益气除痰方对缺氧人脐静脉内皮细胞迁移和侵袭能力的影响

Effects of YQCTF on Migration and Invasion of Hypoxia-induced HUVECs

目的探讨益气除痰方(YQCTF)对缺氧人脐静脉内皮细胞(HUVEC)迁移和侵袭能力的影响及其机制。方法采用氯化钴(Co Cl2)化学诱导建立细胞缺氧模型,加入不同浓度YQCTF干预,设HUVEC常氧(Norm)组,缺氧造模(Hyp)组,缺氧+YQCTF低、中、高剂量(L、M、H)组。采用噻唑蓝(MTT)比色法检测HUVE活力;流式细胞术检测细胞周期;划痕实验测定细胞修复迁移能力;Transwell小室实验测定细胞侵袭能力;Western Blot法检测缺氧诱导因子1α(HIF-1α)、葡萄糖调节蛋白78(GRP78)、细胞外调节蛋白激酶(ERK)、粘着斑激酶(FAK)、p-ERK、p-FAK的蛋白水平;qPCR法检测HIF-1α、GRP78、ERK1、ERK2、FAK的mRNA表达水平。结果 Norm组与Hyp组比较,Hyp组HUVEC无明显细胞增殖(P>0.05),但细胞迁移和侵袭能力增强(P<0.05),上调HIF-1α、GRP78、ERK、FAK蛋白表达(P<0.05,P<0.01)及基因mRNA表达(P<0.01),ERK、FAK磷酸化水平增强(P<0.01);与Hyp组比较,YQCTF可明显抑制缺氧HUVEC增殖(P<0.01,P<0.05),抑制细胞周期有丝分裂(P<0.05,P<0.01),降低细胞迁移和侵袭能力(P<0.01),并可下调HIF-1α、GRP78、ERK、FAK蛋白和基因mRNA表达水平(P<0.05,P<0.01),降低ERK、FAK磷酸化水平(P<0.05,P<0.01),并呈剂量依赖性。结论 YQCTF可抑制缺氧诱导的HUVEC迁移和侵袭,可能与其调控HIF-1α、GRP78、ERK、FAK的蛋白和基因mRNA表达水平有关。

Objective To investigate the effect and mechanism of Yiqi Chutan formula （YQCTF） on migration and invasion of hypoxia-induced human umbilical vein endothelial cells （HUVECs）. Methods Cells were grouped into normoxic （Norm）, hypoxic （Hyp）, hypoxie ＋ YQCTF low concentration （L）, hypoxic ＋ YQCTF medium concentration （M）, and hypoxic ＋ YQCTF high concentration （H）. Cell proliferation was detected by MTT assay and flow cytometry was used to analyze cell cycle. In vitro cell migration and cell invasion abilities were evaluated by cell scratch and transwell chamber assay. Protein levels of HIF-1α , GRP78, ERK, FAK, p-ERK, and p-FAK were detected by Western Blot. The mRNA levels of HIF-1α, GRP78, ERK1, ERK2, and FAK were quantified by qPCR. Results Compared with normoxic group, cell proliferation showed no significant change in hypoxic group （P 〉 0.05）, while cell migration and cell invasion abilities were enhanced （P 〈 0.05） ; and protein expression of HIF-1α , GRP78, ERK, FAK, p-ERK, p-FAK were up-regulated （P 〈 0.05, P 〈 0.01）. Besides, the mRNA levels of HIF-1α, GRP78, ERK1, ERK2, FAK were also increased （P 〈 0.01 ）. On the other hand, when compared with hypoxic control group,the YQCTF could dose-dependently inhibit proliferation and mitosis of HUVECs （P 〈 0.01, P 〈 0.05） , significantly attenuated cell migration and cell invasion abilities （P 〈 0.01 ）. Moreover, YQCTF remarkably down-regulated the protein expression of HIF-1α, GRP78, ERK, FAK, p-ERK, p-FAK, and the mRNA levels of HIF-1α, GRP78, ERK1, ERK2, FAK （P〈 0.01）, also decreased phosphorylation of ERK and FAK （P〈0.01）. Conclusion YQCTF can inhibit migration and invasion abilities of hypoxia-indueed HUVECs, which may be related to regulating mRNA levels and protein expression of HIF-1α, GRP78, ERK and FAK.