摘要
目的:探讨天麻素对D-半乳糖诱导的BV2细胞衰老的保护作用及机制。方法:使用不同浓度(10、20、30和40μg/mL)的D-半乳糖刺激BV2细胞24 h,建立细胞衰老模型,并用CCK-8法筛选出D-半乳糖的最佳造模浓度;实验分为4组:正常组、模型组、Silent mating type information regulation 2 homolog 3(SIRT3)抑制剂+天麻素组和天麻素组;用CCK-8法检测不同浓度(10、20、30、40和50μmol/L)的天麻素对D-半乳糖刺激的BV2细胞活力的影响,并筛选出最佳的天麻素浓度;使用β-半乳糖苷酶(Senescenceβ-Galactosidase,SA-β-Gal)染色检测细胞衰老面积;使用生化法检测各组细胞活性氧(Reactive oxygen species,ROS)水平;使用Enzyme linked immunosorbent assay(ELISA)法检测各组细胞神经炎症因子IL-1β(Interleukin 1β,IL-1β)、IL-6(Interleukin 6,IL-6)和TNF-α(Tumor necrosis factorα,TNF-α)水平;使用免疫荧光法检测各组细胞SIRT3荧光强度;使用Western Blot法检测细胞SIRT3、P16和P21的蛋白表达水平。结果:30μg/mL的D-半乳糖刺激BV2细胞活力极显著降低(P<0.01),引起BV2细胞中SA-β-Gal染色面积和衰老蛋白P16和P21表达极显著增加(P<0.01),细胞中ROS水平和炎症因子IL-1β、IL-6和TNF-α水平极显著增高(P<0.01),细胞内SIRT3蛋白表达极显著降低(P<0.01)。而30μmol/L天麻素能极显著提高D-半乳糖刺激的BV2细胞活力(P<0.01),并且极显著降低细胞SA-β-Gal染色面积和衰老蛋白P16和P21表达水平(P<0.01),极显著降低ROS水平和神经炎症因子IL-1β、IL-6和TNF-α水平(P<0.01),极显著提高细胞内SIRT3蛋白荧光强度和表达水平(P<0.01)。结论:天麻素能够提高D-半乳糖刺激的BV2细胞活力,改善BV2细胞衰老染色和衰老蛋白表达,并降低ROS水平和减缓炎症反应,这可能与天麻素提高SIRT3蛋白表达有关。
Objective:In order to explore the protective effects and mechanism of gastrodin on BV2 cells treated with D-galactose.Methods:The BV2 cells were treated with D-galactose at different concentrations(10,20,30 and 40μg/mL)for 24 h to establish a senescent cell model,and the optimum concentration of D-galactose was selected by CCK-8 method;The cells were divided into control group,model group,silent mating type information regulation 2 homolog 3(SIRT3)inhibitor+gastrodin group and gastrodin group;and the optimum concentration of D-galactose was selected by CCK-8 method;The effects of different concentrations of gastrodin(10,20,30,40 and 50μg/mL)on the viability of BV2 cells treated with D-galactose were detected by CCK-8 method,and the best concentration of gastrodin was selected;The aging area of BV2 cells was detected by Senescenceβ-Galactosidase staining(SA-β-Gal);The level of reactive oxygen species(ROS)in BV2 cells was detected by biochemical method;The levels of neuroinflammatory factor interleukin 1β(IL-1β),interleukin 6(IL-6)and tumor necrosis factorα(TNF-α)were detected by enzyme linked immunosorbent assay(ELISA);The fluorescence intensity of SIRT3 was detected by immunofluorescence method;The protein expression levels of SIRT3,P16 and P21 were detected by Western blot.Results:Treatment with D-galactose at 30μg/mL exerted a significant inhibitory effect on BV2 cell viability,resulting in SA-β-Gal staining area and the expression of aging proteins P16 and P21 increased(P<0.01),ROS level and inflammatory factor IL-1β,IL-6 and TNF-αsignificantly increased(P<0.01),and the expression of SIRT3 protein and fluorescence intensity decreased in BV2 cells(P<0.01).30μmol/L gastrodin significantly increased the of BV2 cells viability treated with D-galactose(P<0.01);Gastrodin reduced SA-β-Gal staining area and the expression levels of aging proteins P16 and P21(P<0.01);Gastrodin significantly decreased the level of ROS and neuroinflammatory factor IL-1β,IL-6 and TNF-α;The expression level of SIRT3 protein
作者
管桐
高丽莎
隋得志
米佳
王斌胜
GUAN Tong;GAO Lisha;SUI Dezhi;MI Jia;WANG Binsheng(School of Integrated Traditional Chinese and Western Medicine,Binzhou Medical University,Yantai 264003,China;School of Pharmacy,Binzhou Medical University,Yantai 264003,China)
出处
《食品工业科技》
CAS
北大核心
2022年第19期410-418,共9页
Science and Technology of Food Industry
基金
国家自然科学基金(31671139):烟台市重点研发计划项目(2017ZH051)。
