Topical Ascorbic Acid Ameliorates Oxidative Stress-Induced Corneal Endothelial Damage via Suppression of Apoptosis and Autophagic Flux Blockage

Yi-Jen Hsueh, Yaa-Jyuhn James Meir, Lung-Kun Yeh, Tze-Kai Wang, Chieh-Cheng Huang, Tsai-Te Lu, Chao-Min Cheng, Wei-Chi Wu, Hung-Chi Chen, Yi-Jen Hsueh, Yaa-Jyuhn James Meir, Lung-Kun Yeh, Tze-Kai Wang, Chieh-Cheng Huang, Tsai-Te Lu, Chao-Min Cheng, Wei-Chi Wu, Hung-Chi Chen

Abstract

Compromised pumping function of the corneal endothelium, due to loss of endothelial cells, results in corneal edema and subsequent visual problems. Clinically and experimentally, oxidative stress may cause corneal endothelial decompensation after phacoemulsification. Additionally, in vitro and animal studies have demonstrated the protective effects of intraoperative infusion of ascorbic acid (AA). Here, we established a paraquat-induced cell damage model, in which paraquat induced reactive oxygen species (ROS) production and apoptosis in the B4G12 and ARPE-19 cell lines. We demonstrate that oxidative stress triggered autophagic flux blockage in corneal endothelial cells and that addition of AA ameliorated such oxidative damage. We also demonstrate the downregulation of Akt phosphorylation in response to oxidative stress. Pretreatment with ascorbic acid reduced the downregulation of Akt phosphorylation, while inhibition of the PI3K/Akt pathway attenuated the protective effects of AA. Further, we establish an in vivo rabbit model of corneal endothelial damage, in which an intracameral infusion of paraquat caused corneal opacity. Administration of AA via topical application increased its concentration in the corneal stroma and reduced oxidative stress in the corneal endothelium, thereby promoting corneal clarity. Our findings indicate a perioperative strategy of topical AA administration to prevent oxidative stress-induced damage, particularly for those with vulnerable corneal endothelia.

Keywords: PI3K/Akt; apoptosis; ascorbic acid; autophagic flux blockage; corneal endothelial cells; oxidative stress.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Oxidative stress induces apoptosis and autophagosome formation in B4G12 and ARPE-19 cells. The HCEC cell line (B4G12) and the RPE cell line (ARPE-19) were treated with paraquat (an oxidative stress inducer, 2 mM for ARPE-19, 0.2 mM for B4G12) for five days. Cell morphology was observed using phase-contrast microscopy. TUNEL assay and immunofluorescence of LC3 were performed to examine apoptosis and autophagosome formation (green color). Nuclei were counterstained with Hoechst 33342 (blue color). The scale bars represent 100 µm.
Figure 2
Figure 2
Ascorbic acid protects B4G12 and ARPE-19 cells from oxidative stress. B4G12 and ARPE-19 were pretreated with ascorbic acid (AA) for two days and further treated with paraquat (2 mM for ARPE-19, 0.2 mM for B4G12) for five days. (A) The time-dependent protective effect of ascorbic acid (1 mM) on B4G12 and ARPE-19 cells was examined on days 0–5. (B) The dose-dependent protective effect of ascorbic acid (0 to 2 mM) on B4G12 and ARPE-19 cells was examined on day five. Cell morphology was observed using phase-contrast microscopy, and images were captured at the same spot on different days. The scale bar represents 100 µm.
Figure 3
Figure 3
Pretreatment with ascorbic acid attenuates oxidative stress-induced apoptosis and autophagic flux blockage in B4G12 and ARPE-19 cells. B4G12 and ARPE-19 cells were cultivated in medium with or without 1 mM of ascorbic acid for two days, followed by the addition of paraquat (2 mM for ARPE-19 and 0.2 mM for B4G12) in the paraquat-treated groups (P only and C + P groups) for five days. (A) Paraquat-induced cellular accumulation of reactive oxygen species (ROS) and rescue by ascorbic acid was detected using ROS fluorescent dye (red color). ROS was induced by paraquat in both the B4G12 and ARPE-19 cells, and this was ameliorated by treatment with ascorbic acid. (B) Paraquat-induced apoptosis and rescue by ascorbic acid was examined using the TUNEL assay (green). (C) Paraquat-induced autophagosome formation and rescue by ascorbic acid was examined using immunofluorescence staining for LC3-II (autophagosome formation biomarker; green). (D) Effects of the paraquat and ascorbic on the protein expression of anti-apoptosis (Bcl-2), pro-apoptosis (lamin A, including cleaved forms), and autophagic flux (LC3 I/II and p62) biomarkers in B4G12 and ARPE-19 cells were probed using Western blotting. Paraquat induced altered protein expression in both B4G12 and ARPE-19 cells, and this could be reversed by ascorbic acid. The scale bars represent 100 µm (A) and 50 µm (B–C). Nuclei were counterstained with Hoechst 33342 (blue; B–D). (n = 3, * p < 0.05, ** p < 0.01).
Figure 3
Figure 3
Pretreatment with ascorbic acid attenuates oxidative stress-induced apoptosis and autophagic flux blockage in B4G12 and ARPE-19 cells. B4G12 and ARPE-19 cells were cultivated in medium with or without 1 mM of ascorbic acid for two days, followed by the addition of paraquat (2 mM for ARPE-19 and 0.2 mM for B4G12) in the paraquat-treated groups (P only and C + P groups) for five days. (A) Paraquat-induced cellular accumulation of reactive oxygen species (ROS) and rescue by ascorbic acid was detected using ROS fluorescent dye (red color). ROS was induced by paraquat in both the B4G12 and ARPE-19 cells, and this was ameliorated by treatment with ascorbic acid. (B) Paraquat-induced apoptosis and rescue by ascorbic acid was examined using the TUNEL assay (green). (C) Paraquat-induced autophagosome formation and rescue by ascorbic acid was examined using immunofluorescence staining for LC3-II (autophagosome formation biomarker; green). (D) Effects of the paraquat and ascorbic on the protein expression of anti-apoptosis (Bcl-2), pro-apoptosis (lamin A, including cleaved forms), and autophagic flux (LC3 I/II and p62) biomarkers in B4G12 and ARPE-19 cells were probed using Western blotting. Paraquat induced altered protein expression in both B4G12 and ARPE-19 cells, and this could be reversed by ascorbic acid. The scale bars represent 100 µm (A) and 50 µm (B–C). Nuclei were counterstained with Hoechst 33342 (blue; B–D). (n = 3, * p < 0.05, ** p < 0.01).
Figure 3
Figure 3
Pretreatment with ascorbic acid attenuates oxidative stress-induced apoptosis and autophagic flux blockage in B4G12 and ARPE-19 cells. B4G12 and ARPE-19 cells were cultivated in medium with or without 1 mM of ascorbic acid for two days, followed by the addition of paraquat (2 mM for ARPE-19 and 0.2 mM for B4G12) in the paraquat-treated groups (P only and C + P groups) for five days. (A) Paraquat-induced cellular accumulation of reactive oxygen species (ROS) and rescue by ascorbic acid was detected using ROS fluorescent dye (red color). ROS was induced by paraquat in both the B4G12 and ARPE-19 cells, and this was ameliorated by treatment with ascorbic acid. (B) Paraquat-induced apoptosis and rescue by ascorbic acid was examined using the TUNEL assay (green). (C) Paraquat-induced autophagosome formation and rescue by ascorbic acid was examined using immunofluorescence staining for LC3-II (autophagosome formation biomarker; green). (D) Effects of the paraquat and ascorbic on the protein expression of anti-apoptosis (Bcl-2), pro-apoptosis (lamin A, including cleaved forms), and autophagic flux (LC3 I/II and p62) biomarkers in B4G12 and ARPE-19 cells were probed using Western blotting. Paraquat induced altered protein expression in both B4G12 and ARPE-19 cells, and this could be reversed by ascorbic acid. The scale bars represent 100 µm (A) and 50 µm (B–C). Nuclei were counterstained with Hoechst 33342 (blue; B–D). (n = 3, * p < 0.05, ** p < 0.01).
Figure 3
Figure 3
Pretreatment with ascorbic acid attenuates oxidative stress-induced apoptosis and autophagic flux blockage in B4G12 and ARPE-19 cells. B4G12 and ARPE-19 cells were cultivated in medium with or without 1 mM of ascorbic acid for two days, followed by the addition of paraquat (2 mM for ARPE-19 and 0.2 mM for B4G12) in the paraquat-treated groups (P only and C + P groups) for five days. (A) Paraquat-induced cellular accumulation of reactive oxygen species (ROS) and rescue by ascorbic acid was detected using ROS fluorescent dye (red color). ROS was induced by paraquat in both the B4G12 and ARPE-19 cells, and this was ameliorated by treatment with ascorbic acid. (B) Paraquat-induced apoptosis and rescue by ascorbic acid was examined using the TUNEL assay (green). (C) Paraquat-induced autophagosome formation and rescue by ascorbic acid was examined using immunofluorescence staining for LC3-II (autophagosome formation biomarker; green). (D) Effects of the paraquat and ascorbic on the protein expression of anti-apoptosis (Bcl-2), pro-apoptosis (lamin A, including cleaved forms), and autophagic flux (LC3 I/II and p62) biomarkers in B4G12 and ARPE-19 cells were probed using Western blotting. Paraquat induced altered protein expression in both B4G12 and ARPE-19 cells, and this could be reversed by ascorbic acid. The scale bars represent 100 µm (A) and 50 µm (B–C). Nuclei were counterstained with Hoechst 33342 (blue; B–D). (n = 3, * p < 0.05, ** p < 0.01).
Figure 3
Figure 3
Pretreatment with ascorbic acid attenuates oxidative stress-induced apoptosis and autophagic flux blockage in B4G12 and ARPE-19 cells. B4G12 and ARPE-19 cells were cultivated in medium with or without 1 mM of ascorbic acid for two days, followed by the addition of paraquat (2 mM for ARPE-19 and 0.2 mM for B4G12) in the paraquat-treated groups (P only and C + P groups) for five days. (A) Paraquat-induced cellular accumulation of reactive oxygen species (ROS) and rescue by ascorbic acid was detected using ROS fluorescent dye (red color). ROS was induced by paraquat in both the B4G12 and ARPE-19 cells, and this was ameliorated by treatment with ascorbic acid. (B) Paraquat-induced apoptosis and rescue by ascorbic acid was examined using the TUNEL assay (green). (C) Paraquat-induced autophagosome formation and rescue by ascorbic acid was examined using immunofluorescence staining for LC3-II (autophagosome formation biomarker; green). (D) Effects of the paraquat and ascorbic on the protein expression of anti-apoptosis (Bcl-2), pro-apoptosis (lamin A, including cleaved forms), and autophagic flux (LC3 I/II and p62) biomarkers in B4G12 and ARPE-19 cells were probed using Western blotting. Paraquat induced altered protein expression in both B4G12 and ARPE-19 cells, and this could be reversed by ascorbic acid. The scale bars represent 100 µm (A) and 50 µm (B–C). Nuclei were counterstained with Hoechst 33342 (blue; B–D). (n = 3, * p < 0.05, ** p < 0.01).
Figure 4
Figure 4
The PI3K/AKT pathway is involved in ascorbic acid-mediated cell protection. (A) Phosphorylation of Akt (Bcl-2 upstream regulator) was detected by Western blotting. Total Akt and β-actin were used as loading controls. Paraquat-suppressed Akt phosphorylation was rescued by ascorbic acid. (B) To examine whether the PI3K/AKT pathway was involved in ascorbic acid-mediated cell protection, LY294002 (a PI3K inhibitor, 50 μmol/L) was added in the culture medium. The cell protection effect was quantified by cell counting. Paraquat-induced cell loss was rescued by pretreatment with ascorbic acid, while the rescue effect of cell loss by ascorbic acid was significantly negated by the addition of LY294002. (n = 3, * p < 0.05, ** p < 0.01).
Figure 4
Figure 4
The PI3K/AKT pathway is involved in ascorbic acid-mediated cell protection. (A) Phosphorylation of Akt (Bcl-2 upstream regulator) was detected by Western blotting. Total Akt and β-actin were used as loading controls. Paraquat-suppressed Akt phosphorylation was rescued by ascorbic acid. (B) To examine whether the PI3K/AKT pathway was involved in ascorbic acid-mediated cell protection, LY294002 (a PI3K inhibitor, 50 μmol/L) was added in the culture medium. The cell protection effect was quantified by cell counting. Paraquat-induced cell loss was rescued by pretreatment with ascorbic acid, while the rescue effect of cell loss by ascorbic acid was significantly negated by the addition of LY294002. (n = 3, * p < 0.05, ** p < 0.01).
Figure 5
Figure 5
Effect of topical ascorbic acid on oxidative stress-induced corneal endothelial damage in a rabbit model. (A) Primary rabbit corneal endothelial cells (in vitro) were cultivated in medium with (A and A + P groups) or without (C and P groups) addition of 1 mM of ascorbic acid for two days, followed by addition of paraquat (0.2 mM) in the paraquat-treated groups (P and A + P groups) for five days. Sloughing of corneal endothelial cells was observed under phase-contrast microscopy. (B) Rabbit corneal tissue specimens (ex vivo) were cultivated in medium with (A and A + P groups) or without 1 mM of ascorbic acid for two days, followed by the addition of paraquat (25 mM) to the P and A + P groups for 15 min. Two days later, the sloughing of corneal endothelial cells was observed using Calcein-AM stain. (C) Rabbit corneas (in vivo) received an application of ascorbic acid (284 mmol/L in BSS solution) or BSS alone for two days (three times per day). After diffusion, ascorbic acid concentrations in the corneal stroma and anterior chambers were examined using the FRASC assay. (n = 3, ** p < 0.01). (D) Rabbit corneas (in vivo) received an application of ascorbic acid (284 mmol/L in BSS solution) or BSS alone for two days (three times per day), followed by intracameral injection of 25 mM paraquat (diluted in BSS) or BSS alone for 15 min. Corneal transparency was assessed using external eye photography on day two. (The scale bar represents 100 µm).

References

    1. Kim S.J., Schoenberger S.D., Thorne J.E., Ehlers J.P., Yeh S., Bakri S.J. Topical Nonsteroidal Anti-inflammatory Drugs and Cataract Surgery. Ophthalmology. 2015;122:2159–2168. doi: 10.1016/j.ophtha.2015.05.014.
    1. Pathengay A., Flynn H.W., Jr., Isom R.F., Miller D. Endophthalmitis outbreaks following cataract surgery: Causative organisms, etiologies, and visual acuity outcomes. J. Cataract. Refract. Surg. 2012;38:1278–1282. doi: 10.1016/j.jcrs.2012.04.021.
    1. Lois N., Wong D. Pseudophakic retinal detachment. Surv. Ophthalmol. 2003;48:467–487. doi: 10.1016/S0039-6257(03)00083-3.
    1. Rosado-Adames N., Afshari N.A. The changing fate of the corneal endothelium in cataract surgery. Curr. Opin. Ophthalmol. 2012;23:3–6. doi: 10.1097/ICU.0b013e32834e4b5f.
    1. Bourne W.M., Nelson L.R., Hodge D.O. Continued Endothelial Cell Loss Ten Years after Lens Implantation. Ophthalmology. 1994;101:1014–1023. doi: 10.1016/S0161-6420(94)31224-3.
    1. Hayashi K., Yoshida M., Manabe S.-I., Hirata A. Cataract surgery in eyes with low corneal endothelial cell density. J. Cataract. Refract. Surg. 2011;37:1419–1425. doi: 10.1016/j.jcrs.2011.02.025.
    1. Yamazoe K., Yamaguchi T., Hotta K., Satake Y., Konomi K., Den S., Shimazaki J. Outcomes of cataract surgery in eyes with a low corneal endothelial cell density. J. Cataract. Refract. Surg. 2011;37:2130–2136. doi: 10.1016/j.jcrs.2011.05.039.
    1. Waring G.O., Bourne W.M., Edelhauser H.F., Kenyon K.R. The Corneal Endothelium. Ophthalmology. 1982;89:531–590. doi: 10.1016/S0161-6420(82)34746-6.
    1. Mishima S. Clinical Investigations on the Corneal Endothelium. Ophthalmology. 1982;89:525–530. doi: 10.1016/S0161-6420(82)34755-7.
    1. Laing R.A., Neubauer L., Oak S.S., Kayne H.L., Leibowitz H.M. Evidence for Mitosis in the Adult Corneal Endothelium. Ophthalmology. 1984;91:1129–1134. doi: 10.1016/S0161-6420(84)34176-8.
    1. Zhu Y.-T., Chen H.-C., Chen S.-Y., Tseng S.C.G. Nuclear p120 catenin unlocks mitotic block of contact-inhibited human corneal endothelial monolayers without disrupting adherent junctions. J. Cell Sci. 2012;125:3636–3648. doi: 10.1242/jcs.103267.
    1. Hsueh Y.-J., Chen H.-C., Wu S.-E., Wang T.-K., Chen J.-K., Ma D.H.-K. Lysophosphatidic acid induces YAP-promoted proliferation of human corneal endothelial cells via PI3K and ROCK pathways. Mol. Ther. Methods Clin. Dev. 2015;2:15014. doi: 10.1038/mtm.2015.14.
    1. Augustin A.J., Dick B.H., Augustin A.J. Oxidative tissue damage after phacoemulsification. J. Cataract. Refract. Surg. 2004;30:424–427. doi: 10.1016/S0886-3350(03)00577-7.
    1. Nemet A.Y., Assia E.I., Meyerstein D., Meyerstein N., Gedanken A., Topaz M. Protective effect of free-radical scavengers on corneal endothelial damage in phacoemulsification. J. Cataract. Refract. Surg. 2007;33:310–315. doi: 10.1016/j.jcrs.2006.10.031.
    1. Murano N., Ishizaki M., Sato S., Fukuda Y., Takahashi H. Corneal Endothelial Cell Damage by Free Radicals Associated With Ultrasound Oscillation. Arch. Ophthalmol. 2008;126:816–821. doi: 10.1001/archopht.126.6.816.
    1. Geffen N., Topaz M., Kredy-Farhan L., Barequet I.S., Farzam N., Assia E.I., Savion N. Phacoemulsification-induced injury in corneal endothelial cells mediated by apoptosis: In vitro model. J. Cataract. Refract. Surg. 2008;34:2146–2152. doi: 10.1016/j.jcrs.2008.08.024.
    1. Nishi Y., Engler C., Na D.R., Kashiwabuchi R.T., Shin Y.J., Cano M., Jun A.S., Chuck R.S. Evaluation of phacoemulsification-induced oxidative stress and damage of cultured human corneal endothelial cells in different solutions using redox fluorometry microscopy. Acta Ophthalmol. 2010;88:e323–e327. doi: 10.1111/j.1755-3768.2010.02024.x.
    1. Lai L.J., Chen Y.F., Wu S., Tsao Y.P., Tsai R.J. Endothelial cell loss induced by phacoemulsification occurs through apoptosis. Chang. Gung Med. J. 2001;24:621–627.
    1. Filomeni G., De Zio D., Cecconi F. Oxidative stress and autophagy: The clash between damage and metabolic needs. Cell Death Differ. 2014;22:377–388. doi: 10.1038/cdd.2014.150.
    1. Tangvarasittichai O., Tangvarasittichai S. Oxidative Stress, Ocular Disease and Diabetes Retinopathy. Curr. Pharm. Des. 2019;24:4726–4741. doi: 10.2174/1381612825666190115121531.
    1. Navarro-Yepes J., Burns M., Anandhan A., Khalimonchuk O., Del Razo L.M., Quintanilla-Vega B., Pappa A., Panayiotidis M.I., Franco R. Oxidative Stress, Redox Signaling, and Autophagy: Cell DeathVersusSurvival. Antioxid. Redox Signal. 2014;21:66–85. doi: 10.1089/ars.2014.5837.
    1. Bartlett H.E., Eperjesi F. An ideal ocular nutritional supplement? Ophthalmic Physiol. Opt. 2004;24:339–349. doi: 10.1111/j.1475-1313.2004.00218.x.
    1. Serbecic N., Beutelspacher S.C. Vitamins Inhibit Oxidant-Induced Apoptosis of Corneal Endothelial Cells. Jpn. J. Ophthalmol. 2005;49:355–362. doi: 10.1007/s10384-005-0209-9.
    1. Serbecic N., Beutelspacher S.C. Anti-oxidative vitamins prevent lipid-peroxidation and apoptosis in corneal endothelial cells. Cell Tissue Res. 2005;320:465–475. doi: 10.1007/s00441-004-1030-3.
    1. Shima N., Kimoto M., Yamaguchi M., Yamagami S. Increased Proliferation and Replicative Lifespan of Isolated Human Corneal Endothelial Cells withl-Ascorbic acid 2-phosphate. Investig. Opthalmol. Vis. Sci. 2011;52:8711–8717. doi: 10.1167/iovs.11-7592.
    1. Rubowitz A., Assia E.I., Rosner M., Topaz M. Antioxidant protection against corneal damage by free radicals during phacoemulsification. Investig. Opthalmol. Vis. Sci. 2003;44:1866–1870. doi: 10.1167/iovs.02-0892.
    1. Park C., Lee H., Hong S.-H., Kim J.-H., Park S.-K., Jeong J.-W., Kim G.-Y., Hyun J.W., Yun S.J., Kim B.W., et al. Protective effect of diphlorethohydroxycarmalol against oxidative stress-induced DNA damage and apoptosis in retinal pigment epithelial cells. Cutan. Ocul. Toxicol. 2019;38:298–308. doi: 10.1080/15569527.2019.1613425.
    1. Nita M., Grzybowski A. The Role of the Reactive Oxygen Species and Oxidative Stress in the Pathomechanism of the Age-Related Ocular Diseases and Other Pathologies of the Anterior and Posterior Eye Segments in Adults. Oxid. Med. Cell. Longev. 2016;2016:1–23. doi: 10.1155/2016/3164734.
    1. Sheu S.-J., Chen J.-L., Bee Y.-S., Lin S.-H., Shu C.-W. ERBB2-modulated ATG4B and autophagic cell death in human ARPE19 during oxidative stress. PLoS ONE. 2019;14:e0213932. doi: 10.1371/journal.pone.0213932.
    1. Mahajan S., Thieme D., Czugala M., Kruse F.E., Fuchsluger T.A. Lamin Cleavage: A Reliable Marker for Studying Staurosporine-Induced Apoptosis in Corneal Tissue. Investig. Opthalmol. Vis. Sci. 2017;58:5802. doi: 10.1167/iovs.17-21830.
    1. Pankiv S., Clausen T.H., Lamark T., Brech A., Bruun J.-A., Outzen H., Overvatn A., Bjørkøy G., Johansen T. p62/SQSTM1 Binds Directly to Atg8/LC3 to Facilitate Degradation of Ubiquitinated Protein Aggregates by Autophagy. J. Boil. Chem. 2007;282:24131–24145. doi: 10.1074/jbc.M702824200.
    1. Lin C.-J., Chen T.-L., Tseng Y.Y., Wu G.-J., Hsieh M.-H., Lin Y.-W., Chen R.-M. Honokiol induces autophagic cell death in malignant glioma through reactive oxygen species-mediated regulation of the p53/PI3K/Akt/mTOR signaling pathway. Toxicol. Appl. Pharmacol. 2016;304:59–69. doi: 10.1016/j.taap.2016.05.018.
    1. Yoon C.K., Yoon S.Y., Hwang J.S., Shin Y.J. O-GlcNAc Signaling Augmentation Protects Human Corneal Endothelial Cells from Oxidative Stress via AKT Pathway Activation. Curr. Eye Res. 2020:1–7. doi: 10.1080/02713683.2019.1686154.
    1. Padua I.R.M., Valdetaro G.P., Lima T.B., Kobashigawa K.K., Silva P.E.S., Aldrovani M., Padua P.P.M., Laus J.L. Effects of intracameral ascorbic acid on the corneal endothelium of dogs undergoing phacoemulsification. Veter- Ophthalmol. 2017;21:151–159. doi: 10.1111/vop.12490.
    1. Brodovsky S.C., Mccarty C.A., Snibson G., Loughnan M., Sullivan L., Daniell M., Taylor H.R. Management of alkali burns. Ophthalmol. 2000;107:1829–1835. doi: 10.1016/S0161-6420(00)00289-X.
    1. Lee C.-Y., Chen H.-T., Hsueh Y.-J., Chen H.-C., Huang C.-C., Meir Y.-J.J., Cheng C.-M., Wu W.-C. Perioperative topical ascorbic acid for the prevention of phacoemulsification-related corneal endothelial damage: Two case reports and review of literature. World J. Clin. Cases. 2019;7:642–649. doi: 10.12998/wjcc.v7.i5.642.
    1. Williams D.L. Oxidative Stress and the Eye. Veter. Clin. North Am. Small Anim. Pr. 2008;38:179–192. doi: 10.1016/j.cvsm.2007.10.006.
    1. Shoham A., Hadziahmetovic M., Dunaief J.L., Mydlarski M.B., Schipper H.M. Oxidative stress in diseases of the human cornea. Free. Radic. Boil. Med. 2008;45:1047–1055. doi: 10.1016/j.freeradbiomed.2008.07.021.
    1. Jurkunas U.V., Bitar M.S., Funaki T., Azizi B. Evidence of Oxidative Stress in the Pathogenesis of Fuchs Endothelial Corneal Dystrophy. Am. J. Pathol. 2010;177:2278–2289. doi: 10.2353/ajpath.2010.100279.
    1. Czarny P., Kasprzak E., Wielgorski M., Udziela M., Markiewicz B., Blasiak J., Szaflik J., Szaflik J.P. DNA damage and repair in Fuchs endothelial corneal dystrophy. Mol. Boil. Rep. 2012;40:2977–2983. doi: 10.1007/s11033-012-2369-2.
    1. Fernández-Pérez J., Ahearne M. Influence of Biochemical Cues in Human Corneal Stromal Cell Phenotype. Curr. Eye Res. 2018;44:135–146. doi: 10.1080/02713683.2018.1536216.
    1. Pugazhenthi S., Nesterova A., Sable C., Heidenreich K.A., Boxer L.M., Heasley L.E., Reusch J.E.-B. Akt/Protein Kinase B Up-regulates Bcl-2 Expression through cAMP-response Element-binding Protein. J. Boil. Chem. 2000;275:10761–10766. doi: 10.1074/jbc.275.15.10761.
    1. Li X., Hu X., Wang J., Xu W., Yi C., Ma R., Jiang H. Inhibition of autophagy via activation of PI3K/Akt/mTOR pathway contributes to the protection of hesperidin against myocardial ischemia/reperfusion injury. Int. J. Mol. Med. 2018;42:1917–1924. doi: 10.3892/ijmm.2018.3794.
    1. Jain A., Lamark T., Sjøttem E., Larsen K.B., Awuh J.A., Øvervatn A., McMahon M., Hayes J., Johansen T. p62/SQSTM1 Is a Target Gene for Transcription Factor NRF2 and Creates a Positive Feedback Loop by Inducing Antioxidant Response Element-driven Gene Transcription. J. Boil. Chem. 2010;285:22576–22591. doi: 10.1074/jbc.M110.118976.
    1. Klionsky D.J., Abeliovich H., Agostinis P., Agrawal D.K., Aliev G., Askew D., Baba M., Baehrecke E.H., Bahr B.A., Ballabio A., et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy. 2007;4:151–175. doi: 10.4161/auto.5338.
    1. Yan X., Zhong X., Yu S., Zhang L., Liu Y., Zhang Y., Sun L., Su J. p62 aggregates mediated Caspase 8 activation is responsible for progression of ovarian cancer. J. Cell. Mol. Med. 2019;23:4030–4042. doi: 10.1111/jcmm.14288.
    1. Young M., Takahashi Y., Khan O., Park S., Hori T., Yun J., Sharma A.K., Amin S., Hu C.-D., Zhang J., et al. Autophagosomal Membrane Serves as Platform for Intracellular Death-inducing Signaling Complex (iDISC)-mediated Caspase-8 Activation and Apoptosis. J. Boil. Chem. 2012;287:12455–12468. doi: 10.1074/jbc.M111.309104.
    1. Nikoletopoulou V., Markaki M., Palikaras K., Tavernarakis N. Crosstalk between apoptosis, necrosis and autophagy. Biochim. Biophys. Acta (BBA) Bioenergy. 2013;1833:3448–3459. doi: 10.1016/j.bbamcr.2013.06.001.
    1. Canadananovic V., Latinovic S., Barišić S., Babić N., Jovanovic S. Age-related changes of vitamin C levels in aqueous humour. Vojn. Pregl. 2015;72:823–826. doi: 10.2298/VSP131212063C.

Source: PubMed

Sponsorer och medarbetare

Medicinska tillstånd

Läkemedelsinterventioner

3
Prenumerera