Maintaining blood retinal barrier homeostasis to attenuate retinal ischemia-reperfusion injury by targeting the KEAP1/NRF2/ARE pathway with lycopene
Graphical abstract
Introduction
Retinal ischemia-reperfusion (I/R) is a leading pathological process involved in different retinal diseases, ranging from glaucoma and diabetic retinopathy to retinal vascular occlusion, causing visual impairments [1], [2], [3], [4], [5]. During I/R, primary ischemia oppresses cells with energy failure, elementary oxidation and calcium influx, which is followed by the overproduction of reactive oxygen species (ROS) generated from re‑oxygenation of reperfusion [5], [6], [7]. The structural integrity and physiological functions of the normal retina are thus corrupted by oxidative stress and cascades of consequent inflammation and variations leading to cell death [1], [8]. Sophisticated vascular systems, including the retinal pigment epithelium (RPE) and retinal microvascular endothelium (RME, the vascular endothelium is considered a special type of single-layer epithelial tissue [9]), form the blood retinal barrier (BRB) to preserve internal environment homeostasis [10], [11]. BRB breakdown resulting from I/R conversely aggregates pathological consequences, which are associated with increased vascular permeability, retinal edema, tissue damage, neurotic retinal injury and visual loss [1], [12], [13]. Consequently, enhancing crisis resistance of the BRB may provide a solution for retinal I/R management.
The nuclear factor erythroid 2-related factor 2 (NRF2) pathway is a possible molecular target in BRB homeostasis maintenance. NRF2 is an emerging regulator of cellular resistance to oxidative stress resulting from different causes, including I/R [14], [15], [16], [17], regularly suppressed by Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (KEAP1) and degraded through ubiquitination-proteasomal protocols [18], [19], [20]. Activated NRF2 is detached from KEAP1 and phosphorylated. It is thus transported into the nucleus, identifying antioxidant response element (ARE) genes with the synergism of small musculoaponeurotic fibrosarcoma proteins (Maf)s. The chain is followed by the production of antioxidants [15], [21], [22], [23]. On the other hand, lycopene (LYC), a member of the carotenoid family, is reported to be an agonist of the NRF2/KEAP1/ARE pathway [24]. Evidence indicates that LYC is capable of protecting different types of epithelium and endothelium from damage [25], [26], [27], [28]. Despite the multiple pharmacological properties of LYC, it was shown that LYC protects against myocardial or neural I/R [29], [30], [31]. However, the role of LYC in retinal I/R remains unknown.
Accordingly, we investigated the possibility of attenuating retinal I/R injury by protecting the BRB through LYC and discovered the intrinsic molecular modulation. LYC attenuated retinal I/R injury in the BRB in vitro and in vivo by activating the KEAP1/NRF2/ARE pathway. This study illustrates the therapeutic potential of the BRB as a target in retinal I/R, giving rise to the protective effect of LYC in retinal protection.
Section snippets
Cell culture and reagents
Human retinal pigment epithelial cell line aRPE-19 cells (American Type Culture Collection, USA) and human retinal microvascular endothelial cell line hRME cells (Cell System, USA) were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; Gibco, Thermo Fisher Scientific, USA) culture medium with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, USA) and 1% penicillin and streptomycin (HyClone, GE Healthcare Life Science, USA). In order to simulate the
Ischemia induced progressive cell death in retinal cells in vitro
To establish retinal I/R models in vitro, we subjected aRPE-19 cells (a retinal pigment epithelium cell line derived from Homo sapiens) and hRME cells (a human retinal microvascular endothelial cell line) to OGD to induce primary ischemia. OGD is a condition where the cells were cultured in PBS dissolved in 15 mM Na2S2O4, which is capable of stably depleting oxygen from a culture medium for more than 1 h (Fig. 1A) [35].
Cellular viability was detected using a MUSE Annexin V-Cell Death kit after
Discussion
Generally occurring in glaucoma, diabetic retinopathy, retinal vascular occlusive diseases and other retinopathies, the retinal I/R triggers cascades leading to physiological dysfunction and cell death, and the intractable visual impairment afterwards threatens civil health globally [1], [2], [3], [4], [5]. The RPE and RME — the basic components of the BRB — are essential to the internal environmental homeostasis of the retina, which is a crux in retinal recovery after I/R [11], [36]. Thus, the
Author contributions
H. H., CD. L. and HX. S. designed the experiments. H. H., XL. K., XB. Z. and YX. Z. performed the experiments. H. C., H. D., and H. T. analyzed the data. LB. Z., JS. Z., HJ. L., and JH. Y. prepared the figs. H. H., XL. K. and XB. Z. wrote the main text. CD. L and HX. S. revised the manuscript. All authors reviewed the manuscript.
Funding and acknowledgement
This study was supported by the National Natural Science Foundation of China (NO. 81670874), Natural Science Foundation of Gurangdong Province of China (NO. 2020A1515010144 & NO. 2021A1515010513), and the Fundamental Research Funds of the State Key Laboratory of Ophthalmology (NO. N/A) and the Open Research Funds of the State Key Laboratory of Ophthalmology (NO. N/A).
Declaration of Competing Interest
No conflict of interest exits in the submission of this manuscript.
References (56)
- et al.
Neuroprotection in relation to retinal ischemia and relevance to glaucoma
Surv. Ophthalmol.
(1999) - et al.
Retinal ischemia: mechanisms of damage and potential therapeutic strategies
Prog. Retin. Eye Res.
(2004) - et al.
Reperfusion injury and reactive oxygen species: the evolution of a concept
Redox Biol.
(2015) - et al.
The molecular basis of retinal ganglion cell death in glaucoma
Prog. Retin. Eye Res.
(2012) - et al.
Blood-retinal barrier in hypoxic ischaemic conditions: basic concepts, clinical features and management
Prog. Retin. Eye Res.
(2008) - et al.
Importance of phase 2 gene regulation in protection against electrophile and reactive oxygen toxicity and carcinogenesis
Adv. Enzym. Regul.
(2003) - et al.
Nrf2 the rescue: effects of the antioxidative/electrophilic response on the liver
Toxicol. Appl. Pharmacol.
(2010) Signal transduction by mitochondrial oxidants
J. Biol. Chem.
(2012)- et al.
Transcriptional regulation of the rat glutathione S-transferase Ya subunit gene. Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidants
J. Biol. Chem.
(1990) - et al.
Natural ingredients-derived antioxidants attenuate H(2)O(2)-induced oxidative stress and have chondroprotective effects on human osteoarthritic chondrocytes via Keap1/Nrf2 pathway
Free Radic. Biol. Med.
(2020)
Lycopene treatment inhibits activation of Jak1/Stat3 and Wnt/beta-catenin signaling and attenuates hyperproliferation in gastric epithelial cells
Nutr. Res.
Tomato paste supplementation improves endothelial dynamics and reduces plasma total oxidative status in healthy subjects
Nutr. Res.
Nano-liposomes of lycopene reduces ischemic brain damage in rodents by regulating iron metabolism
Free Radic. Biol. Med.
Quercetin protects oligodendrocyte precursor cells from oxygen/glucose deprivation injury in vitro via the activation of the PI3K/Akt signaling pathway
Brain Res. Bull.
Retinal metabolism: a comparative look at energetics in the retina
Brain Res.
Mechanisms of ischemic cerebral injury
Resuscitation
ROS and redox signaling in myocardial ischemia-reperfusion injury and cardioprotection
Free Radic. Biol. Med.
Cell permeable ROS scavengers, tiron and tempol, rescue PC12 cell death caused by pyrogallol or hypoxia/reoxygenation
Neurosci. Res.
The b-wave of the electroretinogram as an index of retinal ischemia
Gen. Pharmacol.
The inner blood-retinal barrier: cellular basis and development
Vis. Res.
Inhibition of the Keap1-Nrf2 protein-protein interaction protects retinal cells and ameliorates retinal ischemia-reperfusion injury
Free Radic. Biol. Med.
Cellular stress response and immune signaling in retinal ischemia-reperfusion injury
Front. Immunol.
Pathophysiology of diabetic retinopathy: the old and the new
Diabetes Metab. J.
Association between systemic oxidative stress and visual field damage in open-angle glaucoma
Sci. Rep.
Molecular mechanisms of ischemia-reperfusion injury in brain: pivotal role of the mitochondrial membrane potential in reactive oxygen species generation
Mol. Neurobiol.
Use of mesothelial cells and biological matrices for tissue engineering of simple epithelium surrogates
Front. Bioeng. Biotechnol.
The blood-retinal barriers
Doc. Ophthalmol.
Ischemia-reperfusion injury induces occludin phosphorylation/ubiquitination and retinal vascular permeability in a VEGFR-2-dependent manner
J. Cereb. Blood Flow Metab.
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These authors contributed equally to this work.