Current Eye Research, Early Online, 1–8, 2014 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.959607
RESEARCH REPORT
Mitochondria-Targeted Antioxidant Peptide SS31 Protects Cultured Human Lens Epithelial Cells against Oxidative Stress
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Meng Cai, Jing Li, Shaofen Lin, Xiaoyun Chen, Juan Huang, Xiaoyan Jiang, Lizhu Yang and Yan Luo State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, P.R. China
ABSTRACT Purpose: To investigate the effects of the mitochondria-targeted peptide, SS31, on cultured human lens epithelial cells (HLECs) under conditions of oxidative stress. Methods: Optimal concentrations of SS31 were determined by MTT assay. HLEB-3 cells were first treated with SS31 for 2 h and then with 500 mM t-BHP for 6 h. Cell apoptosis was revealed by Annexin V/PI staining. Morphological changes in nuclei were observed by fluorescence microscopy after Hoechest 33258 fluorescent staining. Reactive oxygen species (ROS) were measured by MitoSOX staining. Changes in mitochondrial membrane potential (D m) were detected using the JC-1 fluorescent dye. Activation of p38 and c-Jun N-terminal kinases (JNKs) were quantified by Western Blot analysis. Results: SS31 protected HLEB-3 cells against t-BHP-induced cell apoptosis, reduced ROS, maintained D ms, and inhibited activation of JNK and p38 pathways. Conclusions: SS31 was able to protect HLEB-3 cells against oxidative damage and, thus, represents a potential new treatment modality for preventing the formation of cataracts and other age-related disorders. Keywords: Antioxidant, cataract, cell model, in vitro, SS31
INTRODUCTION
Furthermore, inadequate surgical facilities and the high cost of artificial IOLs represent major problems for many poor and developing countries.5 Moreover, there are numerous increasing social burdens imposed by the adverse effects of cataracts on visual function and quality of life of the elderly. Since there exist no effective drugs for the prevention and treatment of cataracts, development of effective preventative drugs that target the early phases of cataractogenesis is vital. Increased oxidative stress is caused by factors such as ultraviolet light (UV) light and hydrogen peroxide (H2O2), both of which are major risk factors for cataract development.6 Among the age-related changes that occur in the lens, decreased antioxidants
Age-related cataracts are the leading cause of loss of useful vision among elderly persons,1 affecting 46% of the 180 million visually disabled people worldwide.2 As result of the large increase in demographic aging, the prevalence of this age-related disorder is expected to double3 by the year 2020. At present, the only effective treatment for cataract is extraction of the cataractous lens, with implantation of an artificial intraocular lens (IOL).4 However, cataract surgery carries with it a number of post-operative complications, e.g. posterior capsular opacification, stimulation of chronic inflammation, uncorrectable residual refractive errors, and/or post-operative glare.
Received 27 November 2013; revised 20 July 2014; accepted 18 August 2014; published online 30 September 2014 Correspondence: Yan Luo, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, P.R. China. Tel: +86 020 87330485. Fax: +86 020 87333271. E-mail: [email protected].
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in the aging lens nucleus, along with increased protein modifications, may be causative factors in the etiogenesis of cataract. Lens epithelial cells, which are essential for the growth, differentiation and homeostasis of the lens, are the first to be effected by aqueous-associated aging changes.7 The decreased density of the lens epithelium observed in many species during normal aging8,9 renders the lens nucleus highly susceptible to oxidative damage. Lens epithelial cells play important roles in maintaining the oxidation resistance of the lens nucleus. Mitochondria, a major source of cellular reactive oxygen species (ROS), play crucial protective roles in oxidative stress.10,11 Therefore, procedures that protect the mitochondria via mitochondriatargeted protective mechanisms may be beneficial in the prevention and treatment not only of cataract but also other age-related lens disorders. SS31 (H-D-Arg-Dmt-Lys-Phe-NH2), fortuitously discovered as a novel mitochondria-targeted antioxidant peptide, easily penetrates cell membranes and targets mitochondria in a potential-independent manner.12 SS31 has been shown to lessen the accumulation of mitochondrial ROS in a dose-dependent manner and to prevent oxidative damage in several cell types.13,14 In this study, we exposed a human lens epithelial cell line (HLEB-3) to tert-butylhydroperoxide (t-BHP), organic peroxide used in a variety of oxidation processes, in order to mimic the oxidative damage that occurs in lens epithelial cells during cataractogenesis. We also investigated the protective effect(s) of SS31 against oxidative damage to HLEB-3 cells.
plus SS31 for 6 h and then incubated with 200 ml of 0.5 mg/ml MTT (Sigma-Aldrich, St. Louis, MO) at 37 C for 4 h. The MTT formazan product was dissolved in dimethylsulfoxide (DMSO). Absorbance was measured at 490 nm by a microplate reader (Bio-TEK Instruments, Winooski, VT). To obtain the percentage of surviving cells, the reading was divided by the adjusted absorbance reading of untreated cells in control wells. Three replications of each experiment were accomplished, and each experiment was repeated three times.
Apoptosis Assay The rate of apoptosis of HLEB-3 cells was evaluated using the Annexin V/propidium iodide (PI) double staining assay. Early apoptotic, late apoptotic, and necrotic cells were differentiated using the Annexin V-FLUO staining kit (Roche, Mannheim, Germany). Cells were placed in six-well plates at a density of 1 105 cells per well. Cells were pre-treated with 1 mM SS31 for 2 h and then treated with a combination of 500 mM t-BHP plus SS31 for 6 h. Cells were then collected and centrifuged at 1000g for 5 min at room temperature. After washing the centrifuged pellet with phosphate-buffered saline (PBS), cells were resuspended in 100 ml of Annexin V-FLUO labeling solution (2 ml Annexin V plus 2 ml PI in 100 ml incubation buffer) and incubated in the dark at room temperature for 15 min. An additional 400 ml buffer was then added to each tube and samples were analyzed by flow cytometry (BD-FACS AriaTM Flow Cytometer, BD Biosciences, San Jose, CA).
MATERIALS AND METHODS Nuclear Morphology Cells and Cell Culture HLEB-3 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). HLEB-3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY), supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37 C in a humidified atmosphere of 5% CO2. Cells were used for assays when they attained 75–80% confluence.
Apoptosis of treated HLEB-3 cells was determined according to morphological changes in cell nuclei using Hoechst 33258 staining (Sigma). HLEB-3 cells were seeded on 18 18 mm coverslips, pre-treated with 1 mM SS31 for 2 h, and then treated with a combination of 500 mM t-BHP plus SS31 for 6 h. Cells were then washed twice in PBS and incubated with Hochest 33258 (10 mg/mL) for 10 min. Coverslips were mounted on glass microscope slides with glycerol and immediately viewed under a fluorescence microscope (Leica, Solms, Germany).
MTT Assay Proliferation and cell viability of HLEB-3 cells were evaluated by the MTT assay. Cells were plated in 96-well plates at a density of 1 104 cells/well. After pre-treatment with varying concentrations (10, 100, 1, 10, and 100 mM, and 1 M) of SS31 (Stealth Peptides International Inc., Shanghai, P.R. China) for 2 h, cells were treated with a combination of 500 mM t-BHP
Measurement of D)m Changes in t-BHP-induced mitochondrial membrane potential (D m) were evaluated with the JC-1 fluorescent probe (Molecular Probes, Eugene, OR). HLEB-3 cells, at a density of 1 105 cells/well in six-well plates, were pre-treated with SS31 for 2 h and then Current Eye Research
SS31 protects lens epithelial cells incubated with 500 mM t-BHP for 6 h. Cells were centrifuged at 1000g for 5 min, and the pellets harvested. After washing with PBS, cells were stained with 2.5 mg/ml JC-1 at 37 C for 10 min and analyzed immediately by flow cytometry. D m was expressed as the ratio of mean red fluorescence (JC-1 aggregates) to mean green fluorescence (JC-1 monomers).
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Detection of ROS Production of ROS was evaluated with the MitoSOXTM red superoxide mitochondrial indicator, which is oxidized using superoxide and which exhibits red fluorescence in the mitochondria. Coverslip-seeded cells were pre-treated with SS31 for 2 h and then incubated with 500 mM t-BHP for 6 h. Following this, the contents (50 mg) of one vial of MitoSOXTM was dissolved in 13 ml of DMSO to make a 5 mM MitoSOXTM stock solution. After exposure to 5 mM of MitoSOXTM working solution, live HLEB-3 cells were incubated at 37 C for 10 min. Following a gentle rinse with warm buffer, cells were viewed by fluorescence microscopy.
Western Blot After washing with ice-cold PBS, cells were lysed in 100 ml of lysis buffer (BD Biosciences) combined with a protease inhibitor cocktail (Calbiochem, La Jolla, CA) to extract total protein. Protein samples were mixed with 3 sodium dodecyl sulfate (SDS) sample buffer, separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE) gels, and then transferred to polyvinylidene difluoride membranes. Membranes were blocked in 5% bovine serum albumin (BSA) for 1 h and incubated overnight at 4 C with various primary antibodies. Anti-cJun NH2-terminal kinase (JNK), anti-phosphorylated JNK (p-JNK), anti-p38, and anti-phosphorylated p38 (p-p38) (Thr180/Tyr182) antibodies were purchased from Cell Signaling Technology (Danvers, MA). After washing with 0.1% Tween 20-containing PBS, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Bands were visualized using an enhanced chemiluminescence (ECL) detection system (Cell Signaling Technology). Densitometric analysis was conducted using Version 1.41 Image J software (National Institutes of Health, Bethesda, MD).
Statistical Analysis All data are expressed as mean ± SD. Statistical analyses were performed using Version 16.0 SPSS software (SPSS, Chicago, IL). A two-sample t-test !
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was used to compare normally distributed data. Three independent experiments were performed. A value of p50.05 was considered statistically significant.
RESULTS SS31 Protection of HLEB-3 Cells against t-BHP-Induced Oxidative Injury To determine whether SS31 protects HLEB-3 cells against t-BHP-induced oxidative damage, cell viability and cell apoptosis were examined 6 h after 500 mM t-BHP treatment alone or after co-treatment with SS31 at various concentrations. Incubation with 500 mM t-BHP for 6 h resulted in a significantly low ratio of cell survival. Compared with the t-BHP group, SS31 prevented loss of cell viability in a dosedependent manner (p50.01). The maximum effect of SS31 was observed at a concentration of 1 mM (Figure 1). Consequently, concentrations of 1 mM SS31 were used for all experiments. Annexin V/PI staining was used to determine whether SS31 inhibits t-BHP-induced apoptosis (Figure 2A). The percentages of surviving HLEB-3 cells (Q3) were 79.24 ± 6.08, 98.64 ± 0.24, and 96.03 ± 1.02% in the t-BHP-treated, control, and SS31treated groups, respectively. SS31 caused a decrease in cellular apoptosis and increased cell survival than t-BHP (p50.01), indicating its protective effects against oxidative stress.
FIGURE 1. SS31 protected HLEB-3 cells from t-BHP-induced oxidative injury. 500 mM t-BHP was added to HLEB-3 cells and incubated with various concentrations (10, 100, 1, and10 mM, and 100 mM) of SS31 for 6 h. Cell viability was measured by MTT assay. The survival ratio of HLEB-3 indicates a concentration-dependency in cell viability and is markedly increased under the protection of 1 mM SS31. Data are shown as mean ± standard deviation (n = 3), *p50.01 versus t-BHP.
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FIGURE 2. SS31 significantly decreased apoptosis of HLEB-3 cells. Cells were pre-treated with 1 mM SS31 for 2 h and then treated with a combination of 500 mM t-BHP plus SS31 for 6 h. (A) Cell apoptosis was analyzed by flow cytometry after Annexin V/PI staining. The percentage of surviving HLEB-3 cells (Q3) was much higher in cells of the SS31-treated group than in those of the t-BHP-treated group. (B) When HLEB-3 cells were exposed to 500 mM t-BHP for 6 h, nuclear density decreased and the number of apoptotic cells (arrows) with nuclear fragmentation and condensed chromatin increased. There were fewer cells with apoptotic nuclei in the SS31-treated group.
Hoechst staining showed apoptotic cells with condensed nuclear chromatin and multiple chromatin fragments. As shown in Figure 2(B), cells exposed to 500 mM t-BHP for 6 h demonstrated obvious apoptotic features, including condensed chromatin and multiple chromatin fragments. In contrast, cell nuclei in the control group showed no apoptotic morphology. SS31 treatment in the t-BHP-treated group showed a decrease in apoptotic cells (p50.01), indicating that SS31 protected the HLEB-3 cells against oxidative damage.
SS31-Induced Prevention of Loss of D)m in t-BHP-Treated HLEB-3 Cells To determine if mitochondria are involved in the protective effects of SS31 on t-BHP-induced cell death, the loss of D m in HLEB-3 cells was examined using the JC-1 fluorescent probe. A rapid loss of D m in t-BHP-treated HLEB-3 cells was prevented by SS31 (Figures 3A and B, p50.01). These data suggest that SS31 prevents the loss of D m caused by t-BHP in HLEB-3 cells. Current Eye Research
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SS31 protects lens epithelial cells
FIGURE 3. SS31 prevented loss of D m and decreased ROS production induced by t-BHP in HLEB-3 cells. (A) The D m of HLEB-3 cells was measured by flow cytometry after JC-1 staining. Exposure to 500 mM t-BHP for 6 h resulted in a rapid loss of D m in HLEB-3 cells, while, SS31 inhibited the loss of D m induced by t-BHP. (B) Quantitative analysis of D m in HLEB-3 cells is shown. (C) ROS production was examined under by fluorescence microscopy. Leves of mitochondrial ROS in the control group was very low. ROS production significantly increased 6 h after t-BHP treatment. SS31 treatment decreased t-BHP-induced ROS production in mitochondria. Data are shown as mean ± standard deviation (n = 3), *p50.01 versus t-BHP.
SS31-Induced Reduction of ROS in t-BHP-Treated HLEB-3 Cells t-BHP-induction of ROS was examined by staining with MitoSOXTM Red fluorescent stain. As illustrated !
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by fluorescence microcopy, incubation with 500 mM t-BHP for 6 h resulted in a significant increase in ROS production. However, ROS production was remarkably reduced in the SS31-treated group (Figure 3C, p50.01).
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SS31-Inhibition of JNK and p38 Activation in t-BHP-Treated HLEB-3 Cells
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To examine whether the JNK and p38 pathways contribute to the protective effects of SS31, Western blot analysis was performed. Protein levels of p-JNK and p-p38 in cells of the t-BHP-treated group were significantly greater than those of control group cells, while the total protein levels of JNK and p38 remained unchanged in all groups. Protein levels of p-JNK and p-p38 were much lower in the SS31-treated group than in the t-BHP-treated group (p50.01). Thus, SS31 inhibits activation of JNK and p38 in t-BHP-treated HLEB-3 cells.
DISCUSSION An understanding of the physiological and biochemical changes that occur in cataract formation is constantly being sought. The lens epithelium, which
covers the anterior face of the lens, protects the underlying lens fibers from oxidative injury, as well as transports materials into and out of the deeper layers of the lens.15,16 Lens epithelial cells also play important roles in maintaining the oxidation resistance of the lens. Loss of antioxidants in the lens triggers proteins into undergoing non-enzymatic, post-translational modifications, thus increasing susceptibility of the lens to oxidation. Progressive insolublization of lens crystallins, as well as the formation of high molecular weight aggregates, disturb the short-range order of the lens crystallins, thus leading to increased light-scatter and loss of the transparency.17–19 Since oxidative damage plays a pivotal role in age-related lens alterations, targeting oxidative damage to the lens epithelium offers a potentially significant focus for research in the treatment of cataract. There are a considerable number of in vitro and in vivo studies on cataract-preventing antioxidants. Several herbal drugs and non-steroidal anti-inflammatory drugs with antioxidant properties may be of
FIGURE 4. SS31 inhibited t-BHP-induced activation of the MAPK pathways. (A) Protein levels of JNK, p38, and p-JNK plus p-p38 were measured by Western Blot. When HLEB-3 cells were exposed to 500 mM t-BHP, expression levels of p-JNK and p-p38 increased dramatically. There were significantly decreased levels of p-JNK and p-p38 in the SS31-treated group. (B) Quantitative analysis of expression levels of p-JNK and p-p38 in HLEB-3 cells. Data are shown as mean ± standard deviation (n = 3), *p50.01 versus t-BHP. Current Eye Research
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SS31 protects lens epithelial cells benefit to patients with cataracts. Resveratrol20 and low concentrations of acetylsalicylic acid, aspirin21 protect HLECs against peroxide-induced oxidative damage. In addition, many nutritional or metabolic oxyradical scavengers, e.g. ascorbate22, vitamin E23, pyruvate24, and caffeine appear to attenuate oxidative damage during cataract development.25 However, most efforts to prevent cataracts do not provide significant clinical benefits. Future studies are needed to develop new drugs that prevent the early onset of cataractogenesis. The structure of SS31, a small aromatic-cationic mitochondria-targeted peptide, is highly suitable for penetrating membranes, targeting mitochondria, and reducing generation of mitochondrial ROS.26–28 Our previous study indicated that SS31 can effectively attenuate oxidative injury in human retinal endothelial cells13 and in streptozotocin (STZ)-induced diabetic rats.29 Results of these studies suggest SS31 to be an ideal therapeutic candidate for the treatment of age-related lens disorders. t-BHP, a cell permeable oxidant compound, is more stable than H2O2 in vitro, and is used to induce in vitro models of oxidative damage.30,31 To mimic the oxidative stress that occurs in lens epithelial cells during cataractogenesis, we used t-BHP-treated HLEB-3 cells, which exhibited a high rate of apoptosis, rapid loss of D m, and increased levels of intracellular ROS. SS31 was shown to effectively preserve cell viability of these HLEB-3 cells, prevent the loss of D m, and inhibit production of t-BHP-induced mitochondrial ROS. To investigate the mechanisms by which SS31 exerts it protective effects against oxidative stress in HLEB-3 cells, several potentially involved pathways were examined. Mitogen-activated protein kinase (MAPK) signaling pathways, important upstream regulators of transcriptional factor activities, comprise a phosphorylation-dependent relay of protein activation pathways. MAPK signaling pathways alter transcription and, ultimately, regulate cell proliferation and differentiation.32,33 JNK and p38 cascades are involved in MAPK signaling pathways, and activation of both JNK and p38 are important participants in cell death. These two MAPK signaling pathways are expressed in the mammalian lens, with primary activities in the epithelial layer, and are activated by a variety of environmental stresses, inflammatory cytokines, UV radiation, and growth factors. Grape seed pro-anthocyanidin extract and resveratrol have been reported to reduce oxidative stress in lens epithelial cells via inhibition of the JNK and p38 signaling cascades.34,35 SS31 has also been reported to attenuate high glucose-induced p38 MAPK pathway activation in human neuroblastoma cells.36 Therefore, whether or not t-BHP-induced JNK and p38 pathways in the lens epithelium are inhibited by SS31 was investigated in this study. Our results indicate that t-BHP-induced activation of p38 and !
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JNK in HLEB-3 cells was significantly suppressed when cells were treated with SS31, i.e. SS31 inhibited JNK and p38 pathway-mediated cell death in HLEB-3 cells. Although SS31 was shown to have protective effects on HLEB-3 cells against oxidative damage, its protective effects on the ex vivo and in vivo whole lens are still unclear. Questions that need to be addressed in future studies include (i) the capability of SS31 to penetrate the lens capsule, (ii) the effects of SS31 on cultured lens, and (iii) the effects of SS31 in in vivo animal experiments. Overall, SS31 attenuates t-BHP-induced oxidative damages to HLEB-3 cells by stabilizing the D m, decreasing ROS production, and inhibiting activation of JNK and p38 pathways, and thus represents a potentially viable modality for prevention/treatment of cataract and other oxidative stress-related diseases.
DECLARATION OF INTEREST There are no conflicts of interest to declare for any of the authors. This work was supported by grants from National Basic Research Development Program of China (973 program: 2013CB967000) and the National Natural Science Foundation of China to YAN LUO (81170864).
REFERENCES 1. Asbell PA, Dualan I, Mindel J, Brocks D, Ahmad M, Epstein S. Age-related cataract. Lancet 2005;365:599–609. 2. WHO. Global initiative for the elimination of avoidable blindness. Fact sheet number 213 Geneva: WHO, 2000 Available from http://wwwwhoint/mediacentre/factsheets/fs213/en/ [last accessed 10 Dec 2004]. 3. WHO. Blindness and visual disability: major causes worldwide. Fact sheet number 143. Geneva: WHO, 1997. Available from http://www.who.int/mediacentre/factsheets/fs413/en/ [last accessed 10 Dec 2004]. 4. WHO. Control of major blinding diseases and disorders. Fact sheet number 214. Geneva: WHO, 2000. Available from http:// www.who.int/mediacentre/factsheets/ fs214/en/ [last accessed 10 Dec 2004]. 5. WHO. Blindness and visual disability: socioeconomic aspects. Fact sheet number 145. Geneva: WHO, 1997. Available from http://www.who.int/int-fs/en/ fact145.html [last accessed 10 Dec 2004]. 6. Truscott RJ. Age-related nuclear cataract-oxidation is the key. Exp Eye Res 2005;80:709–725. 7. Zhang Z, Zhang J, Hui Y, Zheng M, Liu X, Kador P, et al. Up-regulation of NDRG2 in senescent lens epithelial cells contributes to age-related cataract in human. PLoS One 2011;6:e26102. 8. Balaram M, Tung WH, Kuszak JR, Ayaki M, Shinohara T, Chylack Jr LT,. Noncontact specular microscopy of human lens epithelium. Invest Ophthalmol Vis Sci 2000;41: 474–481. 9. Pendergrass W, Penn P, Possin D, Wolf N. Accumulation of DNA, nuclear and mitochondrial debris, and ROS at sites of age-related cortical cataract in mice. Invest Ophthalmol Vis Sci 2005;46:4661–4670.
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10. Lenaz G. Role of mitochondria in oxidative stress and ageing. Biochim Biophys Acta 1988;1366:53–67. 11. Ott M, Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria, oxidative stress and cell death. Apoptosis 2007;12:913–922. 12. Szeto HH. Cell-permeable, mitochondrial-targeted, peptide antioxidants. AAPS J 2006;8:E277–283. 13. Li J, Chen X, Xiao W, Ma W, Li T, Huang J, et al. Mitochondria-targeted antioxidant peptide SS31 attenuates high glucose-induced injury on human retinal endothelial cells. Biochem Biophys Res Commun 2011;404:349–356. 14. Chen M, Liu B, Gao Q, Zhuo Y, Ge J. Mitochondriatargeted peptide MTP-131 alleviates mitochondrial dysfunction and oxidative damage in human trabecular meshwork cells. Invest Ophthalmol Vis Sci 2011;52: 7027–7037. 15. Kevin Ml, George JH, Allan EO, David C.B. Human age-related cataract and lens epithelial cells death. Invest Ophthalmol Vis Sci 1998;39:2996–2706. 16. Bhat SP. The ocular lens epithelium. Biosci Rep 2001;21: 537–563. 17. Michael R, Bron AJ. The ageing lens and cataract: a model of normal and pathological ageing. Philos Trans R Soc Lond B Biol Sci 2011;366:1278–1292. 18. Truscott R. Age-related nuclear cataract: a lens transport problem. Ophthalmic Res 2000;32:185–194. 19. Bron AJ, Vrensen GF, Koretz J, Maraini G, Harding JJ. The ageing lens. Ophthalmologica Journal international d’ophtalmologie Int J Ophthalmol 2000;214:86–104. 20. Li G, Luna C, Navarro ID, Epstein DL, Huang W, Gonzalez P, et al. Resveratrol prevention of oxidative stress damage to lens epithelial cell cultures is mediated by forkhead box O activity. Invest Ophthalmol Vis Sci 2011;52:4395–4401. 21. Petersen A, Zetterberg M, Sjostrand J, Palsson AZ, Karlsson JO. Potential protective effects of NSAIDs/ASA in oxidatively stressed human lens epithelial cells and intact mouse lenses in culture. Ophthalmic Res 2005;37: 318–327. 22. Robertson JM, Donner AP, Trevithick JR. A possible role for vitamins C and E in cataract prevention. Am J Clin Nutr 1991;53:346–351. 23. Taylor A, Jacques PF, Chylack Jr LT, Hankinson SE, Khu PM, Rogers G, et al. Long-term intake of vitamins and carotenoids and odds of early age-related cortical and posterior subcapsular lens opacities. Am J Clin Nutr 2002; 75:540–549. 24. Hegde KR, Varma SD. Prevention of cataract by pyruvate in experimentally diabetic mice. Mol Cell Biochem 2005; 269:115–120.
25. Varma S, Kovtun S, Hegde K. Role of ultraviolet irradiation and oxidative stress in cataract formation-medical prevention by nutritional antioxidants and metabolic agonists. Eye Contact Lens 2011;37:233–245. 26. Szeto HH. Mitochondria-targeted cytoprotective peptides for ischemia-reperfusion injury. Antioxid Redox Signal 2008;10:601–619. 27. Zhao K, Luo G, Giannelli S, Szeto H. Mitochondriatargeted peptide prevents mitochondrial depolarization and apoptosis induced by tert-butyl hydroperoxide in neuronal cell lines. Biochem Pharmacol 2005;70: 1796–1806. 28. Zhao K, Zhao G, Wu D, Soong Y, Birk A, Schiller P, et al. Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J Biol Chem 2004;279:34682–34690. 29. Huang J, Li X, Li M, Li J, Xiao W, Ma W, et al. Mitochondria-targeted antioxidant peptide SS31 protects the retinas of diabetic rats. Curr Mol Med 2013;13: 935–945. 30. Haidara K, Morel I, Abalea V, Barre GM, Denizeau F. Mechanism of tert-butylhydroperoxide induced apoptosis in rat hepatocytes: involvement of mitochondria and endoplasmic reticulum. Biochim Biophys Acta 2002;1542: 173–185. 31. Piret JP, Arnould T, Fuks B, Chatelain P, Remacle J, Michiels C. Mitochondria permeability transitiondependent tert-butyl hydroperoxide-induced apoptosis in hepatoma HepG2 cells. Biochem Pharmacol 2004;67: 611–620. 32. Chen Z, Gibson TB, Robinson F, Silvestro L, Pearson G, Xu B, et al. MAP kinases. Chem Rev 2001;101:2449–2476. 33. Roux PP, Blenis J. ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 2004;68:320–344. 34. Zheng Y, Liu Y, Ge J, Wang X, Liu L, Bu Z, et al. Resveratrol protects human lens epithelial cells against H2O2-induced oxidative stress by increasing catalase, SOD-1, and HO-1 expression. Mol Vis 2010;16:1467–1474. 35. Jia Z, Song Z, Zhao Y, Wang X, Liu P. Grape seed proanthocyanidin extract protects human lens epithelial cells from oxidative stress via reducing NF-small ka, CyrillicB and MAPK protein expression. Mol Vis 2011;17: 210–217. 36. Cao M, Jiang J, Du Y, Yan P. Mitochondria-targeted antioxidant attenuates high glucose-induced P38 MAPK pathway activation in human neuroblastoma cells. Mol Med Rep 2012;5:929–934.
Current Eye Research
RESEARCH REPORT
Mitochondria-Targeted Antioxidant Peptide SS31 Protects Cultured Human Lens Epithelial Cells against Oxidative Stress
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Meng Cai, Jing Li, Shaofen Lin, Xiaoyun Chen, Juan Huang, Xiaoyan Jiang, Lizhu Yang and Yan Luo State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, P.R. China
ABSTRACT Purpose: To investigate the effects of the mitochondria-targeted peptide, SS31, on cultured human lens epithelial cells (HLECs) under conditions of oxidative stress. Methods: Optimal concentrations of SS31 were determined by MTT assay. HLEB-3 cells were first treated with SS31 for 2 h and then with 500 mM t-BHP for 6 h. Cell apoptosis was revealed by Annexin V/PI staining. Morphological changes in nuclei were observed by fluorescence microscopy after Hoechest 33258 fluorescent staining. Reactive oxygen species (ROS) were measured by MitoSOX staining. Changes in mitochondrial membrane potential (D m) were detected using the JC-1 fluorescent dye. Activation of p38 and c-Jun N-terminal kinases (JNKs) were quantified by Western Blot analysis. Results: SS31 protected HLEB-3 cells against t-BHP-induced cell apoptosis, reduced ROS, maintained D ms, and inhibited activation of JNK and p38 pathways. Conclusions: SS31 was able to protect HLEB-3 cells against oxidative damage and, thus, represents a potential new treatment modality for preventing the formation of cataracts and other age-related disorders. Keywords: Antioxidant, cataract, cell model, in vitro, SS31
INTRODUCTION
Furthermore, inadequate surgical facilities and the high cost of artificial IOLs represent major problems for many poor and developing countries.5 Moreover, there are numerous increasing social burdens imposed by the adverse effects of cataracts on visual function and quality of life of the elderly. Since there exist no effective drugs for the prevention and treatment of cataracts, development of effective preventative drugs that target the early phases of cataractogenesis is vital. Increased oxidative stress is caused by factors such as ultraviolet light (UV) light and hydrogen peroxide (H2O2), both of which are major risk factors for cataract development.6 Among the age-related changes that occur in the lens, decreased antioxidants
Age-related cataracts are the leading cause of loss of useful vision among elderly persons,1 affecting 46% of the 180 million visually disabled people worldwide.2 As result of the large increase in demographic aging, the prevalence of this age-related disorder is expected to double3 by the year 2020. At present, the only effective treatment for cataract is extraction of the cataractous lens, with implantation of an artificial intraocular lens (IOL).4 However, cataract surgery carries with it a number of post-operative complications, e.g. posterior capsular opacification, stimulation of chronic inflammation, uncorrectable residual refractive errors, and/or post-operative glare.
Received 27 November 2013; revised 20 July 2014; accepted 18 August 2014; published online 30 September 2014 Correspondence: Yan Luo, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, P.R. China. Tel: +86 020 87330485. Fax: +86 020 87333271. E-mail: [email protected].
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in the aging lens nucleus, along with increased protein modifications, may be causative factors in the etiogenesis of cataract. Lens epithelial cells, which are essential for the growth, differentiation and homeostasis of the lens, are the first to be effected by aqueous-associated aging changes.7 The decreased density of the lens epithelium observed in many species during normal aging8,9 renders the lens nucleus highly susceptible to oxidative damage. Lens epithelial cells play important roles in maintaining the oxidation resistance of the lens nucleus. Mitochondria, a major source of cellular reactive oxygen species (ROS), play crucial protective roles in oxidative stress.10,11 Therefore, procedures that protect the mitochondria via mitochondriatargeted protective mechanisms may be beneficial in the prevention and treatment not only of cataract but also other age-related lens disorders. SS31 (H-D-Arg-Dmt-Lys-Phe-NH2), fortuitously discovered as a novel mitochondria-targeted antioxidant peptide, easily penetrates cell membranes and targets mitochondria in a potential-independent manner.12 SS31 has been shown to lessen the accumulation of mitochondrial ROS in a dose-dependent manner and to prevent oxidative damage in several cell types.13,14 In this study, we exposed a human lens epithelial cell line (HLEB-3) to tert-butylhydroperoxide (t-BHP), organic peroxide used in a variety of oxidation processes, in order to mimic the oxidative damage that occurs in lens epithelial cells during cataractogenesis. We also investigated the protective effect(s) of SS31 against oxidative damage to HLEB-3 cells.
plus SS31 for 6 h and then incubated with 200 ml of 0.5 mg/ml MTT (Sigma-Aldrich, St. Louis, MO) at 37 C for 4 h. The MTT formazan product was dissolved in dimethylsulfoxide (DMSO). Absorbance was measured at 490 nm by a microplate reader (Bio-TEK Instruments, Winooski, VT). To obtain the percentage of surviving cells, the reading was divided by the adjusted absorbance reading of untreated cells in control wells. Three replications of each experiment were accomplished, and each experiment was repeated three times.
Apoptosis Assay The rate of apoptosis of HLEB-3 cells was evaluated using the Annexin V/propidium iodide (PI) double staining assay. Early apoptotic, late apoptotic, and necrotic cells were differentiated using the Annexin V-FLUO staining kit (Roche, Mannheim, Germany). Cells were placed in six-well plates at a density of 1 105 cells per well. Cells were pre-treated with 1 mM SS31 for 2 h and then treated with a combination of 500 mM t-BHP plus SS31 for 6 h. Cells were then collected and centrifuged at 1000g for 5 min at room temperature. After washing the centrifuged pellet with phosphate-buffered saline (PBS), cells were resuspended in 100 ml of Annexin V-FLUO labeling solution (2 ml Annexin V plus 2 ml PI in 100 ml incubation buffer) and incubated in the dark at room temperature for 15 min. An additional 400 ml buffer was then added to each tube and samples were analyzed by flow cytometry (BD-FACS AriaTM Flow Cytometer, BD Biosciences, San Jose, CA).
MATERIALS AND METHODS Nuclear Morphology Cells and Cell Culture HLEB-3 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). HLEB-3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY), supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37 C in a humidified atmosphere of 5% CO2. Cells were used for assays when they attained 75–80% confluence.
Apoptosis of treated HLEB-3 cells was determined according to morphological changes in cell nuclei using Hoechst 33258 staining (Sigma). HLEB-3 cells were seeded on 18 18 mm coverslips, pre-treated with 1 mM SS31 for 2 h, and then treated with a combination of 500 mM t-BHP plus SS31 for 6 h. Cells were then washed twice in PBS and incubated with Hochest 33258 (10 mg/mL) for 10 min. Coverslips were mounted on glass microscope slides with glycerol and immediately viewed under a fluorescence microscope (Leica, Solms, Germany).
MTT Assay Proliferation and cell viability of HLEB-3 cells were evaluated by the MTT assay. Cells were plated in 96-well plates at a density of 1 104 cells/well. After pre-treatment with varying concentrations (10, 100, 1, 10, and 100 mM, and 1 M) of SS31 (Stealth Peptides International Inc., Shanghai, P.R. China) for 2 h, cells were treated with a combination of 500 mM t-BHP
Measurement of D)m Changes in t-BHP-induced mitochondrial membrane potential (D m) were evaluated with the JC-1 fluorescent probe (Molecular Probes, Eugene, OR). HLEB-3 cells, at a density of 1 105 cells/well in six-well plates, were pre-treated with SS31 for 2 h and then Current Eye Research
SS31 protects lens epithelial cells incubated with 500 mM t-BHP for 6 h. Cells were centrifuged at 1000g for 5 min, and the pellets harvested. After washing with PBS, cells were stained with 2.5 mg/ml JC-1 at 37 C for 10 min and analyzed immediately by flow cytometry. D m was expressed as the ratio of mean red fluorescence (JC-1 aggregates) to mean green fluorescence (JC-1 monomers).
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Detection of ROS Production of ROS was evaluated with the MitoSOXTM red superoxide mitochondrial indicator, which is oxidized using superoxide and which exhibits red fluorescence in the mitochondria. Coverslip-seeded cells were pre-treated with SS31 for 2 h and then incubated with 500 mM t-BHP for 6 h. Following this, the contents (50 mg) of one vial of MitoSOXTM was dissolved in 13 ml of DMSO to make a 5 mM MitoSOXTM stock solution. After exposure to 5 mM of MitoSOXTM working solution, live HLEB-3 cells were incubated at 37 C for 10 min. Following a gentle rinse with warm buffer, cells were viewed by fluorescence microscopy.
Western Blot After washing with ice-cold PBS, cells were lysed in 100 ml of lysis buffer (BD Biosciences) combined with a protease inhibitor cocktail (Calbiochem, La Jolla, CA) to extract total protein. Protein samples were mixed with 3 sodium dodecyl sulfate (SDS) sample buffer, separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE) gels, and then transferred to polyvinylidene difluoride membranes. Membranes were blocked in 5% bovine serum albumin (BSA) for 1 h and incubated overnight at 4 C with various primary antibodies. Anti-cJun NH2-terminal kinase (JNK), anti-phosphorylated JNK (p-JNK), anti-p38, and anti-phosphorylated p38 (p-p38) (Thr180/Tyr182) antibodies were purchased from Cell Signaling Technology (Danvers, MA). After washing with 0.1% Tween 20-containing PBS, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Bands were visualized using an enhanced chemiluminescence (ECL) detection system (Cell Signaling Technology). Densitometric analysis was conducted using Version 1.41 Image J software (National Institutes of Health, Bethesda, MD).
Statistical Analysis All data are expressed as mean ± SD. Statistical analyses were performed using Version 16.0 SPSS software (SPSS, Chicago, IL). A two-sample t-test !
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was used to compare normally distributed data. Three independent experiments were performed. A value of p50.05 was considered statistically significant.
RESULTS SS31 Protection of HLEB-3 Cells against t-BHP-Induced Oxidative Injury To determine whether SS31 protects HLEB-3 cells against t-BHP-induced oxidative damage, cell viability and cell apoptosis were examined 6 h after 500 mM t-BHP treatment alone or after co-treatment with SS31 at various concentrations. Incubation with 500 mM t-BHP for 6 h resulted in a significantly low ratio of cell survival. Compared with the t-BHP group, SS31 prevented loss of cell viability in a dosedependent manner (p50.01). The maximum effect of SS31 was observed at a concentration of 1 mM (Figure 1). Consequently, concentrations of 1 mM SS31 were used for all experiments. Annexin V/PI staining was used to determine whether SS31 inhibits t-BHP-induced apoptosis (Figure 2A). The percentages of surviving HLEB-3 cells (Q3) were 79.24 ± 6.08, 98.64 ± 0.24, and 96.03 ± 1.02% in the t-BHP-treated, control, and SS31treated groups, respectively. SS31 caused a decrease in cellular apoptosis and increased cell survival than t-BHP (p50.01), indicating its protective effects against oxidative stress.
FIGURE 1. SS31 protected HLEB-3 cells from t-BHP-induced oxidative injury. 500 mM t-BHP was added to HLEB-3 cells and incubated with various concentrations (10, 100, 1, and10 mM, and 100 mM) of SS31 for 6 h. Cell viability was measured by MTT assay. The survival ratio of HLEB-3 indicates a concentration-dependency in cell viability and is markedly increased under the protection of 1 mM SS31. Data are shown as mean ± standard deviation (n = 3), *p50.01 versus t-BHP.
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FIGURE 2. SS31 significantly decreased apoptosis of HLEB-3 cells. Cells were pre-treated with 1 mM SS31 for 2 h and then treated with a combination of 500 mM t-BHP plus SS31 for 6 h. (A) Cell apoptosis was analyzed by flow cytometry after Annexin V/PI staining. The percentage of surviving HLEB-3 cells (Q3) was much higher in cells of the SS31-treated group than in those of the t-BHP-treated group. (B) When HLEB-3 cells were exposed to 500 mM t-BHP for 6 h, nuclear density decreased and the number of apoptotic cells (arrows) with nuclear fragmentation and condensed chromatin increased. There were fewer cells with apoptotic nuclei in the SS31-treated group.
Hoechst staining showed apoptotic cells with condensed nuclear chromatin and multiple chromatin fragments. As shown in Figure 2(B), cells exposed to 500 mM t-BHP for 6 h demonstrated obvious apoptotic features, including condensed chromatin and multiple chromatin fragments. In contrast, cell nuclei in the control group showed no apoptotic morphology. SS31 treatment in the t-BHP-treated group showed a decrease in apoptotic cells (p50.01), indicating that SS31 protected the HLEB-3 cells against oxidative damage.
SS31-Induced Prevention of Loss of D)m in t-BHP-Treated HLEB-3 Cells To determine if mitochondria are involved in the protective effects of SS31 on t-BHP-induced cell death, the loss of D m in HLEB-3 cells was examined using the JC-1 fluorescent probe. A rapid loss of D m in t-BHP-treated HLEB-3 cells was prevented by SS31 (Figures 3A and B, p50.01). These data suggest that SS31 prevents the loss of D m caused by t-BHP in HLEB-3 cells. Current Eye Research
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SS31 protects lens epithelial cells
FIGURE 3. SS31 prevented loss of D m and decreased ROS production induced by t-BHP in HLEB-3 cells. (A) The D m of HLEB-3 cells was measured by flow cytometry after JC-1 staining. Exposure to 500 mM t-BHP for 6 h resulted in a rapid loss of D m in HLEB-3 cells, while, SS31 inhibited the loss of D m induced by t-BHP. (B) Quantitative analysis of D m in HLEB-3 cells is shown. (C) ROS production was examined under by fluorescence microscopy. Leves of mitochondrial ROS in the control group was very low. ROS production significantly increased 6 h after t-BHP treatment. SS31 treatment decreased t-BHP-induced ROS production in mitochondria. Data are shown as mean ± standard deviation (n = 3), *p50.01 versus t-BHP.
SS31-Induced Reduction of ROS in t-BHP-Treated HLEB-3 Cells t-BHP-induction of ROS was examined by staining with MitoSOXTM Red fluorescent stain. As illustrated !
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by fluorescence microcopy, incubation with 500 mM t-BHP for 6 h resulted in a significant increase in ROS production. However, ROS production was remarkably reduced in the SS31-treated group (Figure 3C, p50.01).
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SS31-Inhibition of JNK and p38 Activation in t-BHP-Treated HLEB-3 Cells
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To examine whether the JNK and p38 pathways contribute to the protective effects of SS31, Western blot analysis was performed. Protein levels of p-JNK and p-p38 in cells of the t-BHP-treated group were significantly greater than those of control group cells, while the total protein levels of JNK and p38 remained unchanged in all groups. Protein levels of p-JNK and p-p38 were much lower in the SS31-treated group than in the t-BHP-treated group (p50.01). Thus, SS31 inhibits activation of JNK and p38 in t-BHP-treated HLEB-3 cells.
DISCUSSION An understanding of the physiological and biochemical changes that occur in cataract formation is constantly being sought. The lens epithelium, which
covers the anterior face of the lens, protects the underlying lens fibers from oxidative injury, as well as transports materials into and out of the deeper layers of the lens.15,16 Lens epithelial cells also play important roles in maintaining the oxidation resistance of the lens. Loss of antioxidants in the lens triggers proteins into undergoing non-enzymatic, post-translational modifications, thus increasing susceptibility of the lens to oxidation. Progressive insolublization of lens crystallins, as well as the formation of high molecular weight aggregates, disturb the short-range order of the lens crystallins, thus leading to increased light-scatter and loss of the transparency.17–19 Since oxidative damage plays a pivotal role in age-related lens alterations, targeting oxidative damage to the lens epithelium offers a potentially significant focus for research in the treatment of cataract. There are a considerable number of in vitro and in vivo studies on cataract-preventing antioxidants. Several herbal drugs and non-steroidal anti-inflammatory drugs with antioxidant properties may be of
FIGURE 4. SS31 inhibited t-BHP-induced activation of the MAPK pathways. (A) Protein levels of JNK, p38, and p-JNK plus p-p38 were measured by Western Blot. When HLEB-3 cells were exposed to 500 mM t-BHP, expression levels of p-JNK and p-p38 increased dramatically. There were significantly decreased levels of p-JNK and p-p38 in the SS31-treated group. (B) Quantitative analysis of expression levels of p-JNK and p-p38 in HLEB-3 cells. Data are shown as mean ± standard deviation (n = 3), *p50.01 versus t-BHP. Current Eye Research
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SS31 protects lens epithelial cells benefit to patients with cataracts. Resveratrol20 and low concentrations of acetylsalicylic acid, aspirin21 protect HLECs against peroxide-induced oxidative damage. In addition, many nutritional or metabolic oxyradical scavengers, e.g. ascorbate22, vitamin E23, pyruvate24, and caffeine appear to attenuate oxidative damage during cataract development.25 However, most efforts to prevent cataracts do not provide significant clinical benefits. Future studies are needed to develop new drugs that prevent the early onset of cataractogenesis. The structure of SS31, a small aromatic-cationic mitochondria-targeted peptide, is highly suitable for penetrating membranes, targeting mitochondria, and reducing generation of mitochondrial ROS.26–28 Our previous study indicated that SS31 can effectively attenuate oxidative injury in human retinal endothelial cells13 and in streptozotocin (STZ)-induced diabetic rats.29 Results of these studies suggest SS31 to be an ideal therapeutic candidate for the treatment of age-related lens disorders. t-BHP, a cell permeable oxidant compound, is more stable than H2O2 in vitro, and is used to induce in vitro models of oxidative damage.30,31 To mimic the oxidative stress that occurs in lens epithelial cells during cataractogenesis, we used t-BHP-treated HLEB-3 cells, which exhibited a high rate of apoptosis, rapid loss of D m, and increased levels of intracellular ROS. SS31 was shown to effectively preserve cell viability of these HLEB-3 cells, prevent the loss of D m, and inhibit production of t-BHP-induced mitochondrial ROS. To investigate the mechanisms by which SS31 exerts it protective effects against oxidative stress in HLEB-3 cells, several potentially involved pathways were examined. Mitogen-activated protein kinase (MAPK) signaling pathways, important upstream regulators of transcriptional factor activities, comprise a phosphorylation-dependent relay of protein activation pathways. MAPK signaling pathways alter transcription and, ultimately, regulate cell proliferation and differentiation.32,33 JNK and p38 cascades are involved in MAPK signaling pathways, and activation of both JNK and p38 are important participants in cell death. These two MAPK signaling pathways are expressed in the mammalian lens, with primary activities in the epithelial layer, and are activated by a variety of environmental stresses, inflammatory cytokines, UV radiation, and growth factors. Grape seed pro-anthocyanidin extract and resveratrol have been reported to reduce oxidative stress in lens epithelial cells via inhibition of the JNK and p38 signaling cascades.34,35 SS31 has also been reported to attenuate high glucose-induced p38 MAPK pathway activation in human neuroblastoma cells.36 Therefore, whether or not t-BHP-induced JNK and p38 pathways in the lens epithelium are inhibited by SS31 was investigated in this study. Our results indicate that t-BHP-induced activation of p38 and !
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JNK in HLEB-3 cells was significantly suppressed when cells were treated with SS31, i.e. SS31 inhibited JNK and p38 pathway-mediated cell death in HLEB-3 cells. Although SS31 was shown to have protective effects on HLEB-3 cells against oxidative damage, its protective effects on the ex vivo and in vivo whole lens are still unclear. Questions that need to be addressed in future studies include (i) the capability of SS31 to penetrate the lens capsule, (ii) the effects of SS31 on cultured lens, and (iii) the effects of SS31 in in vivo animal experiments. Overall, SS31 attenuates t-BHP-induced oxidative damages to HLEB-3 cells by stabilizing the D m, decreasing ROS production, and inhibiting activation of JNK and p38 pathways, and thus represents a potentially viable modality for prevention/treatment of cataract and other oxidative stress-related diseases.
DECLARATION OF INTEREST There are no conflicts of interest to declare for any of the authors. This work was supported by grants from National Basic Research Development Program of China (973 program: 2013CB967000) and the National Natural Science Foundation of China to YAN LUO (81170864).
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25. Varma S, Kovtun S, Hegde K. Role of ultraviolet irradiation and oxidative stress in cataract formation-medical prevention by nutritional antioxidants and metabolic agonists. Eye Contact Lens 2011;37:233–245. 26. Szeto HH. Mitochondria-targeted cytoprotective peptides for ischemia-reperfusion injury. Antioxid Redox Signal 2008;10:601–619. 27. Zhao K, Luo G, Giannelli S, Szeto H. Mitochondriatargeted peptide prevents mitochondrial depolarization and apoptosis induced by tert-butyl hydroperoxide in neuronal cell lines. Biochem Pharmacol 2005;70: 1796–1806. 28. Zhao K, Zhao G, Wu D, Soong Y, Birk A, Schiller P, et al. Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J Biol Chem 2004;279:34682–34690. 29. Huang J, Li X, Li M, Li J, Xiao W, Ma W, et al. Mitochondria-targeted antioxidant peptide SS31 protects the retinas of diabetic rats. Curr Mol Med 2013;13: 935–945. 30. Haidara K, Morel I, Abalea V, Barre GM, Denizeau F. Mechanism of tert-butylhydroperoxide induced apoptosis in rat hepatocytes: involvement of mitochondria and endoplasmic reticulum. Biochim Biophys Acta 2002;1542: 173–185. 31. Piret JP, Arnould T, Fuks B, Chatelain P, Remacle J, Michiels C. Mitochondria permeability transitiondependent tert-butyl hydroperoxide-induced apoptosis in hepatoma HepG2 cells. Biochem Pharmacol 2004;67: 611–620. 32. Chen Z, Gibson TB, Robinson F, Silvestro L, Pearson G, Xu B, et al. MAP kinases. Chem Rev 2001;101:2449–2476. 33. Roux PP, Blenis J. ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 2004;68:320–344. 34. Zheng Y, Liu Y, Ge J, Wang X, Liu L, Bu Z, et al. Resveratrol protects human lens epithelial cells against H2O2-induced oxidative stress by increasing catalase, SOD-1, and HO-1 expression. Mol Vis 2010;16:1467–1474. 35. Jia Z, Song Z, Zhao Y, Wang X, Liu P. Grape seed proanthocyanidin extract protects human lens epithelial cells from oxidative stress via reducing NF-small ka, CyrillicB and MAPK protein expression. Mol Vis 2011;17: 210–217. 36. Cao M, Jiang J, Du Y, Yan P. Mitochondria-targeted antioxidant attenuates high glucose-induced P38 MAPK pathway activation in human neuroblastoma cells. Mol Med Rep 2012;5:929–934.
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