摘要
目的探究当归对血管紧张素Ⅱ(angiotensin-2,Ang Ⅱ)诱导损伤的人脐静脉内皮细胞(HUVECs)微小RNA-122(miR-122)与精氨酸转运通路的作用机制。方法将HUVECs随机分为6组:空白组、模型组、对照组和低、中、高3个浓度实验组。除空白组外,其余各组给予终浓度为1×10^-5mol·L^-1的AngⅡ处理;同时,对照组给予1×10^-4mol·L^-1缬沙坦,3个浓度实验组给予5%,10%和20%当归含药血清,培养24 h。以CCK8法检测细胞活性(光密度值),以硝酸还原法检测培养基上清液一氧化氮(NO)水平,以实时荧光定量-PCR法检测细胞miR-122和氨基酸转运体(SLC7A1)基因水平;以miR-122抑制剂和miR-122类似物转染HUVECs,检测转染细胞SLC7A1基因和阳离子氨基酸转运蛋白1(CAT-1)水平;以蛋白质印迹法检测CAT-1蛋白水平。结果miR-122对照组、miR-122类似物组和miR-122抑制剂组细胞的SLC7A1基因表达量分别为1.06±0.12,0.63±0.05和1.46±0.20;这3组的CAT-1蛋白表达量分别为0.84±0.06,0.68±0.04和1.24±0.02,上述指标:miR-122类似物和miR-122抑制剂组与miR-122对照组对比,差异均有统计学意义(均P<0.01)。空白组、模型组、对照组和低、中、高3个浓度实验组的细胞活性分别为0.89±0.02,0.56±0.04,0.69±0.03,0.67±0.03,0.66±0.02和0.70±0.03;这6组的细胞合成NO含量分别为10.66±0.20,8.48±0.31,11.24±0.13,12.67±0.23,15.26±0.31和16.63±0.18;这6组的细胞miR-122表达量分别为1.00±0.09,1.46±0.07,0.38±0.05,0.56±0.09,0.55±0.15和0.45±0.04;这6组的SLC7A1基因表达量分别为1.01±0.19,0.68±0.18,2.32±0.24,0.71±0.12,0.79±0.10和1.07±0.05;这6组的CAT-1蛋白表达量分别为0.88±0.02,0.76±0.03,0.97±0.04,0.97±0.03,0.98±0.04和1.09±0.02。上述指标:对照组和低、中、高3个浓度实验组与模型组对比,miR-122、CAT-1、NO和细胞活性水平的差异均有统计学意义(均P<0.01);对照组和高剂量实验组与模型组对比,SLC7A1基因水平的差异均有统计学意义(均P<0.01)。结论当归可以通过miR-122负向调控其靶基因和蛋白表达水平,通过调控精氨酸转运通路促进NO的合成。
Objective To investigate the effect and mechanism of Angelica sinensis on microRNA-122(miR-122)and mechanism of arginine transport pathway in human umbilical vein endothelial cells(HUVECs)injured by angiotensin-2(Ang Ⅱ).Methods HUVECs were randomly divided into 6 groups:blank group,model group,control group and experimental-L,experimental-M,experimental-H groups.In addition to the blank group,the other groups were given Ang Ⅱ with the final concentration of 1×10^-5mol·L^-1,and then treated with 1×10^-4mol·L^-1 valsartan in control group and different concentrations(5%,10% and 20%)of Angelica containing serum(ACS)for 24 h.The cells activity(OD value)in each group was detected by cell counting kit-8(CCK8).The level of nitric oxide(NO)in supernatant of culture medium was detected by nitrate reduction method.The expression level of miR-122 and solute carrier family 7 member 1(SLC7A1)mRNA in each group were detected by real time fluorescence quantitative-PCR.HUVECs were transfected with miR-122 inhibitor and miR-122 mimics and then the level of SLC7A1 mRNA was detected.The expression level of Cationic amino acid transporter 1(CAT-1)protein was detected by Western blot.Results The expression of SLC7A1 mRNA in the miR-122 control,mimics and inhibitor groups were 1.06±0.12,0.63±0.05,1.46±0.20,respectively;the expression of CAT-1 protein in the three groups were 0.84±0.06,0.68±0.04,1.24±0.02,respectively;comparison between miR-122 inhibitor,miR-122 mimics groups and miR-122 NC group,the difference of the above factors were significant(all P<0.01).The expression of cell activity in blank group,model group,control group and experimental-L,experimental-M and experimental-H groups were 0.89±0.02,0.56±0.04,0.69±0.03,0.67±0.03,0.66±0.02,0.70±0.03,respectively;the expression of NO in the six groups were 10.66±0.20,8.48±0.31,11.24±0.13,12.67±0.23,15.26±0.31,16.63±0.18,respectively;the expression of miR-122 in the six groups were 1.00±0.09,1.46±0.07,0.38±0.05,0.56±0.09,0.55±0.15,0.45±0.04,respectively;the expression of SLC7A1 mRNA in the six groups were 1.01±0.19,0.68±0.18,2.32±0.24,0.71±0.12,0.79±0.10,1.07±0.05,respectively;the expression of CAT-1 protein in the six groups were 0.88±0.02,0.76±0.03,0.97±0.04,0.97±0.03,0.98±0.04,1.09±0.02,respectively;compared between control,three concentration experimental groups and model group,the expression of miR-122,CAT-1,NO and cell activity were significant(all P<0.01);compared between control group,experimental-H group and model group,the difference of the expression of SLC7A1 mRNA was significant(all P<0.01).Conclusion Angelica sinensis can negatively regulate the mRNA and protein expression level of miR-122 target gene through miR-122,and promote the synthesis of NO by regulating the arginine transport pathway.
作者
杨锐
谢青
江华
毛玉娟
何亚丽
伊琳
YANG Rui;XIE Qing;JIANG Hua;MAO Yu-juan;HE Ya-li;YI Lin(School of Traditional Chinese and Western Medicine,Gansu University of Chinese Medicine,Lanzhou 730000,Gansu Province,China)
出处
《中国临床药理学杂志》
CAS
CSCD
北大核心
2020年第20期3228-3232,共5页
The Chinese Journal of Clinical Pharmacology
基金
国家自然科学基金资助项目(81760803)
甘肃中医药大学研究生创新基金资助项目(2019CX19)。
