Effect of Shenfu Injection on Vascular Endothelial Injury Induced by Oxidative Stress and Related Signal Transduction Pathway
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摘要: 目的 研究参附注射液对氧化应激血管内皮损伤的作用及相关信号转导通路的影响。方法 培养血管内皮细胞ECV304，MTT法筛选H2O2造模和参附注射液的干预浓度；DCFH-DA荧光探针检测细胞内活性氧(ROS)含量；Hoechst染色和流式细胞术检测评估细胞凋亡；Western blot法检测Cleaved Caspase-3、ERK、p-ERK、Bcl-2和Bax蛋白的表达。结果 H2O2呈浓度依赖性诱导ECV304细胞死亡；H2O2促进ECV304细胞内ROS含量增加，参附注射液或N-乙酰半胱氨酸(NAC)可拮抗此过程；与对照组比较，H2O2能够诱导ECV304细胞凋亡，增加ERK和Caspase-3的活化，下调Bcl-2/Bax表达；与模型组比较，参附注射液或NAC能拮抗H2O2诱导的ECV304细胞凋亡，抑制ERK与Caspase-3的活化，上调Bcl-2/Bax表达(P < 0.05)。结论 参附注射液通过抑制ERK磷酸化和Caspase-3活化，上调Bcl-2/Bax表达，拮抗H2O2诱导的ECV304细胞凋亡。Abstract: OBJECTIVE To explore the effect of Shenfu injection on vascular endothelial injury induced by oxidative stress and related signal transduction pathway.METHODS Human umbilical vein endothelial cells ECV304 were cultured. The concentration of H2O2 for modeling and Shenfu injection for intervening were determined by methylthiazoletetrazolium (MTT) assay. The intracellular reactive oxygen species level was detected by DCFH-DA fluorescent probe. Cell apoptosis was measured by Hoechst staining and flow cytometry. Western blot was used to determine the expression of Cleaved Caspase-3 (C-Caspase-3), ERK, p-ERK, Bcl-2 and Bax proteins.RESULTS H2O2 induced ECV304 vascular endothelial cell death in a dose-dependent manner. The content of ROS was promoted by H2O2, which could be antagonized by Shenfu injection. Compared with the control group, H2O2 could increase the apoptosis of ECV304 cell, promote the activation of ERK and Caspase-3, and decrease Bcl-2/Bax. Shenfu injection or NAC could reduce H2O2-induced ECV304 vascular endothelial cell apoptosis, inhibit the activation of ERK and Caspase-3, and up-regulate Bcl-2/Bax (P < 0.05).CONCLUSION Shenfu injection can reduce H2O2-induced ECV304 cell apoptosis by decreasing the phosphorylation of ERK, activating Caspase-3, and up-regulating Bcl-2/Bax.
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Key words:
- Shenfu injection /
- oxidative stress /
- vascular endothelium /
- apoptosis /
- signal pathway
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图 1 H2O2和H2O2+参附注射液对ECV304细胞存活率的影响
注：与对照组相比，*P < 0.05，**P < 0.01；与H2O2组相比，#P < 0.05，##P < 0.01。 x±s，n=6。
图 2 参附注射液对H2O2诱导ECV304细胞凋亡Hoechst染色和阳性细胞率的影响
注：与对照组比较，**P < 0.01；与H2O2组比较，##P < 0.01。 x±s，n=3。
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[1] SINGER M, DEUTSCHMAN CS, SEYMOUR CW, et al. The third international consensus definitions for Sepsis and septic shock (Sepsis-3)[J]. JAMA, 2016, 315(8): 801-810. doi: 10.1001/jama.2016.0287 [2] 杨霞, 李香琴, 马晓媛, 等. 脓毒症血管内皮损伤及调节策略的研究进展[J]. 中国中西医结合急救杂志, 2016, 23(1): 108-110. [3] 朱丹丹, 于健. 内皮细胞微粒与脓毒症关系研究进展[J]. 中国实用内科杂志, 2018, 38(S2): 88-91, 94. https://www.cnki.com.cn/Article/CJFDTOTAL-SYNK2018S2014.htm [4] AIRD WC. The role of the endothelium in severe Sepsis and multiple organ dysfunction syndrome[J]. Blood, 2003, 101(10): 3765-3777. http://www.bloodjournal.org/content/101/10/3765.full-text.pdf [5] 朱欣佚, 奚肇庆, 张文曦. 复方薤白胶囊抗内皮细胞凋亡与信号转导通路的研究[J]. 南京中医药大学学报, 2010, 26(5): 359-361. doi: 10.3969/j.issn.1000-5005.2010.05.014 [6] ZHANG N, LIU J, QIU Z, et al. Shenfu injection for improving cellular immunity and clinical outcome in patients with Sepsis or septic shock[J]. Am J Emerg Med, 2017, 35(1): 1-6. doi: 10.1016/j.ajem.2016.09.008 [7] JIN YY, GAO H, ZHANG XY, et al. Shenfu injection inhibits inflammation in patients with acute myocardial infarction complicated by cardiac shock[J]. Chin J Integr Med, 2017, 23(3): 170-175. doi: 10.1007/s11655-016-2749-x [8] EL KAMOUNI S, EL KEBBAJ R, ANDREOLETTI P, et al. Protective effect of argan and olive oils against LPS-induced oxidative stress and inflammation in mice livers[J]. Int J Mol Sci, 2017, 18(10): E2181. doi: 10.3390/ijms18102181 [9] NAGASE M, SAKURAI A, SUGITA A, et al. Oxidative stress and abnormal cholesterol metabolism in patients with post-cardiac arrest syndrome[J]. J Clin Biochem Nutr, 2017, 61(2): 108-117. doi: 10.3164/jcbn.17-30 [10] 郭发先. 参附注射液对H2O2氧化应激诱导人脐静脉血管内皮细胞损伤的保护作用[D]. 南昌: 南昌大学, 2014. [11] 李春玲, 陈静. 参附注射液在ICU脓毒症患者救治中的应用[J]. 中国中医急症, 2014, 23(4): 608-609. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYJZ201404018.htm [12] 邱泽亮, 刘建华, 张宁, 等. 参附注射液对严重脓毒症患者肝肾功能的保护作用[J]. 中药药理与临床, 2013, 29(4): 155-157. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYYL201304054.htm [13] HONG FF, GUO FX, ZHOU Y, et al. Shenfu injection protects human ECV304 cells from hydrogen peroxide via its anti-apoptosis way[J]. J Ethnopharmacol, 2015, 163: 203-209. doi: 10.1016/j.jep.2015.01.032 [14] 张艺森, 徐晓涵, 孙炳伟. 脓毒症时血管内皮细胞与血管平滑肌细胞相互作用的研究进展[J]. 中华危重病急救医学, 2016(2): 180-183. [15] 柴云飞, 雷黎明, 李鹏, 等. 血管内皮细胞损伤与脓毒症休克的相关性[J]. 实用医学杂志, 2017, 33(15): 2529-2532. https://www.cnki.com.cn/Article/CJFDTOTAL-SYYZ201715029.htm [16] SUN M, WANG R, HAN Q. Inhibition of leukotriene B4 receptor 1 attenuates lipopolysaccharide-induced cardiac dysfunction: Role of AMPK-regulated mitochondrial function[J]. Sci Rep, 2017, 7: 44352. http://www.onacademic.com/detail/journal_1000039852337810_30f7.html [17] 戴晓明, 吴慧平, 蒋凤荣, 等. 参附注射液对大鼠肾缺血再灌注损伤作用的研究[J]. 南京中医药大学学报, 2006, 22(6): 380-381. http://xb.njucm.edu.cn/jnutcmns/ch/reader/view_abstract.aspx?file_no=200606170&flag=1 [18] 董雅洁, 高维娟. Bcl-2、bax、Caspase-3在细胞凋亡中的作用及其关系[J]. 中国老年学杂志, 2012, 32(21): 4828-4830. https://www.cnki.com.cn/Article/CJFDTOTAL-ZLXZ201221128.htm [19] 赵明哲, 刘靖华, 李玉花, 等. ERK信号通路的信号转导调控机制[J]. 国际病理科学与临床杂志, 2009, 29(1): 15-19. https://www.cnki.com.cn/Article/CJFDTOTAL-WYSB200901003.htm [20] 陈畅, 唐娟娟, 夏宝妹, 等. 越鞠丸对帕金森病体外模型的神经保护作用研究[J]. 南京中医药大学学报, 2015, 31(2): 156-159. http://xb.njucm.edu.cn/jnutcmns/ch/reader/view_abstract.aspx?file_no=20150215&flag=1 [21] KAWASAKI T, KITAO T, NAKAGAWA K, et al. Nitric oxide-induced apoptosis in cultured rat astrocytes: Protection by edaravone, a radical scavenger[J]. Glia, 2007, 55(13): 1325-1333. doi: 10.1002/glia.20541/pdf -
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