NADPH oxidase participation in cell proliferation of human lens epithelial cells and its expression / 眼科

ABSTRACT

Objective To study whether the inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can inhibit the proliferation of human lens epithelial cells and to investigate which isoforms of NOX, one of the subunits of NADPH oxidase, are expressed in human lens epithelial cells at mRNA level. Design Experimental study. Participants Human lens epithelial cells (SRA 01/04). Methods The cells were divided into four groups EGF group was added with EGF in SRA 01/04, DPI group was added with the inhibitor of NADPH oxidase-diphenylene iodonium (DPI), DPI +EGF group was added with EGF after DPI and control group was added with nothing. Using Microplate reader and Cell Counting Kit-8 to determine OD450 value of SRA 01/04 of each group. The ex- pression of NOX family, including NOX1, NOX2 or gp91phox, NOX3, NOX4 and NOX5, was detected with RT-PCR. The RT-PCR products were sequenced to confirm their identities by NCBI BLAST. Main Outcome Measures OD450 of SRA 01/04, expression of NOX and their sequence alignments with other human tissues. Results After added with CCK-8 for 3.5 h, the OD450 value of EGF group increased 12.0% compared with control group (P=0.000). The OD450 value of DPI group decreased 25.5% compared with control group (P=0.000). The OD450 value of DPI+EGF group decreased 26.1% compared with EGF group (P=0.000). RT-PCR using primers specific for mRNAs of the five isoforms of the NOX proteins documented that mRNA encoding NOX1 through NOX5 were constitutively present in SRA 01/04 cells. The similarity of sequences of NOX1-NOX5 in human lens epithelial cells with other human tissues was 100%, 100%, 99.7%, 100% and 100%, respectively. Conclusions NADPH oxidase complex could promote cell proliferation in human lens epithelial cells. SRA 01/04 cells constitutively produced mRNA encoding five isoforms of NOX proteins, NOX1 was much weaker than the other four NOXs.