摘要
本研究运用细胞生物学研究方法,探讨中草药提取物黄连素对草鱼(Ctenopharyngodon idellus)肝细胞(L8824)活力、氧化应激和细胞凋亡的影响。用不同浓度黄连素(0μmol/L、5μmol/L、25μmol/L、50μmol/L和100μmol/L)分别处理体外培养的草鱼肝细胞Oh、6 h、12 h和24 h,采用CCK-8法检测细胞活力。结果显示,黄连素浓度和时间对细胞活力有显著交互作用(P<0.05),且黄连素浓度对其活力影响差异显著(P<0.05),草鱼肝细胞活力随着黄连素的浓度增加先升高后降低(P<0.05)。在此基础上,设置了黄连素梯度浓度(0μmol/L、5μmol/L、25μmol/L、50μmol/L和100μmol/L)培养草鱼肝细胞12 h后,收集细胞,测定细胞谷丙转氨酶(GPT)及谷草转氨酶(GOT)活性,丙二醛(MDA)及活性氧(ROS)含量、细胞凋亡水平和相关基因mRNA表达量。结果表明,与不添加黄连素组相比,当黄连素浓度为100μmol/L时,细胞GOT和GPT活性、ROS和MDA含量显著(P<0.05)增加。当黄连素的浓度为25μmol/L和50μmol/L时,细胞凋亡水平显著(P<0.05)升高;当黄连素的浓度增加到100μmol/L时,细胞凋亡水平显著(P<0.05)降低。当黄连素的浓度为50μmol/L和100μmol/L时,细胞半胱氨酸蛋白酶-3(caspase-3)mRNA表达量显著(P<0.05)升高。综上所述,当黄连素浓度超过50μmol/L会抑制细胞的生长,引发氧化应激,影响细胞功能。因此,黄连素在草鱼肝细胞培养液中添加的适宜浓度为25～50μmol/L。
Berberine was one kind of Chinese herbal additive, which extracted from the stems and roots of various Berberis species, such as Cortex phellodendri （Huangbai）, Rhizoma coptidis （Huanglian） and so on. It possessed a series of biological activities, including antioxidant, anti-inflammatory, reducing blood glucose and lipid level. In addition, it had been proved that berberine could improve lipid deposition and glucose metabolism in diabetes. Recently, berberine had been applied to aquaculture diets as an herbal feed additive in Asian area. Our laboratory conducted many vivo experiments to study the function of berberine. The current study demonstrated that berber-ine added in high-fat diet could mitigate oxidative stress, inhibit the apoptosis and enhance disease resistance of fish. However, it was found that the growth performance of fish fed with normal diet supplied berberine was sig-nificantly enhanced but immunity was not improved. In order to solve this problem, the study was aimed to ex-plore the effect of different concentrations of berberine on cell activity, oxidative stress and apoptosis of grass carp （Ctenopharyngodon idellus） hepatopancreas cell in vitro experiment. Cultured cells were treated with different concentrations of berberine （0, 5 μmol/L, 25 μmol/L, 50 μmol/L, 100 μmol/L） for 0, 6 h, 12 h, 24 h. Then, the optimal concentration of berberine was showed by detecting cell viability in CCK-8 assay. Results showed that the cell activity was affected by interaction of time and berberine concentration （P〈0.05）. Berberine significantly （P〈0.05） affected cell activity in a dose-dependent manner. According to the cell activity data, the remaining in-dicators were selected to data of 12 hours for analyzing related indicators, including glutamate oxalate transami-nase （GOT） and glutamate pyruvate transaminase （GPT） activity, malondialdehyde （MDA） and ROS content, apoptosis, caspase-3, caspase-8 and caspase-9 mRNA expression. Compared with control group, GOT and GPT activity in group of 100 μmol/L berberine concentration were significantly （P〈0.05） increased. Meanwhile, the content of MDA and ROS were significantly increased with increasing concentration of berberine. The tendency of content of MDA and ROS were same as that of GOT and GPT activity （P〈0.05）. In addition, compared with con-trol group, the total cell apoptosis rate in group of 25 μmol/L and 50 μmol/L berberine concentration were sig-nificantly （P〈0.05） increased while cell apoptosis rate in 100 μmol/L berberine group was significantly （P〈0.05） decreased. At last, the mRNA expression of caspase-3 in group of 50 μmol/L and 100 μmol/L berberine group were significantly enhanced （P〈0.05）. In summary, the safety concentration of berberine in stimulating fish liver cells should be 25？50 μmol/L. When the concentration was more than 50 μmol/L, it would have toxicity to normal growth cells and affect cell function.
作者
陈丹红
陈青青
田红艳
施华娟
刘文斌
CHEN Danhong;CHEN Qingqing;TIAN Hongyan;SHI Huajuan;LIU Wenbin(Key Laboratory of Aquatic Nutrition of Jiangsu Province,College of Animal Science and Technology,Nanjing Agri-cultural University,Nanjing 210095,China)
出处
《中国水产科学》
CAS
CSCD
北大核心
2018年第6期1281-1288,共8页
Journal of Fishery Sciences of China
基金
国家自然科学基金面上项目(31472292)
国家大宗淡水鱼产业技术体系资金项目(CARS-45-14)
关键词
草鱼
肝细胞
黄连素
细胞活力
氧化应激
细胞凋亡
Ctenopharyngodon idellus
hepatopancreas cell
berberine
cell activity
oxidative stress
apoptosis
