半夏有效成分大黄酚对肿瘤坏死因子-α诱导人脑微血管内皮细胞的作用机制
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摘要
目的:研究半夏有效成分大黄酚对肿瘤坏死因子-α(TNF-α)诱导人脑微血管内皮细胞(HBMEC)的作用机制。方法:体外培养HBMEC,用TNF-α诱导HBMEC造模,将细胞分为正常组、模型组、大黄酚组、依达拉奉组。采用噻唑蓝比色(MTT)法检测大黄酚对HBMEC增殖能力的影响,用逆转录PCR法(RT-PCR)检测TNF-α,白细胞介素-6(IL-6),IL-8 mRNA的表达,用酶联免疫吸附法(ELISA)检测TNF-α,IL-6,IL-8,磷酸化氨基末端蛋白激酶(p-JNK)和磷酸化细胞外调节蛋白激酶(pERK)的含量,用蛋白免疫印迹法(Western blot)检测p-JNK,total JNK,p-ERK和total ERK的蛋白表达。结果:在256μmol·L~(-1)浓度时,大黄酚对HBMEC细胞有明显的抑制作用(P<0.01)。与模型组比较,在64~128μmol·L~(-1)浓度时,大黄酚对模型细胞生长有明显的促进作用(P<0.05);大黄酚组TNF-α,IL-6,IL-8 mRNA表达明显下降(P<0.01);大黄酚组TNF-α,IL-6,IL-8,p-JNK和p-ERK的含量明显减少(P<0.05);大黄酚组磷酸化JNK/JNK,磷酸化ERK/ERK表达明显下降(P<0.05)。结论:半夏有效成分大黄酚具有抑制TNF-α诱导HBMEC损伤作用,可能是通过下调ERK/JNK信号通路减少炎症相关因子TNF-α,IL-6和IL-8的表达。
Objective: To study the effect of chrysophanol from pinellia on tumor necrosis factor-α(TNF-α)-induced human cerebral microvascular endothelial cells(HBMEC). Method: HBMECs were cultured in vitro,and modeled with TNF-α. The cells were divided into normal group,model group,chrysophanol group and edaravone group. TNF-α,interleukin-6(IL-6) and IL-8 mRNA expressions were detected by RT-PCR. TNF-α,IL-6, IL-8, p-JNK and p-ERK were detected by enzyme-linked immunosorbent assay(ELISA). Protein expressions of p-JNK, total JNK, p-ERK and total ERK were detected by Western blot. Result: At the concentration of 256 μmol·L~(-1),chrysophanol significantly inhibited HBMEC cells(P < 0. 01). Compared with the model group,chrysophanol had a significant effect on the growth of model cells at concentrations between 64μmol·L~(-1) and 128 μmol·L~(-1)(P < 0. 05). Expressions of TNF-α,IL-6,IL-6,IL-8,p-JNK and p-ERK in chrysophanol group were significantly decreased(P < 0. 05). Expressions of p-JNK/JNK and p-ERK/ERK in chrysophanol group were significantly decreased(P < 0. 05). Conclusion: Chrysophanol has an effect in inhibiting TNF-α-induced HBMEC damage,and may reduce the expressions of TNF-α,IL-6 and IL-8 by down-regulating ERK/JNK signaling pathway.
Objective: To study the effect of chrysophanol from pinellia on tumor necrosis factor-α(TNF-α)-induced human cerebral microvascular endothelial cells(HBMEC). Method: HBMECs were cultured in vitro,and modeled with TNF-α. The cells were divided into normal group,model group,chrysophanol group and edaravone group. TNF-α,interleukin-6(IL-6) and IL-8 mRNA expressions were detected by RT-PCR. TNF-α,IL-6, IL-8, p-JNK and p-ERK were detected by enzyme-linked immunosorbent assay(ELISA). Protein expressions of p-JNK, total JNK, p-ERK and total ERK were detected by Western blot. Result: At the concentration of 256 μmol·L~(-1),chrysophanol significantly inhibited HBMEC cells(P < 0. 01). Compared with the model group,chrysophanol had a significant effect on the growth of model cells at concentrations between 64μmol·L~(-1) and 128 μmol·L~(-1)(P < 0. 05). Expressions of TNF-α,IL-6,IL-6,IL-8,p-JNK and p-ERK in chrysophanol group were significantly decreased(P < 0. 05). Expressions of p-JNK/JNK and p-ERK/ERK in chrysophanol group were significantly decreased(P < 0. 05). Conclusion: Chrysophanol has an effect in inhibiting TNF-α-induced HBMEC damage,and may reduce the expressions of TNF-α,IL-6 and IL-8 by down-regulating ERK/JNK signaling pathway.
引文
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[21]Byun E B,Sung N Y,Park J N,et al.Gammairradiated resveratrol negatively regulates LPS-induced MAPK and NF-kappa B signaling through TLR4 in macrophages[J].Int Immunopharmacol,2015,25(2):249-259.
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[23]郑堰心,张丽,邓虹珠,等.槐定碱、苦参碱和苦豆碱对LPS诱导结肠上皮细胞炎症模型中细胞因子IL-6和TNF-α水平的影响[J].中国实验方剂学杂志,2014,20(8):133-136.
[2]田爱荣.半夏白术天麻汤加减联合依达拉奉治疗急性缺血性脑卒中疗效分析[J].中国实用医药,2014,9(27):161-162.
[3]周红霞,李鱼昆,张运克.从脑缺血后血脑屏障的通透性调节机制探索宣通玄府法的内涵[J].世界中西医结合杂志,2017,12(5):717-720.
[4]YU J,MA M,MA Z,et al.HDAC6 inhibition prevents TNF-alpha-induced caspase 3 activation in lung endothelial cell and maintains cell-cell junctions[J].Oncotarget,2016,7(34):54714-54722.
[5]Good R B,Gilbane A J,Trinder S L,et al.Endothelial to mesenchymal transition contributes to endothelial dysfunction in pulmonary arterial hypertension[J].Am J Pathol,2015,185(7):1850-1858.
[6]张秀芳.半夏白术天麻汤加减联合依达拉奉治疗急性缺血性脑卒中疗效分析[J].实用中医药杂志,2013,29(7):525-526.
[7]潘会朝,郭富礼.中药汤剂当用生半夏[J].时珍国医国药,2003,14(9):28-31.
[8]杨虹,侴桂新,王峥涛,等.半夏的化学成分研究[J].中国中药杂志,2007,42(2):99-101.
[9]傅兴圣,陈菲,刘训红,等.大黄化学成分与药理作用研究新进展[J].中国新药杂志,2011,20(46):1534-1539.
[10]ZHANG Y,HAN Y,ZHAO Y,et al.DT-13 ameliorates TNF-α-induced vascular endothelial hyperpermeability via non-muscle myosinⅡAand the Src/PI3K/Akt signaling pathway[J].Front Immunol,2017,doi:10.3389/fimmu.2017.00925.
[11]李曼霞,董志.脑缺血再灌注时血脑屏障损伤的炎性机制研究进展[J].解放军药学学报,2007,23(1):60-63.
[12]杜继浩,马艳娟.生半夏治疗中风后遗症的体会[J].中国社区医师·医学专业半月刊,2010,15(12):112.
[13]耿黎明,李可法,杨振伟.麻黄附子细辛汤加减治疗冠心病缓慢性心律失常32例[J].河南中医,2010,30(9):852-853.
[14]房亚兰,黄语悠,赵咏梅,等.大黄酚对局灶性脑缺血再灌注小鼠缺血半暗带区环氧化酶2和基质金属蛋白酶-9表达的影响[J].首都医科大学学报,2017,38(1):47-52.
[15]王四海,梁春利,张海红,等.大黄酚对脑缺血/再灌注小鼠脑组织NO的影响和断头抗缺氧作用[J].天津医药,2017,45(6):593-595.
[16]武良玉,鲍秀琦,孙华,等.星形胶质细胞上的清道夫受体与神经炎症的关系[J].中国医学科学院学报,2014,36(3):330-335.
[17]泰文娇,叶旋,鲍秀琦,等.小胶质细胞与脑缺血关系的研究进展[J].药学学报,2012,47(3):346-353.
[18]朱长庚.神经免疫内分泌网络与癫痫发病机理的关系[J].解剖学报,2002,33(3):321-324.
[19]Matsushita K,MENG W,WANG X,et al.Evidence for apoptosis after intercerebral hemorrhage in rat striatum[J].J Cereb Blood Flow Metab,2000,20(2):396-404.
[20]FENG S,YANG Q,LIU M,et al.Edaravone for acute ischaemic stroke[J].Cochrane Database Syst Rev,2011,16(12):D7230.
[21]Byun E B,Sung N Y,Park J N,et al.Gammairradiated resveratrol negatively regulates LPS-induced MAPK and NF-kappa B signaling through TLR4 in macrophages[J].Int Immunopharmacol,2015,25(2):249-259.
[22]Julien P,lsabelle M,Beckley K,et al.TypeⅠIFN triggers R1G-1/TLR3/1/NLRP3-dependent inflammasome activation in influenza a virus infected cells[J].PLo S Pathog,2013,9(4):e1003256.
[23]郑堰心,张丽,邓虹珠,等.槐定碱、苦参碱和苦豆碱对LPS诱导结肠上皮细胞炎症模型中细胞因子IL-6和TNF-α水平的影响[J].中国实验方剂学杂志,2014,20(8):133-136.