Yinxing Mihuan Oral Solution Protect HUVECs Against Hypoxia Reoxygenation Injury Through p38 MAPK Regulating eNOS/NO Signaling Pathway

HAN Xiao; XU Li; LIU Jian-xun; WANG Yi-min; WANG Yong

doi:10.13422/j.cnki.syfjx.2017230079

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Yinxing Mihuan Oral Solution Protect HUVECs Against Hypoxia Reoxygenation Injury Through p38 MAPK Regulating eNOS/NO Signaling Pathway

更新时间：2024-01-04

- Yinxing Mihuan Oral Solution Protect HUVECs Against Hypoxia Reoxygenation Injury Through p38 MAPK Regulating eNOS/NO Signaling Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 23, Issue 23, Pages: 79-84(2017)
- 作者机构：
- 作者简介：
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- DOI：10.13422/j.cnki.syfjx.2017230079
  CLC： R285
- Published：2017
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HAN Xiao, XU Li, LIU Jian-xun, et al. Yinxing Mihuan Oral Solution Protect HUVECs Against Hypoxia Reoxygenation Injury Through p38 MAPK Regulating eNOS/NO Signaling Pathway[J]. Chinese journal of experimental traditional medical formulae, 2017, 23(23): 79-84.
DOI：

HAN Xiao, XU Li, LIU Jian-xun, et al. Yinxing Mihuan Oral Solution Protect HUVECs Against Hypoxia Reoxygenation Injury Through p38 MAPK Regulating eNOS/NO Signaling Pathway[J]. Chinese journal of experimental traditional medical formulae, 2017, 23(23): 79-84. DOI： 10.13422/j.cnki.syfjx.2017230079.

摘要

目的：评估银杏蜜环口服溶液对缺氧复氧损伤人脐静脉内皮细胞（HUVECs）的保护作用；从p38丝裂原活化蛋白激酶（p38 MAPK）及内皮型一氧化氮合酶（eNOS）/一氧化氮（NO）信号途径探讨银杏蜜环口服溶液保护受损HUVECs的药效机制。方法：缺氧缺糖/复氧复糖法建立体外HUVECs缺血再灌注损伤模型。分为正常组、模型组、银杏蜜环口服溶液高质量浓度组（75 mg·L-1，简称银蜜高），低质量浓度组（36 mg·L-1，简称银蜜低），p38 MAPK抑制剂SB203580组，银蜜高+SB203580组，eNOS抑制剂亚硝基左旋精氨酸甲酯（L-NAME）组，银蜜高+L-NAME组。细胞增殖-毒性检测试剂盒（CCK-8）及微量酶标法检测细胞活力及细胞损伤；蛋白免疫印迹法（Western blot）检测磷酸化p38 MAPK（p-p38 MAPK）/p38 MAPK和磷酸化eNOS（p-eNOS）/eNOS蛋白表达；酶联免疫吸附法（ELISA）检测肿瘤坏死因子-α（TNF-α），可溶性细胞间黏附分子-1（sICAM-1），半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）含量；硝酸盐还原酶法检测NO含量。结果：银蜜高、银蜜低组可显著提高缺氧复氧损伤HUVECs活力、降低细胞乳酸脱氢酶（LDH）漏出量（P<0.05，P<0.01）；抑制受损细胞p38的磷酸化（P<0.05），降低细胞培养上清中TNF-α，sICAM-1含量（P<0.05，P<0.01）。在给予银蜜高或（和）SB203580后，受抑制的eNOS磷酸化表达水平显著升高（P<0.05）；银蜜高可显著提高受损细胞NO含量，在加入eNOS抑制剂L-NAME后，银蜜高可拮抗L-NAME降低细胞NO生成和升高Caspase-3的作用（P<0.05）。结论：银杏蜜环口服溶液可提高氧复氧损伤HUVEs活力，具有抗炎、调节内皮系统、抑制细胞凋亡的作用，机制与其作用于p38 MAPK进而调节eNOS-NO信号途径相关。

Abstract

Objective: To study the effect and mechanism of Yinxing Mihuan oral solution on human umbilical vein endothelial cells(HUVECs)injured by hypoxia/reoxygenation (H/R) through p38 mitogen activated protein kinase (p38 MAPK) regulating endothelial nitric oxide synthase(eNOS)/nitric oxide (NO) signaling pathway. Method: Hypoxia ischemia/reperfusion injury model was established by hypoxia/hypoglycemia/reoxygenation on HUVECs. Cultured HUVECs were divided into control group

H/R group

Yinxing Mihuan oral solution high(75 mg·L-1)

low dose group(36 mg·L-1)

p38 MAPK inhibitor SB203580

Yinxing Mihuan oral solution high dose+SB203580

eNOS inhibitor L-nitro-arginine methylester(L-NAME)

Yinxing Mihuan oral solution high dose +L-NAME. Cell viability and cell injury were measured by cell counting kit-8 (CCK-8) and enzyme labeling method. Protein expression of phosphorylating-p38 MAPK (p-p38 MAPK)/p38 MAPK and phosphorylating-eNOS (p-eNOS)/eNOS were detected by Western blot. The levels of tumor necrosis factor-α (TNF-α)

soluble intercellular adhesion molecule-1(sICAM-1)

cysteine aspartate protease-3(Caspase-3)were determined by enzyme-linked immunosorbent assay (ELISA). NO content was measured by nitrate reductase method. Result: Yinxing Mihuan oral solution high and low dose group could significantly improve cell viability and reduce lactate dehydrogenase (LDH) content(P<0.05

P<0.01)

inhibit p-p38 MAPK expression on injured HUVECs

decrease TNF-α

sICAM-1 levels in cell culture supernatant(P<0.05

P<0.01). The inhibited p-eNOS expression was significantly increased after administration of Yinxing Mihuan oral solution high dose and (or) SB203580(P<0.05). Yinxing Mihuan oral solution high dose significantly increased the NO content of damaged HUVECs. After the addition of the eNOS inhibitor L-NAME

Yinxing Mihuan oral solution high dose could antagonize the effects of L-NAME on decreasing NO production and Caspase-3 elevation in injured HUVECs. Conclusion: Yinxing Mihuan oral solution can prevent HUVECs against H/R injury by resisting inflammatory

regulating the endothelial system and inhibiting apoptotic through p38 MAPK regulating eNOS/NO pathway.

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