切换至 "中华医学电子期刊资源库"

中华危重症医学杂志(电子版) ›› 2021, Vol. 14 ›› Issue (04) : 281 -289. doi: 10.3877/cma.j.issn.1674-6880.2021.04.004

论著

白藜芦醇对脓毒症血管内皮屏障功能的保护作用及其机制研究
郭智东1, 吴锦鸿1, 赵雪1, 吴泽生1, 王弋1,()   
  1. 1. 310006 杭州,浙江大学医学院附属杭州市第一人民医院急诊科
  • 收稿日期:2021-03-23 出版日期:2021-08-31
  • 通信作者: 王弋
  • 基金资助:
    杭州市医药卫生科技计划项目(B20200683)

Protective effect and related mechanism of resveratrol on vascular endothelial barrier dysfunction in sepsis

Zhidong Guo1, Jinhong Wu1, Xue Zhao1, Zesheng Wu1, Yi Wang1,()   

  1. 1. Department of Emergency Medicine, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China
  • Received:2021-03-23 Published:2021-08-31
  • Corresponding author: Yi Wang
引用本文:

郭智东, 吴锦鸿, 赵雪, 吴泽生, 王弋. 白藜芦醇对脓毒症血管内皮屏障功能的保护作用及其机制研究[J/OL]. 中华危重症医学杂志(电子版), 2021, 14(04): 281-289.

Zhidong Guo, Jinhong Wu, Xue Zhao, Zesheng Wu, Yi Wang. Protective effect and related mechanism of resveratrol on vascular endothelial barrier dysfunction in sepsis[J/OL]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2021, 14(04): 281-289.

目的

探讨白藜芦醇在脂多糖诱导的血管内皮屏障功能损伤中的保护作用及其机制。

方法

将人脐静脉内皮细胞(HUVECs)分成对照组、脓毒症组及干预组。脓毒症组及干预组使用100 μg/mL脂多糖刺激HUVECs制作脓毒症血管内皮细胞损伤模型,干预组在建模前给予白藜芦醇进行预处理。应用细胞计数试剂盒8(CCK-8)及5-乙炔基-2’脱氧尿嘧啶核苷(EdU)细胞增殖试剂盒检测细胞增殖能力,采用划痕实验及Transwell法检测细胞迁移能力,计算各组单核细胞对内皮细胞的黏附率,采用Western-blotting检测CyclinD1、含IQ模序的三磷酸鸟苷酶活化蛋白1(IQGAP1)、VE-cadherin、claudin-5、细胞间黏附分子1(ICAM-1)及血管细胞黏附分子1(VCAM-1)的蛋白表达水平,并比较各组细胞内活性氧的含量及核因子κB(NF-κB)入核率。

结果

CCK-8及EdU检测发现,各组细胞增殖能力的比较,差异均有统计学意义(F = 92.460,P< 0.001;F = 40.910,P<0.001);划痕试验和Transwell法检测发现,各组细胞迁移能力的比较,差异均有统计学意义(F = 27.050,P<0.001;F = 56.000,P<0.001);且对照组及干预组的增殖能力及迁移能力均显著高于脓毒症组(P均<0.05)。各组细胞间单核细胞对内皮细胞的黏附率(F = 19.240,P = 0.035),CyclinD1(F = 115.900,P<0.001)、IQGAP1(F = 116.500,P<0.001)、VE-cadherin (F = 108.500,P = 0.042)、claudin-5(F = 117.100,P = 0.028)、ICAM-1(F = 43.920,P<0.001)、VCAM-1(F = 41.450,P = 0.046)的蛋白表达水平,细胞内活性氧的含量(F = 30.960,P = 0.035)及NF-κB入核率(F = 33.410,P = 0.028)的比较,差异均有统计学意义。且进一步两两比较发现,与对照组及干预组比较,脓毒症组细胞的CyclinD1、IQGAP1、VE-cadherin及claudin-5蛋白表达水平均显著降低,而单核细胞对内皮细胞的黏附率,ICAM-1及VCAM-1蛋白表达水平,细胞内活性氧的含量及NF-κB入核率均显著升高(P均<0.05)。

结论

白藜芦醇通过抑制脂多糖诱导下活性氧聚集,减弱NF-κB激活及入核来保护脓毒症血管内皮屏障功能。

Objective

To investigate the protective effect and related mechanism of resveratrol on lipopolysaccharide-induced vascular endothelial barrier dysfunction in sepsis.

Methods

The human umbilical vein endothelial cells (HUVECs) were divided into control, sepsis and intervention groups. The cells in the sepsis group and intervention group were stimulated with 100 μg/mL lipopolysaccharide to establish a vascular endothelial cell injury model of sepsis, and the cells in the intervention group were pretreated with resveratrol before modeling. The cell reproductive activity was detected by the cell counting kit-8 (CCK-8) and 5- ethynyl-2'-deoxyuridine (EdU), the cell migration ability was detected by the scratch test and Transwell chamber, and the adhesion rate of HUVECs to THP-1 cells was calculated in each group. The protein expression levels of CyclinD1, IQ motif containing guanosine triphosphatase activating protein 1 (IQGAP1), VE-cadherin, claudin-5, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were examined by Western-blotting, and the content of reactive oxygen species and the transfer rate of nuclear factor-kappaB (NF-κB) into the nucleus were compared among the three groups.

Results

The CCK-8 and EdU showed that the cell reproductive activity among the three group was significantly different (F= 92.460, P< 0.001; F = 40.910, P<0.001). The scratch test and Transwell chamber revealed that the cell migration ability among the three group was significantly different (F = 27.050, P<0.001; F = 56.000, P<0.001). The cell reproductive activity and cell migration ability in the control group and intervention group were higher than those in the sepsis group (all P<0.05). The adhesion rate of HUVECs to THP-1 cells (F= 19.240, P= 0.035), the protein expression of CyclinD1 (F= 115.900, P<0.001), IQGAP1 (F = 116.500, P<0.001), VE-cadherin (F = 108.500, P = 0.042), claudin-5 (F = 117.100, P = 0.028), ICAM-1 (F = 43.920, P<0.001) and VCAM-1 (F = 41.450, P = 0.046), the content of reactive oxygen species (F = 30.960, P = 0.035) and the transfer rate of NF-κB into the nucleus (F = 33.410, P = 0.028) among the three group were significantly different. Further pairwise comparison revealed that in the sepsis group, the protein expression of CyclinD1, IQGAP1, VE-cadherin and claudin-5 decreased obviously, while the adhesion rate of HUVECs to THP-1 cells, the protein expression of ICAM-1 and VCAM-1, the content of reactive oxygen species and the transfer rate of NF-κB into the nucleus increased markedly as compared with control and intervention groups (all P<0.05).

Conclusion

Resveratrol can protect the vascular endothelial barrier function in sepsis by inhibiting the accumulation of lipopolysaccharide-induced reactive oxygen species and decreasing the activation and entry of NF-κB.

图1 不同浓度白藜芦醇处理24 h后HUVECs的细胞活性比较(n = 5 × 105个/mL)
图2 CCK-8检测3组HUVECs增殖能力的比较(n = 5 × 103个/mL)
图3 Edu检测3组HUVECs增殖能力的变化(n = 5 × 106个/mL)
图4 3组HUVECs中CyclinD1蛋白表达水平的比较(n = 5 × 106个/mL)
图5 划痕实验检测3组HUVECs的细胞迁移能力变化(n = 5 × 106个/mL)
图6 Transwell法检测3组HUVECs的细胞迁移能力变化(n= 5 × 105个/mL)
图7 3组HUVECs中IQGAP1蛋白表达水平的比较(n= 5 × 106个/mL)
图8 3组HUVECs的单核细胞对内皮细胞的黏附能力变化(n = 5 × 106个/mL)
图9 3组HUVECs中VE-cadherin、claudin-5、ICAM-1及VCAM-1蛋白表达水平比较(n = 5 × 106个/mL)
图10 3组HUVECs中活性氧水平及NF-κB入核比率的变化(n= 5 × 106个/mL)
1
Cecconi M, Evans L, Levy M, et al. Sepsis and septic shock[J]. Lancet, 2018, 392 (10141): 75-87.
2
van der Poll T, van de Veerdonk FL, Scicluna BP, et al. The immunopathology of sepsis and potential therapeutic targets[J]. Nat Rev Immuno, 2017, 17 (7): 407-420.
3
Hattori Y, Hattori K, Suzuki T, et al. Recent advances in the pathophysiology and molecular basis of sepsis-associated organ dysfunction: novel therapeutic implications and challenges[J]. Pharmacol Ther, 2017 (177): 56-66.
4
Merx MW, Weber C. Sepsis and the heart[J]. Circulation, 2007, 116 (7): 793-802.
5
Hasan GM, Al-Eyadhy AA, Temsah MA, et al. Feasibility and efficacy of sepsis management guidelines in a pediatric intensive care unit in Saudi Arabia: a quality improvement initiative[J]. Int J Qual Health Care, 2018, 30 (8): 587-593.
6
Keck T, Balcom JH 4th, Fernández-del Castillo C, et al. Matrix metalloproteinase-9 promotes neutrophil migration and alveolar capillary leakage in pancreatitis-associated lung injury in the rat[J]. Gastroenterology, 2002, 122 (1): 188-201.
7
Toledo AG, Golden G, Campos AR, et al. Proteomic atlas of organ vasculopathies triggered by staphylococcus aureus sepsis[J]. Nat Commun, 2019, 10 (1): 4656.
8
Heun Y, Pircher J, Czermak T, et al. Inactivation of the tyrosine phosphatase SHP-2 drives vascular dysfunction in sepsis[J]. EBioMedicine, 2019 (42): 120-132.
9
Feng L, Ren J, Li Y, et al. Resveratrol protects against isoproterenol induced myocardial infarction in rats through VEGF-B/AMPK/eNOS/NO signalling pathway[J]. Free Radic Res, 2019, 53 (1): 82-93.
10
Bollmann F, Art J, Henke J, et al. Resveratrol post-transcriptionally regulates pro-inflammatory gene expression via regulation of KSRP RNA binding activity[J]. Nucleic Acids Res, 2014, 42 (20): 12555-12569.
11
Liu B, Ghosh S, Yang X, et al. Resveratrol rescues SIRT1-dependent adult stem cell decline and alleviates progeroid features in laminopathy-based progeria[J]. Cell Metab, 2012, 16 (6): 738-750.
12
Coté CD, Rasmussen BA, Duca FA, et al. Resveratrol activates duodenal Sirt1 to reverse insulin resistance in rats through a neuronal network[J]. Nat Med, 2015, 21 (5): 498-505.
13
Alghetaa H, Mohammed A, Sultan M, et al. Resveratrol protects mice against SEB-induced acute lung injury and mortality by miR-193a modulation that targets TGF-β signalling[J]. J Cell Mol Med, 2018, 22 (5): 2644-2655.
14
Armstrong SM, Mubareka S, Lee WL. The lung microvascular endothelium as a therapeutic target in severe influenza[J]. Antiviral Res, 2013, 99 (2): 113-118.
15
Schnoor M, García Ponce A, Vadillo E, et al. Actin dynamics in the regulation of endothelial barrier functions and neutrophil recruitment during endotoxemia and sepsis[J]. Cell Mol Life Sci, 2017, 74 (11): 1985-1997.
16
Kohno T, Urao N, Ashino T, et al. IQGAP1 links PDGF receptor-β signal to focal adhesions involved in vascular smooth muscle cell migration: role in neointimal formation after vascular injury[J]. Am J Physiol Cell Physiol, 2013, 305 (6): C591-C600.
17
Yamaoka-Tojo M, Tojo T, Kim HW, et al. IQGAP1 mediates VE-cadherin-based cell-cell contacts and VEGF signaling at adherence junctions linked to angiogenesis[J]. Arterioscler Thromb Vasc Biol, 2006, 26 (9): 1991-1997.
18
Tian Y, Tian X, Gawlak G, et al. Role of IQGAP1 in endothelial barrier enhancement caused by OxPAPC[J]. Am J Physiol Lung Cell Mol Physiol, 2016, 311 (4): L800-L809.
19
Tanos BE, Perez Bay AE, Salvarezza S, et al. IQGAP1 controls tight junction formation through differential regulation of claudin recruitment[J]. J Cell Sci, 2015, 128 (5): 853-862.
20
Tanos BE, Yeaman C, Rodriguez-Boulan E. An emerging role for IQGAP1 in tight junction control[J]. Small GTPases, 2018, 9 (5): 375-383.
21
Sullivan DP, Dalal PJ, Jaulin F, et al. Endothelial IQGAP1 regulates leukocyte transmigration by directing the LBRC to the site of diapedesis[J]. J Exp Med, 2019, 216 (11): 2582-2601.
[1] 郝玥萦, 毛盈譞, 张羽, 汪佳旭, 韩林霖, 匡雯雯, 孟瑶, 杨秀华. 超声引导衰减参数成像评估肝脂肪变性及其对心血管疾病风险的预测价值[J/OL]. 中华医学超声杂志(电子版), 2024, 21(08): 770-777.
[2] 曹雯佳, 刘学兵, 罗安果, 钟释敏, 邓岚, 王玉琳, 李赵欢. 超声矢量血流成像对2型糖尿病患者颈动脉壁剪切应力的研究[J/OL]. 中华医学超声杂志(电子版), 2024, 21(07): 709-717.
[3] 杨成鹏. 以旋股外侧动脉前支为蒂的股前外侧皮瓣临床应用研究[J/OL]. 中华损伤与修复杂志(电子版), 2025, 20(01): 92-92.
[4] 王卫东. 贲门周围血管离断与左膈下静脉的保留[J/OL]. 中华普通外科学文献(电子版), 2024, 18(06): 464-464.
[5] 吴晖, 佴永军, 施雪松, 魏晓为. 两种解剖入路下行直肠癌侧方淋巴结清扫的效果比较[J/OL]. 中华普外科手术学杂志(电子版), 2025, 19(01): 40-43.
[6] 王学虎, 赵渝. 复杂腹壁疝手术中血管损伤并发症的预防和处理[J/OL]. 中华疝和腹壁外科杂志(电子版), 2024, 18(06): 616-619.
[7] 周正阳, 陈凯, 仇多良, 邵乐宁, 吴浩荣, 钟丰云. 腹腔镜腹股沟疝修补术后出血原因分析及处理[J/OL]. 中华疝和腹壁外科杂志(电子版), 2024, 18(06): 660-664.
[8] 朱佳琳, 方向, 贵诗雨, 黄丹, 周小雨, 郭文恺. 大鼠切口疝腹膜前间隙补片修补术后血清中VEGF 和Ang-1 的表达情况[J/OL]. 中华疝和腹壁外科杂志(电子版), 2024, 18(06): 703-707.
[9] 帖璇, 苏晓乐, 王利华. 抗中性粒细胞胞质抗体相关性血管炎治疗研究进展[J/OL]. 中华肾病研究电子杂志, 2024, 13(06): 345-351.
[10] 陈惠英, 邱敏珊, 邵汉权. 脓毒症诱发肠黏膜屏障功能损伤的风险因素模型构建与应用效果[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(05): 448-452.
[11] 石佳娜, 钱琳艳, 姬凯悦, 祁金文, 胡情, 孙佳斌. 从PVAT 白色脂肪棕色化角度探讨中药在防治动脉粥样硬化中的应用[J/OL]. 中华临床医师杂志(电子版), 2024, 18(09): 853-858.
[12] 李金星, 陈志宏, 孙铭均, 孙岩岩, 党树伟, 李广恩, 彭书奇, 付大伟, 李宏伟, 李国东. 基于多层螺旋CT 的腹腔干解剖变异与新分型研究[J/OL]. 中华临床医师杂志(电子版), 2024, 18(08): 756-763.
[13] 张家珺, 张海涛, 苏菲菲. 径向壁应变分析在冠心病诊疗中的应用[J/OL]. 中华临床医师杂志(电子版), 2024, 18(07): 687-691.
[14] 张克, 杨占奇, 闫维, 张二明, 向平超. 持续气道正压通气对阻塞性睡眠呼吸暂停综合征患者发生心脑血管事件的影响[J/OL]. 中华临床医师杂志(电子版), 2024, 18(05): 433-440.
[15] 王璇, 娜扎开提·尼加提, 雒洋洋, 蒋升. 皮肤晚期糖基化终末产物浓度与2型糖尿病微血管并发症的相关性[J/OL]. 中华临床医师杂志(电子版), 2024, 18(05): 447-454.
阅读次数
全文


摘要