Expressions And Intervention Of NF-κB And ELAM-1in Oxidative Stress Induced By H₂O₂in Trabecular Meshwork Cells

Objective:To evaluate the effects of nuclear transcription factors-kappa B (NF-κB) and endothelial leukocyte adhesion molecule-1(ELAM-1) in oxidative stress induced by hydrogen peroxide (H2O2) in rat trabecular meshwork(TM) cells.At the same time,we used alpha lipoic acid (alpha LA) as the intervention therapy to investigate the protection mechanism for the injured TM cells.Method:1. Primary culture and identification of TM cells:TM cells were cultured using enzyme digestion method with eye tissues of5-week-old Wistar rats. SABC immunocytochemical method was used to identify fibronectin (FN),laminin(LN) and neuron specific enolase(NSE).2.Cell grouping:After one night of serum starvation,The cells were divided into five groups:normal control group(A); H2O2group(B):the cells were cultured with800μM H2O2; H2O2and a-LA group(C):C1:the cells were cultured with800μM H2O2and100μM a-LA; C2:the cells were cultured with800μM H2O2and500μM a-LA; C3:the cells were cultured with800μM H2O2and1000μM a-LA. All the cells was cultured at37℃under5%CO2for2hours.3.Detection of NF-κB and ELAM-1:We observed the morphological changes of the TM cells in each group under optical microscope, and detected and compared the expression of NF-κB and ELAM-1among5groups by immunocytochemistry and Real-time PCR.Results:1.We successfully cultured rat TM cells.Immunocytochemical analysis revealed that these cells were positive for FN,LN and NSE expression.2. Compared with group A, the cells of group B retracted, gap widened and density decreased; And with the increase of concentration of α-LA, the morphology change of group C tended to be not obvious gradually compared with group A.3. NF-κB and ELAM-1levels revealed a high significant increase in group B with respect to the group A (pNF-κB=0.000;pELAM-1=0.000); With the increase of concentration of a-LA, the expressions of NF-κB and ELAM-1of group C decreased gradually compared with group B(pNF-κB=0.036、0.000、0.000;p ELAM-1=0.016、0.00020.000).There was no significant difference in the expressions of NF-κB between group C3and group A(p=0.155),but ELAM-1level was still higher in group C3than that in group A(p=0.004).Conclusions:1. Oxidative stress activates NF-κB obviously,which promotes ELAM-1expression lead to the morphological changes and cell deaths on trabecular meshwork cells;2. ELAM-1may be promoted through a variety of ways including the NF-κB pathway;3. α-LA can inhibit the activation of NF-κB, which reduces the expression of ELAM-1, the oxidative stress damage to the TM cells are reduced eventually;4. With the increase of concentration of α-LA, the expression of NF-κB and ELAM-1decreased gradually.1000μM a-LA can interdict the activation of NF-κB induced by800μM H2O2,but can not interdict the increase of ELAM-1.