Exploration of the protective mechanism of Siwu Humai Decoction on homocysteine-induced injury in HUVEC

XU Meng-jiao; YUAN Wen-wen; WANG Yu-qing; XU Jie; JIN Xiao; YUAN Fang; HAN Ran; YANG Bao-zhong; ZHANG Fan-fan

doi:10.16025/j.1674-1307.2022.11.009

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Exploration of the protective mechanism of Siwu Humai Decoction on homocysteine-induced injury in HUVEC

更新时间：2023-02-15

- Exploration of the protective mechanism of Siwu Humai Decoction on homocysteine-induced injury in HUVEC
- Beijing Journal of Traditional Chinese Medicine Vol. 41, Issue 11, Pages: 1245-1251(2022)
- 作者机构：
  
  1.北京中医药大学东方医院外四科，北京 100078
  2.北京中医药大学东方医院检验科，北京 100078
- 作者简介：
- 基金信息：
- DOI：10.16025/j.1674-1307.2022.11.009
  CLC：
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徐孟娇，袁文雯，王雨晴，等.四五护脉汤对同型半胱氨酸损伤后人脐静脉内皮细胞的保护机制探讨［J］.北京中医药，2022，41（11）：1245-1251.

XU Meng-jiao,YUAN Wen-wen,WANG Yu-qing,et al.Exploration of the protective mechanism of Siwu Humai Decoction on homocysteine-induced injury in HUVEC[J]. Beijing Journal of Traditional Chinese Medicine,2022,41(11):1245-1251.
徐孟娇，袁文雯，王雨晴，等.四五护脉汤对同型半胱氨酸损伤后人脐静脉内皮细胞的保护机制探讨［J］.北京中医药，2022，41（11）：1245-1251. DOI： 10.16025/j.1674-1307.2022.11.009.

XU Meng-jiao,YUAN Wen-wen,WANG Yu-qing,et al.Exploration of the protective mechanism of Siwu Humai Decoction on homocysteine-induced injury in HUVEC[J]. Beijing Journal of Traditional Chinese Medicine,2022,41(11):1245-1251. DOI： 10.16025/j.1674-1307.2022.11.009.

摘要

目的,2,观察四五护脉汤对同型半胱氨酸（homocysteinemia，HCY）诱导人脐静脉内皮细胞（human umbilical vein endothelial cell，HUVEC）损伤的影响及机制。,方法,2,将HUVEC常规培养传代后，MTT法检测细胞活力并确定药物的最佳实验浓度。将细胞分组，A组为正常HUVEC，B~F组加入125 μmol/L的HCY 100 μL制备细胞损伤模型，培养48 h后，C组加入31.25 μmol/L NaHS 100 μL，D、E、F组分别加入1.6、2.2、2.8 g/L四五护脉汤100 μL，继续培养48 h。MTT法检测细胞活力，流式细胞术检测细胞凋亡，Transwell法检测细胞迁移，硝酸还原酶法检测细胞培养液NO水平，Western blot法检测细胞磷脂酰肌醇3-激酶-蛋白激酶B（PI3K）、磷酸化AKT蛋白（p-AKT）抗体、小窝蛋白-1（Caveolin-1，CAV-1）、内皮型一氧化氮合酶（endothelial nitric oxide synthase，eNOS）蛋白表达，RT-PCR法检测细胞内eNOS mRNA表达，BCA测定细胞黏附率。,结果,2,HCY、NaHS最佳实验浓度分别为125、31.25 μmol/L，四五护脉汤干预浓度为1.6、2.2、2.8 g/L。HCY干预后，HUVEC活力下降，凋亡率上升，迁移率下降，NO水平降低，eNOS、PI3K、p-AKT蛋白相对表达量降低，CAV-1蛋白相对表达量升高，eNOS mRNA相对表达量降低，单核细胞黏附率升高（,P,<,0.05）；而四五护脉汤可逆转上述变化（,P,<,0.05），且呈浓度依赖性。,结论,2,四五护脉汤能够通过调节PI3K-AKT-eNOS信号通路，提高NO表达，减少内皮细胞凋亡，提高内皮细胞活力、迁移，抑制单核细胞黏附，从而减少内皮细胞损伤，达到保护内皮细胞的目的。

Abstract

Objective,2,To observe the effects and mechanism of Siwu Humai Decoction on the injury of human umbilical vein endothelial cells （HUVEC） induced by homocysteine （HCY）.,Methods,2,After conventional culture and passage of HUVEC，MTT assay was used to detect the cell viability and determine the best experimental concentration of the drug. The cells were divided into groups： group A was normal HUVEC， group B to F was added with 125 μmol/L HCY 100 μL to make cell injury model， after 48 hours of culture， group C was added with 31.25 μmol/L NaHS 100 μL， and groups D， E and F were added with 1.6， 2.2 and 2.8 g/L Siwu Humai Decoction 100 μL respectively， and continued to culture.MTT method was used to detect cell viability of each group， flow cytometry to detect apoptosis， transwell method to detect cell migration rate in each group；nitrate reductase method to detect NO level in cell culture medium. Western blot to detect PI3K， p-AKT， CAV-1， eNOS protein relative expression in each group， and BCA measurement of endothelial cell-monocyte adhesion rate.,Results,2,The optimal concentrations of HCY and NaHS were 125 μmol/L and 31.25 μmol/L， respectively. The intervention concentration of Siwu Humai Decoction was selected as 1.6， 2.2， and 2.8 g/L. After HCY intervention， the cell viability values were decreased， apoptosis rate increased， migration rate decreased， NO level decreased，eNOS，PI3K，p-AKT protein relative expression decreased， CAV-1 protein relative expression increased， eNOS mRNA relative ct value decreased and adhesion rate with monocytes increased， while Siwu Humai Decoction could reverse the above expressions（,P,<,0.05），and is concentration-dependent.,Conclusion,2,Siwu Humai Decoction can protect HUVEC after HCY injury by regulating the PI3K-AKT-eNOS signaling pathway， increasing NO expression， reducing endothelial cell apoptosis， and improving endothelial cell viability and migration， inhibiting monocyte adhesion， thus reducing endothelial cell damage and achieving the purpose of protecting endothelial cells.

关键词

深静脉血栓四五护脉汤同型半胱氨酸内皮细胞PI3K-AKT-eNOS信号通路

Keywords

Deep vein thrombosisSiwu Humai Decoctionhomocysteineendothelial cell functionPI3K-AKT-eNOS signaling pathway

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