[The study on inhabiting endothelial cell aging by targeted silencing of p22phox].
The aim of the study was to determine the importance and possible mechanism of NAD (P)H oxidase subunits P (superscript 22phox) involved in human umbilical endothelial cell lines ECV-304 aging by special short interference RNA (siRNA). Three siRNAs targeting p22phox were designed and synthesized in vitro, which were used to transfect ECV-304 cultured in vitro for selecting the most powerful and most suitable transfection concentration and time. The cell line ECV-304 was divided into three groups: control group, angiotensin II (Ang II) group, siRNA group, and Ang II + siRNA group. Cell aging was identified by beta-gal stain. Reactive oxygen species (ROS) and NO level in cells and medium were measured. RT-PCR and Western blot were used to analyze mRNA and protein expression of NAD(P)H oxidase subunit p22phox. Among the 3 siRNAs, siRNA-1 was the most powerful on gene silence with 50 nmol/L transfection concentration at 24 h and 36 h. The number of positive cells stained by beta-gal were increased in ECV-304 stimulated with Ang II, and p22phox mRNA and protein expression were increased in aging ECV-304 stimulated with Ang II, which had lower NO and higher ROS. Compared with Ang II group, ROS level was decreased and NO level was increased in Ang+siRNA group with decreased aging level. The result of the present study suggested that siRNA could induce NAD(P)H oxidase subunit p22phox gene silence, Ang II could induce ECV-304 aging cultured in vitro, and the possible pathway of endothelial cell aging is that Ang II upregulates p22phox expression, and then enhances the cell ROS level.
Li
Hong
H
Department of Cardiology, the Affiliated Hangzhou Hospital, Nanjing Medical University, Hangzhou 310006, China. rainbowheart2003@126.com
Bai
Xiao-Juan
XJ
Liu
Qiang
Q
Wang
Ning-Fu
NF
D000375:Aging / Q000187:drug effects; D000804:Angiotensin II / Q000187:drug effects*; D015153:Blotting, Western; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects*; D042783:Endothelial Cells / Q000187:drug effects* / Q000473:pathology; D015870:Gene Expression; D006801:Humans; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D000804:Angiotensin II; D009569:Nitric Oxide; D019255:NADPH Oxidases; C086986:CYBA protein, human