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Y. Wang, M.F. Lou; The Role of P22phox in Reactive Oxygen Species Generation in Human Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1896.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: NADPH oxidase has been confirmed as a major source of reactive oxygen species (ROS) generation in different cell types. It comprises a plasma membrane–bound cytochrome b558, which is a heterodimer of one p22phox and one gp91phox subunit, and at least four other cytosolic subunits. During activation in response to various stimuli, the cytosolic subunits translocate to and associate with cytochrome b558 by binding to p22phox and finally activate NADPH oxidase. This study is to identify the role of p22phox in the function of ROS generation in human lens epithelial cells (HLE–B3). Methods: Total RNA of the HLE–B3 cell was extracted and purified, and then the reverse transcription was performed by using random hexamer primers and AMV reverse transcriptase in thermocycler. The p22phox mRNA expression PCR was performed with specific primers: 5’–CCATGGGGCAGATCGAGTGGG–3’ and 5’–CACACGACCTCGTCGG TCAC. The p22phox PCR product was cloned into pCR3.1 expression vector (Invitrogen), and the orientation of the insert was identified by restriction enzymatic digestion and agarose gel electrophoresis. The vector with inverted insert (p22–KD) was used for knockdown experiments whereas the vector with normal–oriented insert (p22–OE) used for overexpression. The transfection of p22–OE or p22–KD into HLE–B3 cells was performed by electroporation, and the analysis of p22phox protein expression was accomplished by Western blot. The NADPH oxidase enzyme activity was assayed by luminometer using lucigenin as the substrate. Results: We have successfully manipulated the expression of p22 phox in the HLE–B3 cells. Western blot analysis confirmed that cells transfected with p22–OE vector showed elevated p22 phox expression while cells transfected with p22–KD vector had a decreased p22 phox expression. The enzyme activity of NADPH oxidase was correspondingly decreased in p22–KD–transfected HLE–B3 cells and increased in p22–OE–transfected cells. Conclusions: The results suggest that the expression of p22phox can positively regulate the ROS generation by affecting NADPH oxidase activity in human lens epithelial cells.
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