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。
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.
作者
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
WANG Yanling;CHEN Shanbin;ZHAO Wenmei;YIN Liping;LI Jinxue;ZHANG Haipeng;ZHANG Jinping;HUANG Cui;LYU Jiawei;WU Yun;XUE Jie(College of Food Science and Pharmacy,Xinjiang Agricultural University,Urumqi 830052,China;China National Research Institute of Food&Fermentation Industries Co.Ltd,Beijing 100015,China;National Joint International Research Center for Alcohol Quality and Safety,Beijing 100015,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2024年第4期118-125,共8页
Food and Fermentation Industries
基金
新疆维吾尔自治区重大科技专项(2022A02002-2)。
作者简介
第一作者:王妍凌,硕士研究生;共同通信作者:武运,教授,E-mail:wuyunster@sina.com;共同通信作者:薛洁教,授级高级工程师,E-mail:lxxuejie@126.com。
