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Updated: May 19, 2026
Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death
Published on: December 27, 2016
Inhibition of p38 mitogen-activated protein kinase phosphorylation decrease tert-butyl hydroperoxide-induced
Yuxia Yang1, Xing Liu, Jingjing Huang
1State Key Laboratory of Ophthalmology, Department of Glaucoma, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
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.
Insights
Oxidative stress triggers apoptosis in human trabecular meshwork cells, a key factor in primary open-angle glaucoma. Inhibiting p38 mitogen-activated protein kinase (p38MAPK) protects these cells from damage.
Area of Science:
- Ophthalmology
- Cell Biology
- Biochemistry
Background:
- Oxidative stress is implicated in primary open-angle glaucoma (POAG) pathogenesis.
- Cell death in trabecular meshwork (TM) cells contributes to POAG progression.
- The precise molecular mechanisms underlying TM cell apoptosis remain unclear.
Purpose of the Study:
- To investigate the role of p38 mitogen-activated protein kinase (p38MAPK) in tert-butyl hydroperoxide (tBHP)-induced apoptosis of human trabecular meshwork (iHTM) cells.
- To elucidate the involvement of p38MAPK signaling in oxidative stress-induced TM cell death.
Main Methods:
- iHTM cells were exposed to tBHP with or without SB203580, a p38MAPK inhibitor.
- Cell viability was assessed using MTT assays.
- Reactive oxygen species (ROS) levels, proteasome activity, and apoptosis were quantified.
- Western blot analysis was used to measure phospho-p38MAPK levels.
Main Results:
- tBHP significantly increased intracellular ROS and induced apoptosis in iHTM cells.
- Pretreatment with SB203580 improved cell viability, reduced proteasome inactivation, and decreased apoptosis.
- tBHP exposure led to increased phosphorylation of p38MAPK, which was attenuated by SB203580.
Conclusions:
- Activation of p38MAPK is crucial in mediating tBHP-induced apoptosis of iHTM cells.
- Targeting p38MAPK may offer a therapeutic strategy for POAG.
- Further research into p38MAPK signaling pathways in TM cells could illuminate POAG pathogenesis.
