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[The study on inhabiting endothelial cell aging by targeted silencing of p22phox]
Hong Li1, Xiao-Juan Bai, Qiang Liu
1Department of Cardiology, the Affiliated Hangzhou Hospital, Nanjing Medical University, Hangzhou 310006, China. rainbowheart2003@126.com
Abstract:
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.
Insights
Short interference RNA (siRNA) silences NAD(P)H oxidase subunit p22phox, reducing angiotensin II-induced endothelial cell aging. This suggests p22phox upregulation enhances reactive oxygen species, contributing to cell aging.
Area of Science:
- Endothelial cell biology
- Molecular mechanisms of aging
- Oxidative stress pathways
Background:
- Endothelial cell aging is a critical factor in vascular health.
- NAD(P)H oxidase, particularly the p22phox subunit, is implicated in cellular processes.
- Understanding the role of p22phox in endothelial aging is crucial for developing interventions.
Purpose of the Study:
- To investigate the role of NAD(P)H oxidase subunit p22phox in the aging of human umbilical endothelial cells (ECV-304).
- To explore the potential mechanism by which p22phox influences endothelial cell aging.
- To evaluate the efficacy of short interference RNA (siRNA) in modulating p22phox expression and its effect on cell aging.
Main Methods:
- Design and synthesis of three siRNAs targeting p22phox.
- Transfection of ECV-304 cells with siRNA to determine optimal conditions.
- Experimental groups: control, angiotensin II (Ang II) stimulation, siRNA treatment, and Ang II + siRNA.
- Assessment of cell aging using beta-galactosidase staining.
- Measurement of reactive oxygen species (ROS) and nitric oxide (NO) levels.
- Analysis of p22phox mRNA and protein expression via RT-PCR and Western blot.
Main Results:
- siRNA-1 demonstrated the most effective p22phox gene silencing at a concentration of 50 nmol/L.
- Angiotensin II stimulation increased beta-gal positive cells, indicating enhanced ECV-304 aging.
- Ang II upregulated p22phox mRNA and protein expression, accompanied by increased ROS and decreased NO levels.
- Co-treatment with Ang II and siRNA significantly reduced ROS, increased NO, and decreased the aging level compared to the Ang II group.
Conclusions:
- Short interference RNA effectively silences the NAD(P)H oxidase subunit p22phox in endothelial cells.
- Angiotensin II induces ECV-304 cell aging in vitro.
- The mechanism involves Ang II-induced upregulation of p22phox, leading to elevated cellular ROS levels, which contributes to endothelial cell aging.
