蓝莓渣多酚对肝星状细胞的增殖与自噬作用
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(1.江苏省农业科学院农产品加工研究所 南京 210014;2.克孜勒苏柯尔克孜自治州林业工作管理站 新疆阿图什 845350;3.南京市溧水区供销合作总社 南京 211200)

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新疆自治区重点研发计划项目(2023B02028)


Effects of Blueberry Pomace Polyphenols on Proliferation and Autophagy of Hepatic Stellate Cells
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(1.Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014;2.Forestry Management Station, Kizilsu Kirgiz Autonomous Prefecture, Atushi 845350, Xinjiang;3.Nanjing Lishui District Supply and Marketing Cooperative, Nanjing 211200)

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    摘要:

    为了提高蓝莓全资源利用率,降低其加工副产物对环境的危害。通过高效液相色谱串联质谱法(HPLC-MS/MS)分析超高压提取、超声提取和溶剂提取技术对蓝莓渣多酚(BRP)组分及含量的影响。以活化的HSC-T6细胞为模型,通过噻唑蓝法和乳酸脱氢酶法检测BRP对HSC-T6细胞存活率和细胞毒性的影响,透射电镜和吖啶橙染色法观察BRP对HSC-T6细胞自噬的诱导作用,蛋白免疫印迹法(Western blot)检测BRP对HSC-T6细胞自噬相关蛋白的表达量,酶联免疫法(ELISA)分析HSC-T6细胞的纤维化程度。结果表明:BRP中有14种组分,3种提取方法中的矢车菊素-3-O-葡萄糖苷占比最高,分别为57.530%,58.703%,54.830%。3种提取方法所得BRP的组分种类相同,而含量存在较大差异。超高压提取法所得BRP含量显著高于常规溶剂法和超声辅助提取法(P<0.05)。BRP能显著抑制HSC-T6细胞增殖(P<0.05),且在一定浓度范围呈现时间-剂量效应关系;通过上调自噬相关蛋白Beclin-1和LC3-II表达,下调p62蛋白表达,诱导细胞发生自噬;同时BRP显著下调HSC-T6细胞中COL-Ⅰ、COL-Ⅲ、TNF-β1的分泌量(P<0.05)。结论:超高压萃取的BRP组分总含量最高,生物活性功能最强,BRP可通过抑制活化的HSC-T6细胞增殖,诱导细胞自噬从而发挥其抗纤维化效果。

    Abstract:

    In order to improve the utilization rate of blueberry resources and reduce the environmental harm of its processing by-products. The effects of ultra-high pressure extraction, ultrasonic extraction, and solvent extraction on the composition and content of polyphenol (BRP) in blueberry residue were investigated by high performance liquid chromatography (HPLC-MS/MS). Activated HSC-T6 cells were used as models to detect the effects of BRP on the survival rate and cytotoxicity of HSC-T6 cells by thiazole blue and lactate dehydrogenase methods. The autophagy induction effect of BRP on HSC-T6 cells was observed by transmission electron microscopy and acridine orange staining. The expression level of autophagy related proteins in HSC-T6 cells was detected by Western blot. The degree of fibrosis in HSC-T6 cells was analyzed by enzyme-linked immunoassay (ELISA). The results showed that there were 14 components in BRP, and cyanidin-3-O-glucoside accounts for the highest proportion among the three extraction methods, which are 57.530%, 58.703% and 54.830% respectively. The BRP components obtained by the three extraction methods were the same, but the content was different. The BRP content of ultra-high pressure extraction method was significantly higher than that of conventional solvent method and ultrasound-assisted extraction method (P < 0.05). BRP significantly inhibited the proliferation of HSC-T6 cells (P < 0.05), and showed a time-dose effect relationship within a certain concentration range. Autophagy was induced by up-regulating the expression of Beclin-1 and LC3-II, and down-regulating the expression of p62. And, BRP significantly decreased the secretion levels of COL-Ⅰ, COL-Ⅲ and TNF-β1 in HSC-T6 cells (P < 0.05). In conclusion, the total content of BRP components extracted by ultrahigh pressure extraction is the highest and the biological activity is the strongest, and BRP could induce autophagy by inhibiting the proliferation of activated HSC-T6 cells, thus exerting its anti-fibrosis effect.

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马剑,赖光荣,胡一阳,贾飞鸿,马艳弘,刘海燕.蓝莓渣多酚对肝星状细胞的增殖与自噬作用[J].中国食品学报,2024,24(9):105-113

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  • 收稿日期:2023-09-23
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  • 在线发布日期: 2024-10-21
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