丹参多糖对免疫性肝损伤相关炎性因子的体外调节
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摘要
为了探究丹参多糖对导致免疫性肝损伤相关炎性因子的体外调节作用,在体外用LPS刺激共培养的小鼠正常肝细胞NCTC1469和小鼠巨噬细胞RAW246.7建立体外肝细胞损伤模型,LPS单一刺激RAW246.7建立炎症模型。MTT法测定丹参多糖对NCTC1469和RAW246.7活性的安全范围;微板法测定NCTC1469与RAW246.7共培养细胞上清液中ALT的活性;Diff-Quik染色观察RAW246.7的形态变化;ELISA测定RAW246.7细胞上清液中TNF-α、IL-6、IL-10和IL-1β水平;Griss法测定细胞上清液中NO的含量。结果显示,与空白组相比,LPS组能显著性提高NCTC1469和RAW246.7共培养中ALT的含量,刺激RAW246.7细胞变形,RAW246.7细胞液中TNF-α、IL-6、IL-10、IL-1β和NO的释放量显著性升高(P<0.01)。丹参多糖各组(2.0,1.0,0.5g/L)能显著性降低共培养细胞液中ALT的活性(P<0.05),并能改善LPS导致的RAW246.7的变形程度,显著性降低RAW246.7细胞液中TNF-α、IL-6、IL-10、IL-1β和NO的释放量(P<0.01)。结果表明,丹参多糖能有效抑制炎症反应,并通过减少炎性因子的释放达到缓解免疫性肝损伤的效果。
The aim of the present study was to explore the effects of salvia miltiorrhiza polysaccharides on inflammatory factors in immuneologic liver injury in vitro.Co-cultured NCTC1469 and RAW246.7 cells were stimulated with LPS to establish a hepatocyte injury model and LPS single stimulation RAW246.7 to establish an inflammatory model in vitro.Safety scope of salvia miltiorrhiza polysaccharides on the activity of NCTC1469(mouse normal hepatocytes)and RAW246.7(mouse macrophages)was measured by MTT,the activity of ALT in Nctc1469 co-cultured with raw246.7 supernatant was measured by microplate method.The morphological changes of RAW246.7 was observed by Diff-Quik staining.TNF-α,IL-6,IL-10 and IL-1βwere determined by ELISA;Griss methods was used to detect the content of NO.Results showed that compared with the normal group,the activity of ALT,the content of TNF-α,IL-6,IL-10 and IL-1βand expression of NO were increased.Salvia miltiorrhiza polysaccharides(2.0,1.0,0.5 g/L)could obviously reduce the activities of ALT,the content of TNF-α,IL-6,IL-10,IL-1βand NO compared with model group.The results drew the conclusion that salvia miltiorrhiza polysaccharides can effectively inhibit the inflammatory response,and alleviate the harm of immunologic liver injur by reducing the release of inflammatory cytokines.
The aim of the present study was to explore the effects of salvia miltiorrhiza polysaccharides on inflammatory factors in immuneologic liver injury in vitro.Co-cultured NCTC1469 and RAW246.7 cells were stimulated with LPS to establish a hepatocyte injury model and LPS single stimulation RAW246.7 to establish an inflammatory model in vitro.Safety scope of salvia miltiorrhiza polysaccharides on the activity of NCTC1469(mouse normal hepatocytes)and RAW246.7(mouse macrophages)was measured by MTT,the activity of ALT in Nctc1469 co-cultured with raw246.7 supernatant was measured by microplate method.The morphological changes of RAW246.7 was observed by Diff-Quik staining.TNF-α,IL-6,IL-10 and IL-1βwere determined by ELISA;Griss methods was used to detect the content of NO.Results showed that compared with the normal group,the activity of ALT,the content of TNF-α,IL-6,IL-10 and IL-1βand expression of NO were increased.Salvia miltiorrhiza polysaccharides(2.0,1.0,0.5 g/L)could obviously reduce the activities of ALT,the content of TNF-α,IL-6,IL-10,IL-1βand NO compared with model group.The results drew the conclusion that salvia miltiorrhiza polysaccharides can effectively inhibit the inflammatory response,and alleviate the harm of immunologic liver injur by reducing the release of inflammatory cytokines.
引文
[1]KELLY A R,NATALIE S.Co-culture of hepatocytes and kupffer cells as an in vitro model ofinflammation and drug-induced hepatotoxicity[J].J Pharm Sci,2016,105:950-964.
[2]胡宝吉.红景天苷预处理对刀豆蛋白A诱导小鼠肝损伤的作用及机制探讨[D].上海:第二军医大学,2014.
[3]郑加嘉,黄清松,吴蔓,等.茵栀黄注射液对小鼠免疫性肝损伤炎性因子含量的影响[J].成都中医药大学学报,2012,35(4):8-10.
[4]张可锋,高雅,钟明利,等.七味净肝灵对卡介苗联合脂多糖诱导的小鼠免疫性肝损伤的保护作用及其机制[J].中国医院药学杂志,2016,36(13):1062-1065.
[5]周群,吴宝明,孟晓明,等.Kv1.3在LPS诱导的小鼠急性肝损伤模型中表达变化[J].安徽医科大学学报,2016,51(5):649-655.
[6]徐哲.TLR4siRNA防治D-GalN/LPS诱导的小鼠急性肝损伤的实验研究[D].上海:第四军医大学,2007.
[7]李宁,李丽,王月影,等.母体铅暴露对仔鼠大脑皮层组织中IL-1β和TNF-α表达的影响[J].中国兽医学报,2017,37(2):308-311.
[8]王旭,张子英,李培锋,等.牛磺鹅去氧胆酸对TGR5介导的NR8383细胞TNF-ɑ的影响[J].中国兽医学报,2016,36(1):108-111.
[9]李宁,乔明武,吕丰收,等.生命早期铅暴露对小鼠海马内IGF-1表达的影响[J].中国兽医学报,2016,36(1):148-151.
[10]赵翠,刘玲红,王开斌,等.貉IgG克隆及Fc活性多肽功能分析[J].中国兽医学报,2017,37(4):710-716.
[11]曾亚龙,阚兴池,吴艳成,等.PYY3-36对LPS诱导的BV-2细胞中促炎酶类及促炎细胞因子的影响[J].中国兽医学报,2017,37(9):1736-1742.
[12]BASAK C,PATHAK S K,BHATTACHARYYA A,et al.NF-kappaB-and C/EBPbeta-driven interleukinlbeta gene expression and PAK1-mediated caspase-1activation play essential roles in interleukin-lbeta release from Helicobacter pylori lipopolysaccharidestimulated macrophages[J].J Biol Chem,2005,280:4279-4288.
[13]LI M,JIN X K,LI W D.The protective effect of DQHS on ConA-induced liver injury in mice and its mechanism[J].Chin Pharmaeol Bull,2009,25(5):630-633.
[14]GULER R,OLLEROS M L,VESIN D,et al.Inhibition of inducible nitric oxide synthase protects against liver injury induced by mycobacterial infection and endotoxins[J].J Hepatol,2004,41:773-781.
[15]SASS G,KOERBER K,BANG R,et al.Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice[J].J Clin Invest,2001,107:439-447.
[2]胡宝吉.红景天苷预处理对刀豆蛋白A诱导小鼠肝损伤的作用及机制探讨[D].上海:第二军医大学,2014.
[3]郑加嘉,黄清松,吴蔓,等.茵栀黄注射液对小鼠免疫性肝损伤炎性因子含量的影响[J].成都中医药大学学报,2012,35(4):8-10.
[4]张可锋,高雅,钟明利,等.七味净肝灵对卡介苗联合脂多糖诱导的小鼠免疫性肝损伤的保护作用及其机制[J].中国医院药学杂志,2016,36(13):1062-1065.
[5]周群,吴宝明,孟晓明,等.Kv1.3在LPS诱导的小鼠急性肝损伤模型中表达变化[J].安徽医科大学学报,2016,51(5):649-655.
[6]徐哲.TLR4siRNA防治D-GalN/LPS诱导的小鼠急性肝损伤的实验研究[D].上海:第四军医大学,2007.
[7]李宁,李丽,王月影,等.母体铅暴露对仔鼠大脑皮层组织中IL-1β和TNF-α表达的影响[J].中国兽医学报,2017,37(2):308-311.
[8]王旭,张子英,李培锋,等.牛磺鹅去氧胆酸对TGR5介导的NR8383细胞TNF-ɑ的影响[J].中国兽医学报,2016,36(1):108-111.
[9]李宁,乔明武,吕丰收,等.生命早期铅暴露对小鼠海马内IGF-1表达的影响[J].中国兽医学报,2016,36(1):148-151.
[10]赵翠,刘玲红,王开斌,等.貉IgG克隆及Fc活性多肽功能分析[J].中国兽医学报,2017,37(4):710-716.
[11]曾亚龙,阚兴池,吴艳成,等.PYY3-36对LPS诱导的BV-2细胞中促炎酶类及促炎细胞因子的影响[J].中国兽医学报,2017,37(9):1736-1742.
[12]BASAK C,PATHAK S K,BHATTACHARYYA A,et al.NF-kappaB-and C/EBPbeta-driven interleukinlbeta gene expression and PAK1-mediated caspase-1activation play essential roles in interleukin-lbeta release from Helicobacter pylori lipopolysaccharidestimulated macrophages[J].J Biol Chem,2005,280:4279-4288.
[13]LI M,JIN X K,LI W D.The protective effect of DQHS on ConA-induced liver injury in mice and its mechanism[J].Chin Pharmaeol Bull,2009,25(5):630-633.
[14]GULER R,OLLEROS M L,VESIN D,et al.Inhibition of inducible nitric oxide synthase protects against liver injury induced by mycobacterial infection and endotoxins[J].J Hepatol,2004,41:773-781.
[15]SASS G,KOERBER K,BANG R,et al.Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice[J].J Clin Invest,2001,107:439-447.