬,林逸婷,等.健脾清热活血方介导keap-1-Nrf2信号调控claudin-2表达干预溃疡性结肠炎模型大鼠实验研究[J].中国中医急症,2021,30(11):1924-1928. LUO S J,CHEN X F,LIN Y T,et al.Study of Jianpi Qingre Huoxue decoction mediates keap-1-Nrf2 pathway regulation of claudin-2 expression to intervene ulcerative colitis[J]. Journal of Emergency in Traditional Chinese Medicine,2021,30(11):1924-1928.(in Chinese)
[10] 孙志良,盛忠梅,刘湘新,等.复方地锦草乳剂对仔猪黄白痢及腹泻的疗效试验[J].畜牧兽医杂志,1997,16(4):28-30. SUN Z L,SHENG Z M,LIU X X,et al.Efficacy of compound Euphorbia humifusa emulsion on yellow and white diarrhea and diarrhea in piglets[J]. Journal of Animal Husbandry and Veterinary Medicine,1997,16(4):28-30.(in Chinese)
[11] 李涛,饶鸿宇,王永刚.中草药饲料添加剂防治仔猪腹泻的研究进展[J].饲料研究,2021,44(9):139-142. LI T,RAO H Y,WANG Y G.Research progress of traditional Chinese medicine on control of piglet diarrhea[J]. Feed Research,2021,44(9):139-142.(in Chinese)
[12] 杜培培.地锦草总黄酮对T2DM大鼠糖脂代谢、炎性因子及氧化应激的干预作用[J].菏泽医学专科学校学报,2021,33(4):1-4. DU P P.Based on the complications of diabetes to explore the effect of total flavone of Euphorbia officinalis on the metabolism of glucose and lipid,inflammatory factors and oxidative stress of T2DM rats[J]. Journal of Heze Medical College,2021,33(4):1-4.(in Chinese)
[13] 程艳刚,李国艳,谭金燕,等.地锦草总黄酮提取工艺优选及其抑制α-葡萄糖苷酶活性研究[J].辽宁中医药大学学报,2018,20(9):54-58. CHENG Y G,LI G Y,TAN J Y,et al.Optimization of extraction technology for total flavonoids from Euphorbia humifusa willd.and study on its inhibitory effects on α-glucosidase[J]. Journal of Liaoning University of Traditional Chinese Medicine,2018,20(9):54-58.(in Chinese)
[14] 钟树林,卢智健,张婷,等.脐血IL-1β、IL-6、IL-8、IL-10、TNF-α与母体、胎儿炎症反应及早产预后的相关性[J].海南医学,2016,24:4036-4039. ZHONG S L,LU Z J,ZHANG T,et al.Correlation analysis of IL-1 β,IL-6,IL-8,IL-10,TNF-α with maternal,fetal inflammatory response and prognosis of preterm birth[J]. Hainan Medical Journal,2016,24:4036-4039.(in Chinese)
[15] 霍星华,王胜林,程忠刚,等.大豆异黄酮对过氧化氢诱导IPEC-J2细胞氧化应激的影响[J].广东畜牧兽医科技,2021,46(5):37-41. HUO X H,WANG S L,CHENG Z G,et al.Effects of soy isoflavones on oxidative stress in IPEC-J2 cells induced by hydrogen peroxide[J]. Guangdong Journal of Animal and Veterinary Science, 2021,46(5):37-41.(in Chinese)
[16] QIU Z,HE Y,MING H,et al.Lipopolysaccharide (LPS) aggravates high glucose-and hypoxia/reoxygenation-induced injury through activating ROS-dependent NLRP3 inflammasome-mediated pyroptosis in H9C2 cardiomyocytes[J]. Journal of Diabetes Research,2019,2019:8151836.
[17] LIANG J,LI L,SUN Y,et al.The protective effect of activating Nrf2/HO-1 signaling pathway on cardiomyocyte apoptosis after coronary microembolization in rats[J]. BMC Cardiovascular Disorders,2017,17(1):272.
[18] 王甜甜,陈淳媛,杨雷,等.Nrf2/HO-1信号轴在氧化应激性疾病中的机制[J].中南大学学报(医学版),2019,44(1):74-80. WANG T T,CHEN C Y,YANG L,et al.Role of Nrf2/HO-1 signal axis in the mechanisms for oxidative stress-relevant diseases[J]. Journal of Central South University (Medical Science),2019,44(1):74-80.(in Chinese)
[19] YANG C C,HSIAO L D,WANG C Y,et al.HO-1 upregulation by kaempferol via ROS-dependent Nrf2-ARE cascade attenuates lipopolysaccharide-mediated intercellular cell adhesion molecule-1 expression in human pulmonary alveolar epithelial cells[J]. Antioxidants (Basel),2022,11(4):782.
[20] 姜淑妍.大豆异黄酮对高温下体外培养奶牛乳腺上皮细胞增殖和抗氧化能力的影响[J].中国饲料,2020,15:29-32. JIANG S Y.Effects of isoflavones on proliferation and antioxidation of bovine mammary epithelial cells cultures in vitro under high temperature[J]. China Feed,2020,15:29-32.(in Chinese)
[21] 彪雅宁,张睦清,韩雪,等.当归芍药散对非酒精性脂肪肝大鼠Keap1/Nrf2信号通路的调节作用[J].中药药理与临床,2022,38(1):13-18. BIAO Y N,ZHANG K Q,HAN X,et al.Danggui Shaoyao san regulates Keap1/Nrf2 signaling pathway in rats with nonalcoholic fatty liver disease[J]. Pharmacology and Clinics of Chinese Materia Medica,2022,38(1):13-18.(in Chinese)