Inhibition of p38 mitogen-activated protein kinase phosphorylation decrease tert-butyl hydroperoxide-induced apoptosis in human trabecular meshwork cells

Purpose: Oxidative stress induced trabecular meshwork cells death is believed to be involved in the pathogenesis and progression of primary open-angle glaucoma (POAG). However, the intrinsic mechanism is yet to be clarified. This study is to investigate the role of p38 mitogen-activated protein kinase (p38MAPK) in tert-butyl hydroperoxide (tBHP)-induced apoptosis of human trabecular meshwork (iHTM) cells. Methods: The human trabecular meshwork cells were treated with tBHP for 1 or 2 h with or without pretreatment of SB203580, an inhibitor of MAP kinase homologs. Cell viability was analyzed using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2h-tetrazolium bromide assay. Reactive oxygen species (ROS) levels were determined using dihydrodichlorofluorescein staining, and the chymotrypsin-like protease activities were measured using the Suc-LLVY-aminoluciferin substrate. Cell apoptosis was analyzed by Hoechst 33258 staining and annexin V-PI labeling. The protein level of phospho-p38 was measured using western blot analysis. Results: The intracellular ROS increased more than 50 fold and more than 100 fold after tBHP exposure for 1 h and 2 h, respectively (p<0.05). However, there was no difference in ROS levels between SB203580(-) and SB203580(+) cells (p>0.05). In 1 h tBHP treatment group, the cell viability was significantly improved in SB203580(+) cells (81.08%+/- 1.93%) compared to the SB203580(-) cells (69.35%+/- 1.52%), the chymotrypsin-like proteasome inactivation decreased in SB203580(+) cells (60.94%+/- 0.55%) compared to the SB203580(-) cells (70.59%+/- 0.88%), and apoptosis was impoved in SB203580(+) cells (12.75%+/- 1.91%) compared to the SB203580(-) (28.23%+/- 3.23%) (p<0.05). In 2 h tBHP treatment group, cell viability improved in SB203580(+) cells (76.72%+/- 2.11%) compared to SB203580(-) cells (57.88%+/- 2.20%), chymotrypsin-like proteasome inactivation was improved in SB203580(+) cells (62.99%+/- 0.41%) compared to SB203580(-) cells (74.93%+/- 0.54%), and apoptosis was improved in SB203580(+) cells (20.40%+/- 3.44%) compared to SB203580(-) cells (39.20%+/- 5.91%) (p<0.05). Phosphorylation of p38MAPK was significantly increased after tBHP exposure in SB203580 (-) cells and decreased sharply in SB203580(+) cells than that of control group (p<0.05). While there was no difference on the original form of p38MAPK among SB203580(-) and SB203580(+) cells after tBHP exposure and control group (p>0.05). Conclusions: Activation of p38MAPK plays an important role in tBHP-induced apoptosis of iHTM cells. Further study on the mechanisms of p38MAPK in human TM cell apoptosis may help to illuminate the pathogenesis of POAG.