食品与发酵工业 >

2024 , Vol. 50 >Issue 4: 118 - 125

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.035591

研究报告

新疆葡萄酒中的酚类物质对LO2细胞自噬的影响

  • 王妍凌 ,
  • 陈杉彬 ,
  • 赵文梅 ,
  • 尹丽萍 ,
  • 黎进雪 ,
  • 张海鹏 ,
  • 张金萍 ,
  • 黄翠 ,
  • 吕嘉伟 ,
  • 武运 ,
  • 薛洁
展开
  • 1(新疆农业大学 食品科学与药学学院,新疆 乌鲁木齐,830052)
    2(中国食品发酵工业研究院有限公司,北京,100015)
    3(国家酒类品质与安全国际联合研究中心,北京,100015)
第一作者:硕士研究生(武运教授和薛洁教授级高级工程师为共同通信作者,E-mail:wuyunster@sina.com;lxxuejie@126.com)

收稿日期: 2023-03-23

  修回日期: 2023-04-04

  网络出版日期: 2024-03-15

基金资助

新疆维吾尔自治区重大科技专项(2022A02002-2)

收起

Effect of phenolic substances in Xinjiang wine on the activity of autophagy in LO2 cells

  • WANG Yanling ,
  • CHEN Shanbin ,
  • ZHAO Wenmei ,
  • YIN Liping ,
  • LI Jinxue ,
  • ZHANG Haipeng ,
  • ZHANG Jinping ,
  • HUANG Cui ,
  • LYU Jiawei ,
  • WU Yun ,
  • XUE Jie
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  • 1(College of Food Science and Pharmacy, Xinjiang Agricultural University, Urumqi 830052, China)
    2(China National Research Institute of Food & Fermentation Industries Co.Ltd, Beijing 100015, China)
    3(National Joint International Research Center for Alcohol Quality and Safety, Beijing 100015, China)

Received date: 2023-03-23

  Revised date: 2023-04-04

  Online published: 2024-03-15

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摘要

为了探究新疆葡萄酒酚类物质对肝细胞自噬的影响,该研究使用体外培养的人正常肝细胞(LO2),通过免疫印迹、实时荧光定量、细胞免疫荧光、流式细胞术等方法观察LO2细胞中微管相关蛋白1A/1B-轻链3(microtubule-associated proteins 1A/1B light chain3,LC3)和螯合体1(sequestosome 1, P62)的水平变化,初步评估新疆葡萄酒中酚类物质对LO2细胞自噬的影响作用。免疫印迹结果发现,橙皮苷、表儿茶素没食子酸酯,可显著提升LO2细胞中LC3蛋白的表达水平,提示橙皮苷和表儿茶素没食子酸酯或对LO2细胞自噬活性有一定影响作用。进一步通过实时荧光定量方法观察橙皮苷和表儿茶素没食子酸酯对LC3和P62蛋白mRNA的影响作用发现,橙皮苷、表儿茶素没食子酸酯处理细胞后,显著上调了LC3蛋白的mRNA水平,同时降低了P62蛋白的mRNA水平;细胞免疫荧光染色显示橙皮苷、表儿茶素没食子酸酯处理LO2细胞后,绿色荧光蛋白(green fluorescent protein, GFP)和红色荧光蛋白(red fluorescent protein, RFP)标记的LC3蛋白荧光强度增强,提示细胞内形成自噬小体;流式细胞术结果进一步发现,GFP-LC3/RFP-LC3荧光强度比值减小,表明自噬小体成熟。综上所述,橙皮苷、表儿茶素没食子酸酯对LO2细胞自噬活性具有一定的促进作用,但其作用机理尚需要进一步深入研究。

关键词： 新疆葡萄酒; 细胞自噬; 酚类物质; 肝脏保护; LO2细胞

本文引用格式

王妍凌 , 陈杉彬 , 赵文梅 , 尹丽萍 , 黎进雪 , 张海鹏 , 张金萍 , 黄翠 , 吕嘉伟 , 武运 , 薛洁 . 新疆葡萄酒中的酚类物质对LO2细胞自噬的影响[J]. 食品与发酵工业, 2024 , 50(4) : 118 -125 . DOI: 10.13995/j.cnki.11-1802/ts.035591

Abstract

In order to explore the effect of phenolic substances in Xinjiang wine on autophagy of hepatocytes, this study used human normal hepatocytes (LO2) cultured in vitro to observe the changes in the levels of LC3 protein and P62 protein in LO2 cells by immunoblotting, real-time fluorescence quantification, cellular immunofluorescence, and flow cytometry, and preliminarily evaluate the effect of phenolic substances in Xinjiang wine on autophagy of LO2 cells. Immunoblotting results showed that hesperidin and epicatechin gallate significantly increased the expression level of LC3 protein in LO2 cells, suggesting that hesperidin and epicatechin gallate may have a certain impact on the autophagic activity of LO2 cells. Further, the effects of hesperidin and epicatechin gallate on LC3 and P62 mRNA were observed using real-time fluorescence quantitative methods. It was found that treatment with hesperidin and epicatechin gallate significantly upregulated the mRNA level of LC3. At the same time, it decreased the mRNA level of P62 protein; Cellular immunofluorescence staining showed that after treating LO2 cells with hesperidin and epicatechin gallate, the fluorescence intensity of GFP and RFP labeled LC3 protein increased, indicating the formation of autophagosomes within the cells; Flow cytometry further revealed that the fluorescence intensity ratio of GFP-LC3/RFP-LC3 decreased, indicating the maturation of autophagosomes. In summary, hesperidin and epicatechin gallate have a certain promoting effect on the autophagic activity of LO2 cells, but their mechanism needs further research.

Key words： Xinjiang wine; cell autophagy; phenolic substances; liver protection; LO2 cells

参考文献

[1] BERTELLI A A E.Wine, research and cardiovascular disease:Instructions for use[J].Atherosclerosis, 2007, 195(2):242-247.
[2] 金洪艳, 裴立楠.葡萄酒中的营养物质分析[J].食品安全导刊, 2020(21):43.
JIN H Y, PEI L N.Analysis of nutrients in wine[J].Food Safety Guide, 2020(21):43.
[3] 杨志伟, 王圣仪, 齐鹏宇, 等.UPLC-MS/MS测定葡萄酒中29种单体酚方法的建立[J].食品科学, 2019, 40(24):214-219.
YANG Z W, WANG S Y, QI P Y, et al.Establishment of ultra-high performance liquid chromatography-tandem mass spectrometry method for determination of 29 monophenols in wine[J] Food Science, 2019, 40 (24):214-219.
[4] 侍朋宝, 崔彦志, 张昂, 等.闪蒸对昌黎产区‘马瑟兰’葡萄酒单体酚类物质的影响[J].食品科学, 2022, 43(21):102-110.
SHI P B, CUI Y Z, ZHANG A, et al.Effect of flash vacuum expansion on monomeric phenolics in ‘Marselan’ wine from Changli[J] Food Science, 2022, 43 (21):102-110.
[5] 王华, 田雪林, 杨晨露, 等.葡萄酒与健康[J].中国酿造, 2022, 41(3):1-5.
WANG H, TIAN X L, YANG C L, et al.Wine and health[J] China Brewing, 2022, 41 (3):1-5.
[6] BHARRHAN S, KOUL A, CHOPRA K, et al.Catechin suppresses an array of signalling molecules and modulates alcohol-induced endotoxin mediated liver injury in a rat model[J].PLoS One, 2011, 6(6):e20635.
[7] LEE S Y, LEE J, LEE H N, et al.Relative protective activities of quercetin, quercetin-3-glucoside, and rutin in alcohol-induced liver injury[J].Journal of Food Biochemistry, 2019, 43(11):e13002.
[8] CHEN J, XUAN Y H, LUO M X, et al.Kaempferol alleviates acute alcoholic liver injury in mice by regulating intestinal tight junction proteins and butyrate receptors and transporters[J].Toxicology, 2020, 429:152338.
[9] 赵昊. 新疆地产葡萄酒对健康大鼠肠道菌群及肝细胞自噬影响的研究[D].乌鲁木齐:新疆农业大学, 2021.
ZHAO H.Study on the effect of Xinjiang native wine on intestinal flora and hepatocyte autophagy of liver cells in healthy rats[D] Urumqi:Xinjiang Agricultural University, 2021.
[10] 赵昊, 宋晶晶, 于佳俊, 等.不同产区葡萄酒多酚物质抗氧化活性差异及相关性分析[J].食品与发酵工业, 2021, 47(6):84-91.
ZHAO H, SONG J J, YU J J, et al.Differences and correlation analysis of polyphenols and antioxidant activity in different wines regions[J] Food and Fermentation Industries, 2021, 47 (6):84-91.
[11] ZHOU Z T, ZHONG W C, LIN H Y, et al.Hesperidin protects against acute alcoholic injury through improving lipid metabolism and cell damage in zebrafish larvae[J].Evidence-Based Complementray and Alternative Medicine, 2017, 2017:7282653.
[12] ZHANG Y F, LU Y X, JI H, et al.Anti-inflammatory, anti-oxidative stress and novel therapeutic targets for cholestatic liver injury[J].Bioscience Trends, 2019, 13(1):23-31.
[13] BISHEHSARI F, MAGNO E, SWANSON G, et al.Alcohol and gut-derived inflammation[J].Alcohol Research: Current Reviews, 2017, 38(2):163-171.
[14] ESCLATINE A, CHAUMORCEL M, CODOGNO P.Macroautophagy signaling and regulation[J].Current Topics in Microbiology and Immunology, 2009, 335:33-70.
[15] MARIÑO G, NISO-SANTANO M, BAEHRECKE E H, et al.Self-consumption:The interplay of autophagy and apoptosis[J].Nature Reviews. Molecular Cell Biology, 2014, 15(2):81-94.
[16] CHOI A M K, RYTER S W, LEVINE B.Autophagy in human health and disease[J].The New England Journal of Medicine, 2013, 368(19):1845-1846.
[17] RAUTOU P E, MANSOURI A, LEBREC D, et al.Autophagy in liver diseases[J].Journal of Hepatology, 2010, 53(6):1123-1134.
[18] SONG Y, ZHAO Y Y, WANG F, et al.Autophagy in hepatic fibrosis[J].BioMed Research International, 2014(8):436242.
[19] 李一澍, 姚逸萍, 黄和强, 等.萜烯类化合物基于细胞自噬的初步探究[J].食品与发酵工业, 2022, 48(16):42-49.
LI Y S, YAO Y P, HUANG H Q, et al.Preliminary study of terpenes based on autophagy[J] Food and Fermentation Industries, 2022, 48 (16):42-49.
[20] IKARI S, LU S L, HAO F K, et al.Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan[J].PLoS One, 2020, 15(3):e0230156.
[21] JIANG P D, MIZUSHIMA N.LC3-and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells[J].Methods, 2015, 75:13-18.
[22] MEYER G, CZOMPA A, REBOUL C, et al.The cellular autophagy markers Beclin-1 and LC3B-Ⅱ are increased during reperfusion in fibrillated mouse hearts[J].Current Pharmaceutical Design, 2013, 19(39):6912-6918.
[23] BAERGA R, ZHANG Y, CHEN P H, et al.Targeted deletion of autophagy-related 5 (atg5) impairs adipogenesis in a cellular model and in mice[J].Autophagy, 2009, 5(8):1118-1130.
[24] MIZUSHIMA N, YOSHIMORI T.How to interpret LC3 immunoblotting[J].Autophagy, 2007, 3(6):542-545.
[25] YOUSEFI S, PEROZZO R, SCHMID I, et al.Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis[J].Nature Cell Biology, 2006, 8(10):1124-1132.
[26] TANIDA I, MINEMATSU-IKEGUCHI N, UENO T, et al.Lysosomal turnover,but not a cellular level, of endogenous LC3 is a marker for autophagy[J].Autophagy, 2005, 1(2):84-91.
[27] KIMURA S, NODA T, YOSHIMORI T.Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3[J].Autophagy, 2007, 3(5):452-460.
[28] KAIZUKA T, MORISHITA H, HAMA Y, et al.An autophagic flux probe that releases an internal control[J].Molecular Cell, 2016, 64(4):835-849.
[29] TABESHPOUR J, HOSSEINZADEH H, HASHEMZAEI M, et al.A review of the hepatoprotective effects of hesperidin, a flavanon glycoside in citrus fruits, against natural and chemical toxicities[J].DARU Journal of Pharmaceutical Sciences, 2020, 28(1):305-317.
[30] YIN X M, DING W X, GAO W T.Autophagy in the liver[J].Hepatology, 2008, 47(5):1773-1785.
[31] CZAJA M J, DING W X, DONOHUE T M JR, et al.Functions of autophagy in normal and diseased liver[J].Autophagy, 2013, 9(8):1131-1158.
[32] LI S L, ZHU J J, PAN L, et al.Potential protective effect of hesperidin on hypoxia/reoxygenationinduced hepatocyte injury[J].Experimental and Therapeutic Medicine, 2021, 22(1):764.
[33] RUSSELL R C, YUAN H X, GUAN K L.Autophagy regulation by nutrient signaling[J].Cell Research, 2014, 24(1):42-57.
[34] MALHI H, GORES G J.Cellular and molecular mechanisms of liver injury[J].Gastroenterology, 2008, 134(6):1641-1654.
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