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Regulation of iNOS expression by NF-κB in human lens epithelial cells treated with high levels of glucose.

PURPOSE
To explore the regulation of inducible nitric oxide synthase (iNOS) expression by nuclear factor kappa B (NF-κB) in human lens epithelial cells (LECs) treated with high levels of glucose, and to elucidate the impact of this in the pathogenesis of cataracts associated with diabetes.


METHODS
LECs (SRA01/04) were cultured in vitro. NF-κB nuclear translocation and iNOS expression were measured at different glucose concentrations and at various time points, and the optimal concentration for detecting changes in the patterns of NF-κB nuclear translocation and iNOS expression was chosen. As a specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) was used to assess the effect of inhibiting NF-κB. Western blotting and inverted fluorescence microscopy were used to monitor the nuclear translocation of NF-κB. PCR and Western blotting were used to measure iNOS expression. Using the University of California, Santa Cruz database and the TFSEARCH program, we searched the DNA sequence upstream of iNOS for the core binding sequence for NF-κB. Chromatin immunoprecipitation (ChIP) was used to measure the binding of NF-κB.


RESULTS
The nuclear translocation of NF-κB was measured upon glucose treatment, and the concentration of NF-κB in the nucleus was found to peak at 25 to 30 minutes of treatment with 25 mM glucose. iNOS mRNA and protein levels also increased significantly in a time- and concentration-dependent manner and iNOS mRNA and protein reached their peak values after 8 hours of treatment with 25 mM glucose. The binding of NF-κB to the promoter of the iNOS gene was enhanced in the 25 mM glucose group compared with the 5.5 mM glucose group or the 25 mM glucose + 100 μL PDTC group, and this difference was statistically significant (P < 0.05).


CONCLUSIONS
NF-κB regulates iNOS expression in a time- and concentration-dependent manner. Under high glucose conditions, NF-κB is activated and rapidly translocates to the nucleus, leading to increased binding to the iNOS promoter and a consequent increase in iNOS expression. The findings of this study provide important experimental evidence that clarifies the pathogenesis of cataracts associated with diabetes and contributes to the search for therapeutic targets of these cataracts.