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(0~2000 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CoCl2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CoCl2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,200μmol/L CoCl2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。
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).Methods Firstly,Fugan formula extract was incubated with HHSEC in different doses(0~2000 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 CoCl2 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,vW F 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,200μmol/L CoCl2 can induce hypoxia injury model of HHSEC,and in the model group the expression of miR-424 and HIF-1αincreased,cell migration and lumen formation increased,and the expression of vW F 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-1αdecreased,the cell migration and tube formation were inhibited and the expression of vW F 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.
作者
王清兰
张风
雷扬
刘成海
陶艳艳
WANG Qinglan;ZHANG Feng;LEI Yang;LIU Chenghai;TAO Yanyan(Institute of Liver Diseases,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Teaching and research Section of TCM Classics-Shanghan and Jingui,School of Basic Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Shanghai Key Laboratory of Clinical Chinese Medicine,Shanghai 201203,China;Key Laboratory of Liver and Kidney Diseases,Ministry of Education,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)
出处
《上海中医药杂志》
2020年第1期68-73,96,共7页
Shanghai Journal of Traditional Chinese Medicine
基金
国家自然科学基金资助项目(81270053,81573810)
上海市自然科学基金项目(16ZR1437800)
上海市自然科学基金项目(16ZR1437800).
