Citation: WANG Li-Ying, SU Hai-Yan, WANG Xuan, ZHOU Yan, YANG Shi-Jie. Role of Reactive Oxygen Species by Fluorescence Analysis in Proliferation of Myofibroblasts Induced by Angiotensin Ⅱ[J]. Chinese Journal of Analytical Chemistry, ;2015, 43(12): 1870-1875. doi: 10.11895/j.issn.0253-3820.150331 shu

Role of Reactive Oxygen Species by Fluorescence Analysis in Proliferation of Myofibroblasts Induced by Angiotensin Ⅱ

1.
¹ Physiology Department of Basic Medicine College of Beihua University, Jilin 132013, China;

Corresponding author: ZHOU Yan,
Received Date: 23 April 2015
Available Online: 18 August 2015
Fund Project: 本文系973重大基础研究基金资助课题(No.2007CB512006) (No.2007CB512006)国家自然科学基金项目(No.30873066/C180102) (No.30873066/C180102)博士启动基金(No.199500033) (No.199500033)吉林市科技计划项目(No.201536058)资助 (No.201536058)

Fluorescence analysis was performed to explore the role of reactive oxygen species(ROS) in the proliferation of myofibroblasts(myoFbs) induced by angiotensin Ⅱ(AngⅡ). Primary cultures of neonatal rat myoFbs were obtained by enzymatic dissociation of Wistar rat neonates, and myoFbs were cultured under 10% fetal bovine serum. MyoFbs of 2-3 generation cultured in vitro were used in experiments and divided into three groups which were treated by AngⅡ, AngⅡ +N-acetyl-L-cysteine(NAC), and normal culture medium, respectively. MyoFbs were cultured for 12, 24 and 36 h. In the experiment, the proliferation rate of myoFbs induced by AngⅡ under different concentrations of 3-(4,5)-dimethylthiazo(-z-yl)-3,5-diphenyl-etrazoliumromide(MTT) was detected, the ROS levels of myoFbs were detected by fluorescent probes in 2',7'-dichlorofluorescein fluorescence Huang Shuang acetate(DCFH-DA), and the contents and levels of oxygen free radicals(·OH) in three groups were detected by spectrophotometry, immunocytochemical staining and fluorescence confocal. Immunocytochemical staining and fluorescence confocal analysis was used to measure membrane translocation and expression of p-protein kinase Cα(p-PKCα). The results showed that myoFbs incubated with AngⅡ(10^-8-10^-6 mol/L) for 24 h increased the proliferation rate(the value of η(%) was 170.15) and ROS, especially the content of·OH(94.53±1.68) reached the highest level. The immunocytochemistry, confocal fluorescence staining and image analysis result showed that AngⅡ could increase the translocation and expression of p-PKCα in membrane(p<0.001vs control group); NAC could inhibit the proliferation of myoFbs induced by AngⅡ(the value of η(%) was 117.05), decrease the content of ROS and the levels of·OH(75.57±1.48), and inhibit the translocation and expression of p-PKCα in membrane.
- N-Acetyl-L-cysteine,
- AngiotensinⅡ,
- Oxygen free racical,
- Myofibroblasts,
- Protein kinase Cα,
- Reactive oxygen species
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