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Yiguo Qiu, Lifei Tao, Peizeng Yang, Qiuhong Li, Bo Lei; Downregulating p22phox ameliorates inflammation in Angiotensin II induced oxidative stress by regulating MAPK and NF-κB pathways in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):839.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress and inflammation are interrelated biological events and both have been identified to play important roles in the pathological process of age-related macular degeneration (AMD). The purpose of this study is to investigate the anti-inflammation effect and the mechanism of downregulating p22phox in Angiotensin II (Ang II) induced oxidative stress in ARPE-19 cells.
ARPE-19 cells were transfected with p22phox siRNA (P) followed by stimulation with Ang II for 48 hours. The mRNA levels of p22phox, inflammatory cytokines, IκBα and NOX1, 2, 4 were analyzed by real time PCR. The protein expression of p22phox, p65 and the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK1/2) and c-Jun N terminal kinase (JNK) were detected by Western Blotting. The reactive oxygen species (ROS) was investigated by DCFH-DA assay with flow cytometry. Protein concentrations of IL-6, IL-8 and MCP-1 in the supernatant were measured by ELISA. The signal transduction mechanisms involved in cytokine production were evaluated using inhibitors for MAPK and NF-κB pathways.
The expressions of p22phox, IL-6, IL-8 and MCP-1 remarkably decreased in p22phox siRNA transfected and Ang II treated (P plus Ang II) group at both mRNA and protein levels. The mRNA expressions of NOX1, 2, 4 were reduced in P plus Ang II group than Ang II group (p<0.05, p<0.01). The ROS production was decreased in P plus Ang II group than Ang II group (p<0.01). The mRNA level of IκBα was significantly increased and the protein level of p65 decreased in P plus Ang II group (p<0.05, p<0.001). Downregulating p22phox decreased the phosphorylation of p38, ERK1/2 and JNK stimulated by Ang II. The inhibitory experiments showed that MAPK and NF-κB pathways were involved in the inhibitory effect of downregulating p22phox on cytokine production.
Downregulating p22phox shows anti-inflammation effect in Ang II induced oxidative stress in ARPE-19 cells by regulating MAPK and NF-κB pathways. These results support the notions that p22phox plays an important role in inflammation accompanied by oxidative stress and p22phox may provide a novel therapeutic target for oxidative induced inflammation.
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