抗氧化菌株的筛选及其对HepG2细胞氧化应激损伤的保护作用

Screening of antioxidant strains and its protective effect on oxidative stress damage of HepG2 cells

摘要:

【背景】机体的氧化损伤会引发多种疾病和机体衰老，研究发现益生菌可以改善宿主的代谢水平，提高宿主的抗氧化能力，为抗氧化产品开发提供新方向。【目的】筛选具有良好抗氧化效果的菌株，为抗氧化药物以及保健品的开发和研究提供菌种材料。【方法】将从江西省宜春市温汤长寿村地区的健康人群粪便中筛选出的菌株进行16S rRNA基因鉴定，挑取其中6株益生菌，通过测定其培养液上清中的超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)酶活及1,1-二苯基-2-三硝基苯肼(1,1-dipheny-2-picrylhydrazyl,DPPH)自由基清除能力，选取一株抗氧化能力最佳的菌株进行后续细胞实验。建立HepG2细胞氧化应激损伤模型，使用CCK-8法评估其培养液上清对HepG2细胞氧化应激损伤的保护效果，通过试剂盒测定培养液上清对细胞中SOD酶活的保护作用，并进一步验证其培养液上清的抗氧化效果。【结果】筛选出的6株益生菌培养液上清中，菌株H0661表现出最佳的抗氧化能力，其培养液上清中GSH-Px酶活为586.11 U/mL (P<0.01)，SOD酶活为1 278.00 U/mL (P<0.01)，DPPH自由基的清除能力达到40.5%。结合16S rRNA基因鉴定结果，将其命名为木糖葡萄球菌(Staphylococcus xylosus) TG022。木糖葡萄球菌TG022培养液上清浓度为15%时对HepG2细胞氧化应激损伤的保护效果最佳(P<0.01)。此外，木糖葡萄球菌TG022培养液上清能有效保护HepG2细胞氧化应激损伤中SOD活性(P<0.01)。经木糖葡萄球菌TG022培养液上清处理后可以有效提高HepG2细胞的抗氧化活性(P<0.05)。【结论】本研究筛选出一株木糖葡萄球菌TG022，其培养液上清表现出良好的抗氧化效果，为研发抗氧化药物和保健品提供了一个潜在的菌株材料。

Abstract:

[Background] The oxidative damage of the body can cause a variety of diseases and body aging. It had been found that probiotics can improve the metabolic level and the antioxidant capacity of the host. This provided a new direction for the development of antioxidant products. [Objective] To screen the strains with good antioxidant effects and provide strain materials for the development and research of antioxidant drugs and nutraceuticals. [Methods] The strains isolated from the feces of healthy people in Changshou Village, Wentang Town, Yichun City, Jiangxi Province were identified by 16S rRNA gene sequencing. We selected six strains of probiotics and then determined the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity of the culture supernatants of the strains. One strain with the best antioxidant capacity was selected for subsequent cellular experiments. A model of oxidative stress damage in HepG2 cells was established, and the protective effect of the culture supernatant of the selected strain on the cell model was evaluated by the CCK-8 method. The protective effect of the culture supernatant on SOD in the cells was determined by the enzyme assay kit, and the antioxidant effect of the culture supernatant was further verified. [Results] Among the six strains of probiotics, strain H0661 had the best antioxidant capacity with GSH-Px activity of 586.11 U/mL (P<0.01), SOD activity of 1 278.00 U/mL (P<0.01), and DPPH radical scavenging capacity of 40.5%. The strain was identified based on the 16S rRNA gene sequencing results and named Staphylococcus xylosus TG022. The culture supernatant of this strain at the concentration of 15% (volume fraction) showed the best protective effect against oxidative stress in HepG2 cells (P<0.01). In addition, the culture supernatant of S. xylosus TG022 was effective in protecting the SOD activity of the HepG2 cells exposed to oxidative stress (P<0.01). Moreover, the antioxidant activity of HepG2 cells was increased after treatment with the culture supernatant of S. xylosus TG022 (P<0.05). [Conclusion] S. xylosus TG022 was screened out, and its culture supernatant had strong antioxidant effects. This study provided a candidate strain for the development of antioxidant drugs and nutraceuticals.