Effect Of Selenium Supplement And Peroxynitrite On Nitration And Phosphorylation Of Src Protein In Human Len Epithelial Cells

Cataract, as a leading cause of blindness in the world, has become a hot spot in the eyestudy. The senile or diabetic cataract is related to the damage of lens epithelial cells caused byoxidative stress, although the pathology of cataract is very complex. Peroxynitrite is a strongoxidizing and nitrating agent, which may influence numerous signal transduction pathwaysand differentiation of lens epithelial cells (LECs) through activating the Src kinase because oftyrosine residue nitration. In this paper, effects of peroxynitrite on tyrosine nitration andphosphorylation and the inhibition of selenium on tyrosine nitration were investigated in hLECsby Western Blotting. The main results are as follows:(1) Exposure of hLECs to peroxynitrite (100~500μM) led to a dose-dependent increase oftyrosine nitration and decrease of tyrosine phosphorylation in proteins extracted from hLECs,indicating protein tyrosine nitration by peroxynitrite can downregulate the proteinphosphorylation. Selenium supplement may enhance the anti-oxidation damage of hLE cells, andinhibits protein tyrosine nitration. Selenium supplement (25nM) obviously attenuates theinhibition of phosphorylation by tyrosine nitration.(2) When hLE cells were exposed to various concentration (100~500μM) of peroxynitrite,the expression level of Src protein and Y418phosphorylation were detected by western blotting.The results showed that high concentration peroxynitrite significantly reduced the expressionlevel of Src protein; however, the level of Y418phosphorylation was increased. It is possible thatthe increase may be related with cell compensatory. What is more, selenium supplement(25~50nM) improve Src protein expression, in parallel with decrease of Y418phosphorylation;however, at high concentration selenium (100nM) elevated Y418phosphorylation level,suggesting selenite of high concentration may induce oxidation stress.These preliminary results suggest that peroxynitrite-induced tyrosine nitration downregulatesphosphorylation of protein tyrosine and the appropriate concentration selenium may inhibitstyrosine nitration by eliminating peroxynitrite, but this conclusion remains to be confirmed bymore experiments in future.