title: Inhibition of p38 mitogen-activated protein kinase phosphorylation decrease tert-butyl hydroperoxide-induced apoptosis in human trabecular meshwork cells.
abstract: OBJECTIVE
  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.
authors.affiliation: State Key Laboratory of Ophthalmology, Department of Glaucoma, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
authors.forename: Yuxia
authors.initials: Y
authors.lastname: Yang
authors.forename: Xing
authors.initials: X
authors.lastname: Liu
authors.forename: Jingjing
authors.initials: J
authors.lastname: Huang
authors.forename: Yimin
authors.initials: Y
authors.lastname: Zhong
authors.forename: Zhen
authors.initials: Z
authors.lastname: Mao
authors.forename: Hui
authors.initials: H
authors.lastname: Xiao
authors.forename: Mei
authors.initials: M
authors.lastname: Li
authors.forename: Yehong
authors.initials: Y
authors.lastname: Zhuo
mesh_terms: D017304:Annexin A5; D017209:Apoptosis / Q000187:drug effects; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D053818:Chymases / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
chemical_list: D017304:Annexin A5; D007093:Imidazoles; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D017382:Reactive Oxygen Species; D020122:tert-Butylhydroperoxide; D048051:p38 Mitogen-Activated Protein Kinases; D053818:Chymases; C093642:SB 203580