骆驼蓬多糖调节细胞自噬及巨噬细胞炎症反应的活性研究
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摘要
目的探讨骆驼蓬多糖调节细胞自噬及巨噬细胞炎症反应的活性。方法用不同浓度的骆驼蓬多糖干预,诱导NRK细胞自噬,用激光共聚焦显微镜观察自噬体,筛选出骆驼蓬多糖诱导细胞自噬的最佳干预浓度和时间范围;利用RAW264.7细胞,以1μg/mL脂多糖(lipopolysaccharides, LPS)建立体外细胞炎症模型;在骆驼蓬多糖诱导细胞自噬的最佳浓度和时间下进行干预,以CCK-8试验检测细胞活性,用Hoechst和PI双重染色方法观察细胞凋亡,用ELISA试剂盒检测炎症因子IL-1β和IL-6的分泌量。结果骆驼蓬多糖对NRK细胞诱导自噬的最佳时间为12 h,浓度范围为50～100μg/mL。骆驼蓬多糖干预LPS诱导的RAW264.7细胞炎症模型后,对细胞活力均无明显影响,诱导细胞凋亡,呈剂量依赖性,细胞培养液中IL-6和IL-1β分泌量降低,与模型组有显著差异(P(27)0.05)。结论骆驼蓬多糖能抑制LPS刺激诱导的炎症因子的释放,诱导细胞自噬和凋亡,在50～100μg/mL范围内对巨噬细胞RAW264.7具有良好的免疫调节作用。
Objective To investigate the activity of Peganum harmala polysaccharides in regulating autophagy and macrophage inflammatory response. Methods Different concentrations of Peganum harmala polysaccharides were used to induce autophagy in NRK cells. Autophagosomes were observed by laser confocal microscopy, and the optimal concentration and time range of Peganum harmala polysaccharides for autophagy inducing were screened. In vitro cytotoxic model was established by 1 μg/mL lipopolysaccharides(LPS) using RAW264.7 cells. Intervention was carried out at the optimal concentration and time of autophagy induced by Peganum harmala polysaccharide. Cell viability was measured by CCK-8 assay, apoptosis was observed by Hoechst and PI double staining, and the secretion of inflammatory factors IL-1β and IL-6 was detected by ELISA kit. Results The optimal time for autophagy induced by Peganum harmala polysaccharide to NRK cells was 12 h, and the concentration ranged from 50 μg/mL to 100 μg/mL. After treatment with LPS-induced RAW264.7 cell inflammation model, polysaccharides of Peganum harmala had no significant effect on cell viability, and induced apoptosis in a dose-dependent manner. The secretion of IL-6 and IL-1β in cell culture medium decreased, which was significantly different from the model group(P(27)0.05). Conclusion Peganum harmala polysaccharide can inhibit the release of inflammatory factors induced by LPS stimulation, induce autophagy and apoptosis, and have a good immunomodulatory effect on macrophage RAW264.7 in a range of 50-100 μg/mL.
Objective To investigate the activity of Peganum harmala polysaccharides in regulating autophagy and macrophage inflammatory response. Methods Different concentrations of Peganum harmala polysaccharides were used to induce autophagy in NRK cells. Autophagosomes were observed by laser confocal microscopy, and the optimal concentration and time range of Peganum harmala polysaccharides for autophagy inducing were screened. In vitro cytotoxic model was established by 1 μg/mL lipopolysaccharides(LPS) using RAW264.7 cells. Intervention was carried out at the optimal concentration and time of autophagy induced by Peganum harmala polysaccharide. Cell viability was measured by CCK-8 assay, apoptosis was observed by Hoechst and PI double staining, and the secretion of inflammatory factors IL-1β and IL-6 was detected by ELISA kit. Results The optimal time for autophagy induced by Peganum harmala polysaccharide to NRK cells was 12 h, and the concentration ranged from 50 μg/mL to 100 μg/mL. After treatment with LPS-induced RAW264.7 cell inflammation model, polysaccharides of Peganum harmala had no significant effect on cell viability, and induced apoptosis in a dose-dependent manner. The secretion of IL-6 and IL-1β in cell culture medium decreased, which was significantly different from the model group(P(27)0.05). Conclusion Peganum harmala polysaccharide can inhibit the release of inflammatory factors induced by LPS stimulation, induce autophagy and apoptosis, and have a good immunomodulatory effect on macrophage RAW264.7 in a range of 50-100 μg/mL.
引文
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[2]Ahn CB,ChoYS,Je JY.Purification and anti-inflammatory action of tripeptide from salmon pectoral fin byproduct protein hydrolysate[J].Food Chem,2015,(168):151-156.
[3]赵婷,王长虹,王峥涛.骆驼蓬属植物中生物碱类化学成分及其药理活性研究进展[J].国际药学研究杂志,2010,37(5):333-339.Zhao T,Wang CH,Wang YT.Research progress on chemical constituents and pharmacological activities of alkaloids in Peganum chinensis[J].Int JPharm Res,2010,37(5):333-339.
[4]李越鲲.骆驼蓬地上部分化学成分的研究[D].杨凌:西北农林科技大学,2005.Li YZ.Research on the chemical composition of the upper part of Pegana chinensis[D].Yangling:Northwest A&F University,2005.
[5]Ruan S,Jia F,Li J.Potential antitumor effect of harmine in the treatment of thyroid cancer[J].Evid Bas Compl Altern Med,2017,(4):9402615.
[6]Wang J,Hu S,Nie S,et al.Reviews on mechanisms of in vitro antioxidant activity of polysaccharides[J].Oxid Med Cell Longev,2016,2016(64):1-13.
[7]张鸣,常国华,陈映全.骆驼蓬粗多糖抗氧化性的研究[J].中国食品添加剂,2015,99(22):85-88.Zhang M,Chang GH,Chen YQ.Study on antioxidant activity of Polygonum cuspidatum polysaccharides[J]Chin Food Add,2015,99(22):85-88.
[8]Nathan C.Points of control in inflammation[J].Nature,2002,420(6917):846-852.
[9]Nabavi SF,Braidy N,Orhan IE,et al.Rhodiola rosea L.and Alzheimer’s disease:from farm to pharmacy[J].Phytother Res,2016,30(4):532-539.
[10]Conti B,Tabarean I,Andrei C,et al.Cytokines and fever[J].Front Biosci,2004,9(6):1433-1449.
[11]张梦雅,王春丽,杜先华,等.血根碱通过STAT3通路对LPS致RAW264.7细胞炎症的保护作用[J].中药新药与临床药理,2017,28(6):714-718.Zhang MY,Wang CL,Du XH,et al.The protective effect of blood root base on LPS-induced RAW264.7 cell inflammation through STAT3pathway[J].Tradit Chin Med Clin Pharm,2017,28(6):714-718.
[12]Rhule A,Navarro S,Smith JR,et al.Panax notoginsengattenuates LPS-induced pro-inflammatory mediators in RAW264.7 cells[J].JEthnopharm,2006,106(1):121-128.
[13]麻冬滨,东方,季宇彬.多糖结构修饰研究进展[J].黑龙江科技信息,2013,(3):53-54.Ma DB,Dong F,Ji YB.Research progress on polysaccharide structure modification[J].Heilongjiang Sci Technol Inf,2013,(3):53-54.
[14]Aboughe-Angone S,Nguema-Ona E,Boudjeko T,et al.Plant cell wall polysaccharides as immmunomodulators of the immune system[J].Curr Top Phytochem,2011,(10):1-16.
[15]Boudjeko T,Megnekou R,Woguia AL,et al.Antioxidant and immunomodulatory properties of polysaccharides from Allanblackia floribunda oliv stem bark and Chromolaena odorata(L)king and H.E.robins leaves[J].BMC Res Note,2015,8(1):759.
[16]安晓娟,冯琳,宋红平,等.植物多糖的结构分析及药理活性研究进展[J].中国药学杂志,2012,47(16):1271-1275.An XJ,Feng L,Song HP,et al.Progress in structural analysis and pharmacological activity of plant polysaccharides[J].Chin Pharm J,2012,47(16):1271-1275.
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[18]Kim HS,Kim YJ,Lee HK,et al.Activation of macrophages by polysaccharide isolated from Paecilomyces cicadae through toll-like receptor 4[J].Food Chem Toxicol,2012,50(9):3190-3197.
[19]李敬双,刘英姿,唐雨顺,等.苜蓿多糖对小鼠淋巴细增殖和NK细胞活性影响的研究[J].中国农学通报,2012,28(32):89-93.Li JS,Liu YZ,Tang YS,et al.Effect of Lycium Barbarum polysaccharide on lymphocyte proliferation and NK cell activity in mice[J].Chin Agric Sci Bull,2012,28(32):89-93.
[20]徐贤柱,饶华,蔡险峰,等.杜仲叶多糖提取及对小鼠免疫功能影响研究[J].时珍国医国药,2013,(3):541-542.Xu XZ,Rao H,Cai XF,et al.Study on the extraction of polysaccharides from Eucommia Ulmoides Leaves and its effect on mouse immune function[J].Lishizhen Med Mater Med Res,2013,(3):541-542.
[21]尚庆辉,解玉怀,张桂国,等.植物多糖的免疫调节作用及其机制研究进展[J].动物营养学报,2015,27(1):49-58.Shang QH,Xie YH,Zhang GG,et al.Research progress on immunomodulatory effects and mechanisms of plant polysaccharides[J].JAnim Nutr,2015,27(1):49-58.
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[25]Mizushima N,Komatsu M.Autophagy:renovation of cells and tissues[J].Cell,2011,(147):728-741.
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