食品科学 ›› 2020, Vol. 41 ›› Issue (13): 174-183.doi: 10.7506/spkx1002-6630-20190710-134

• 营养卫生 • 上一篇    下一篇

灵芝菌丝体多糖对人皮肤成纤维细胞氧化应激损伤的防护机制

张佳婵,邵卿,王倩,王昌涛,赵丹,李萌,孙宝国,刘继涛   

  1. (1.北京工商大学理学院,植物资源研究开发北京市重点实验室,北京 100048;2.北京工商大学 北京食品营养与人类健康高精尖创新中心,北京 100048;3.北京工商大学食品与健康学院,北京市食品添加剂工程技术研究中心,北京 100048;4.云南白药集团股份有限公司,云南 昆明 650000)
  • 出版日期:2020-07-15 发布日期:2020-07-29
  • 基金资助:
    云南省生物医药重大科技专项(2018ZF005)

Extraction and Purification of Ganoderma lucidum Polysaccharides and Mechanistic Study of Their Protective Effect against Oxidative Stress Injury in Human Skin Fibroblast Cells

ZHANG Jiachan, SHAO Qing, WANG Qian, WANG Changtao, ZHAO Dan, LI Meng, SUN Baoguo, LIU Jitao   

  1. (1. Beijing Key Laboratory of Plant Resource Research and Development, School of Science, Beijing Technology and Business University, Beijing 100048, China; 2. Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing 100048, China; 3. Beijing Engineering and Technology Research Center of Food Additives, School of Food and Health, Beijing Technology and Business University, Beijing 100048, China; 4. Yunnan Baiyao Group Co. Ltd., Kunming 650000, China)
  • Online:2020-07-15 Published:2020-07-29

摘要: 灵芝多糖(Ganoderma lucidum polysaccharide,GLP)是灵芝(Ganoderma lucidum)的重要活性物质之一,拥有优异的保健功效,具有较高的研究价值。本实验以菌种G055的GLP为研究对象,对其进行提取纯化并探究其对人皮肤成纤维细胞氧化应激损伤的防护机制。通过Sevag法脱蛋白,采用DEAE-52离子交换层析得到GLP I和GLP II两组分,通过正交试验确定GLP的提取纯化工艺,GLP的最优提取条件为料液比1∶35(m/V)、浸提温度65 ℃、浸提时间1.5 h,在该条件下进行验证,GLP提取率为(47.70±0.50)%,再现性良好;通过建立以H2O2诱导的人皮肤成纤维细胞氧化应激损伤模型,围绕细胞抗氧化酶体系以及Kelch-like ECH-associated protein-1(Keap1)-核因子-E2相关因子(nuclear factor-erythroid 2-related factor 2,Nrf2)/antioxidant response element(ARE)信号通路,探究GLP及其组分(GLP I和GLP II)在防护损伤方面的作用效果及机制。损伤前的保护处理和损伤后的修复处理下,GLP及其两组分均能够显著提高细胞的超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶水平(P<0.05),降低活性氧以及脂质过氧化产物丙二醛的水平;GLP激活了Keap1-Nrf2/ARE信号通路的关键调控因子Nrf2,抑制了负调控因子Keap1,进而促进了下游抗氧化酶基因(NQO1和HO-1)的表达。综上,GLP的防护作用与其抗氧化酶水平及Keap1-Nrf2/ARE信号通路密切相关。

关键词: 灵芝菌丝体多糖, 提取纯化, 防护, 氧化应激损伤, Keap1-Nrf2/ARE信号通路

Abstract: Ganoderma lucidum polysaccharides (GLPs), one of the most important bioactive substances in Ganoderma lucidum, have excellent health benefits and therefore attract great research interest. The purpose of this study was to explore the extraction and purification of GLPs derived from Ganoderma lucidum strain G055 and to evaluate the mechanism underlying the protective effect of GLPs against oxidative stress injury in human skin fibroblast (HSF) cells. The crude polysaccharides were deproteinated by the Sevag method and separated into two fractions named GLP I and GLP II by DEAE-52 ion exchange chromatography. Using an orthogonal array design, the optimal extraction conditions were determined as 1:35 (m/V), 65 ℃ and 1.5 h for solid-to-solvent ratio, extraction temperature and time, respectively. Experiments performed under these conditions gave GLP extraction yield of (47.70 ± 0.50)% with good reproducibility. The protective effect of the crude GLPs as well as GLP I and GLP II on H2O2-induced oxidative stress injury in HSF cells was evaluated and the underlying mechanism was studied from the perspective of the antioxidant enzyme system and the Keap1 (Kelch-like ECH-associated protein-1)-Nrf2 (NF-E2-related factor 2)/ARE (antioxidant response element) signaling pathway. In the experiments evaluating their effects on preventing and repairing oxidative stress injury in HSF cells, we found that all three polysaccharides could significantly improve the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) (P < 0.05), and decrease the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), a product of lipid peroxidation, in cells. Furthermore, they could activate the key regulatory factor Nrf2 in the Keap1-Nrf2/ARE signaling pathway and inhibit the negative regulatory factor Keap1 thereby promoting the expression of downstream antioxidant enzyme genes (NQO1 and HO-1). Therefore, the protective and repairing effect of GLPs may be closely related to regulating the levels of antioxidant enzymes and activating the Keap1-Nrf2/ARE signaling pathway.

Key words: Ganoderma lucidum polysaccharides, extraction and purification, protection, oxidative stress damage, Kelch-like ECH-associated protein-1-nuclear factor-erythroid 2-related factor 2/antioxidant response element signaling pathway

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