摘要
目的:探讨川乌提取物对人脐静脉内皮细胞(HUVECs)增殖和凋亡的影响及其机制。方法:分别用2.5 g/L(AR 2.5 g/L组)、5.0 g/L(AR 5.0 g/L组)和7.5 g/L(AR 7.5 g/L组)川乌提取物处理HUVECs(购自中国科学院上海细胞库),对照组(Con)未行川乌提取物处理。将微小RNA(miRNA,miR)-NC(miR-NC组)、miR-589(miR-589组)、anti-miR-NC(anti-miR-NC组)和anti-miR-589(anti-miR-589组)分别转染至HUVECs中;将anti-miR-NC、anti-miR-589、pcDNA3.1、pcDNA3.1-蛋白激酶B1(Akt1)转染至HUVECs细胞后用5.0 g/L的川乌提取物处理,分别标记为AR 5.0 g/L+anti-miR-NC组、AR 5.0 g/L+anti-miR-589组、AR 5.0 g/L+pcDNA3.1组和AR 5.0 g/L+pcDNA3.1-Akt1组。噻唑蓝(MTT)法检测各组细胞增殖;流式细胞术检测细胞凋亡;实时定量反转录聚合酶链反应(RT-qPCR)检测miR-589的表达水平;蛋白质印迹法(Western blot)检测蛋白表达;双荧光素酶报告基因检测实验检测miR-589和Akt1的靶向关系。两组间比较采用 t检验。多组间比较采用SNK- q检验。 结果:不同浓度川乌提取物组HUVECs细胞增殖活性、细胞周期蛋白(Cyclin) D1、B细胞淋巴瘤/白血病-2(bcl-2)和miR-589表达均显著低于Con组( F=107.822、86.321、136.234, P<0.05),凋亡率、p21和bcl-2相关X蛋白(bax)蛋白表达显著高于Con组( F=95.875、124.365、107.541, P<0.05)。miR-589组HUVECs增殖活性、Cyclin D1和bcl-2蛋白表达显著高于miR-NC组( t=15.358、31.109、8.971, P<0.05),凋亡率、p21和bax蛋白表达显著低于miR-NC组( t=23.124、21.175、11.364, P<0.05),抑制miR-589表达可逆转川乌提取物对HUVECs增殖和凋亡的作用。miR-589可靶向调控Akt1的表达。 结论:川乌提取物可抑制HUVECs增殖并促进细胞凋亡,可能与调控miR-589/Akt1有关。
Objective To study the effect of the extract of Aconiti Radix(AR)on proliferation and apoptosis of human umbilical vein endothelial cells(HUVECs)and its mechanism.Methods HUVECs were treated with AR at different concentrations of 2.5,5.0 and 7.5 g/L,and HUVECs without AR treatment served as control(Con)group.The miR-NC,miR-589,anti-miR-NC,and anti-miR-589 were transfected into HUVECs cells,respectively.The anti-miR-NC,anti-miR-589,pcDNA3.1 and pcDNA3.1-protein kinase B1(Akt1)were transfected into HUVECs and treated with 5 g/L of AR as AR-5.0 g/L+anti-miR-NC group,AR-5.0 g/L+anti-miR-589 group,AR-5.0 g/L+pcDNA3.1 group and AR 5.0 g/L+pcDNA3.1-Akt1 group respectively.Methyl thiazolyl tetrazolium(MTT)assay was used to detect cell proliferation.Flow cytometry was used to detect apoptosis.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect miR-589 expression.Western blotting was used to detect protein expression,and dual luciferase reporter assay was used to detect the targeting relationship between miR-589 and Akt1.Results The proliferation activity,Cyclin D1,B cell lymphoma/leukemia-2(bcl-2)and miR-589 expression in 2.5 g/L,5.0 g/L and 7.5 g/L groups were significantly reduced as compared with those in Con group(F=107.822,86.321,136.234,P<0.05),while apoptosis rate,p21 and bcl-2 associated X protein(bax)protein were significantly up-regulated(F=95.875,124.365,107.541,P<0.05).The proliferation activity,Cyclin D1 and bcl-2 expressions in miR-589 group were significantly increased as compared with those in miR-NC group(t=15.358,31.109,8.971,P<0.05),and the apoptosis rate,p21 and bax protein expression were significantly reduced as compared with those in miR-NC group(t=23.124,21.175,11.364,P<0.05).Inhibition of miR-589 expression and over-expression of Aktl reversed the proliferation inhibition and apoptosis-promoting effects of AR extract on HUVECs.Conclusion The extract of AR can inhibit the proliferation of HUVECs and promote apoptosis,which may be related to regulation of miR-589/Akt1.
作者
程鑫
胥雄飞
孙晓磊
刘勇
Cheng Xin;Xu Xiongfei;Sun Xiaolei;Liu Yong(Department of Vascular Surgery,the Affiliated Hospital of Southwest Medical University,Luzhou 646000,China;Department of General Surgery,the Affiliated Hospital of Ya’an Polytechnic College,Ya’an 625000,China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2020年第5期803-806,共4页
Chinese Journal of Experimental Surgery
关键词
川乌
人脐静脉内皮细胞
增殖
脱噬作用
机制
Aconiti radix
Human umbilical vein endothelial cells
Proliferation
Apoptosis
Mechanism
