Fugan formula inhibits angiogenesis of hepatic sinusoidal endothelial cells by regulating miR-424 expression in vitro

WANG Qinglan; ZHANG Feng; LEI Yang; LIU Chenghai; TAO Yanyan

doi:10.16305/j.1007-1334.2020.01.017

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Fugan formula inhibits angiogenesis of hepatic sinusoidal endothelial cells by regulating miR-424 expression in vitro

更新时间：2022-08-31

- Fugan formula inhibits angiogenesis of hepatic sinusoidal endothelial cells by regulating miR-424 expression in vitro
- Shanghai Journal of Traditional Chinese Medicine Vol. 54, Issue 1, Pages: 68-73(2020)
- 作者机构：
  
  1. 上海中医药大学附属曙光医院肝病研究所,上海,201203
  2. 上海中医药大学基础医学院伤寒金匮教研室,上海,201203
  3. 上海市中医临床重点实验室,上海,201203
  4. 肝肾疾病病证教育部重点实验室,上海中医药大学),上海,201203
- 作者简介：
- 基金信息：
- DOI：10.16305/j.1007-1334.2020.01.017
  CLC：
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WANG Qinglan, ZHANG Feng, LEI Yang, et al. Fugan formula inhibits angiogenesis of hepatic sinusoidal endothelial cells by regulating miR-424 expression in vitro. [J]. Shanghai Journal of Traditional Chinese Medicine 54(1):68-73(2020)
DOI：

WANG Qinglan, ZHANG Feng, LEI Yang, et al. Fugan formula inhibits angiogenesis of hepatic sinusoidal endothelial cells by regulating miR-424 expression in vitro. [J]. Shanghai Journal of Traditional Chinese Medicine 54(1):68-73(2020) DOI： 10.16305/j.1007-1334.2020.01.017.

摘要

目的：观察复肝丸调控miR-424表达以抑制肝窦内皮细胞血管新生的作用及其机制。方法：以不同剂量(0～2 000 mg/L)的复肝丸与人肝窦内皮细胞(HHSEC)共孵育,CCK8法检测复肝丸对细胞活力的影响,分析复肝丸对细胞无毒性作用的剂量范围,并以此作为后续药效及机制评价的剂量。以CoCl,2,诱导HHSEC血管新生模型,分为正常组、模型组、miR-424 抑制剂组及复肝丸组,除正常组外,其余各组以CoCl,2,诱导细胞血管新生,miR-424 抑制剂组及复肝丸组分别以miR-424 抑制剂及复肝丸(200 mg/L)与细胞共孵育24 h。采用CCK8法检测细胞活力与增殖情况；Transwell观察细胞迁移变化情况；Matrigel管腔生成实验评价细胞管腔形成情况；定量RT-PCR检测miR-424、HIF-1α、vWF、CD31基因表达；Western blot法检测HIF-1α、vWF、CD31蛋白表达。结果：复肝丸剂量小于400 mg/L对HHSCE无明显细胞毒性。与正常组比较,200 μmol/L CoCl,2,可诱导肝窦内皮细胞缺氧损伤模型,且模型组miR-424、HIF-1α表达增加,细胞迁移和管腔生成增多,vWF、CD31表达增加(P,<,0.01)；与模型组比较,miR-424抑制剂与200 mg/L复肝丸干预后,miR-424与HIF-1α表达均不同程度降低,细胞迁移和管腔生成受抑制,vWF、CD31表达降低(P,<,0.01)；且复肝丸组综合疗效与miR-424抑制剂相当。结论：复肝丸具有良好的抑制肝窦内皮细胞血管新生的作用,其作用机制与抑制miR-424表达相关。

Abstract

Objective:To observe the effect and mechanism of Fugan formula on regulating miR-424 expression to inhibit the angiogenesis of human hepatic sinusoidal endothelial cells (HHSEC). MethodsFirstly, Fugan formula extract was incubated with HHSEC in different doses (0～2 000 mg/L). The effect of Fugan formula on the cell activity was detected by CCK8 method, the range of the non-toxic effect of Fugan formula on the cell was analyzed and the dosage was used for the follow-up efficacy and mechanism evaluation. Then HHSEC were divided into the normal group,the CoCl,2, model group, the miR-424 inhibitor group and the Fugan formula group (200 mg/L). After 24 h incubation, cell viability and proliferation were detected by CCK8. Cell migration was observed by Transwell and tube formation was evaluated by Matrigel. The expression of miR-424, HIF-1α, vWF and CD31 were detected by RT-PCR and Western Blot. Results:Fugan formula of 0~400 mg/L had no obvious cytotoxicity. Compared with the normal group, 200 μmol/L CoCl,2, can induce hypoxia injury model of HHSEC, and in the model group the expression of miR-424 and HIF-1 α increased, cell migration and lumen formation increased, and the expression of vWF and CD31 increased (P,<,0.01); Compared with the model group, after the intervention of miR-424 inhibitor and 200 mg/L Fugan formula, the expression of miR-424 and HIF-1 α decreased, the cell migration and tube formation were inhibited and the expression of vWF and CD31 decreased (P,<,0.01); and the comprehensive effect of Fugan formula was similar to that of miR-424 inhibitor. Conclusion:Fugan formula could inhibit angiogenesis of HHSEC and its mechanism is related to the inhibition of miR-424 expression.

关键词

miRNA-424血管新生肝窦内皮细胞复肝丸

Keywords

miR-424angiogenesishepatic sinusoidal endothelial cells (HSEC)Fugan formula

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