Elsevier

Cellular Signalling

Volume 88, December 2021, 110153
Cellular Signalling

Maintaining blood retinal barrier homeostasis to attenuate retinal ischemia-reperfusion injury by targeting the KEAP1/NRF2/ARE pathway with lycopene

https://doi.org/10.1016/j.cellsig.2021.110153Get rights and content

Highlights

  • LYC is found to stabilize the BRB homeostasis in retinal I/R for the first time.

  • LYC protects against retinal I/R injury via increasing NRF2.

  • Antioxidants are the major facors in KEAP1/NRF2/ARE pathway activation by LYC.

Abstract

Retinal ischemia-reperfusion (I/R) often results in intractable visual impairments, where blood retinal barrier (BRB) homeostasis mediated by retinal pigment epithelium (RPE) and retinal microvascular endothelium (RME) is crucial. However, strategies targeting the BRB are limited. Thus, we investigated the inconclusive effect of lycopene (LYC) in retinal protection under I/R. LYC elevated cellular viability and reversed oxidative stress in aRPE-19 cells/hRME cells under I/R conditions based on oxygen-glucose deprivation (OGD) in vitro. Molecular analysis showed that LYC promoted NRF2 expression and enhanced the downstream factors of the KEAP1/NRF2/ARE pathway: LYC increased the activities of antioxidants, including SOD and CAT, whereas it enhanced the mRNA expression of HO-1 (ho-1) and NQO-1 (nqo-1). The activation resulted in restrained ROS and MDA. On the other hand, LYC ameliorated the damage to retinal function and morphology in a mouse I/R model, which was established by unilateral ligation of the left pterygopalatine artery/external carotid artery and reperfusion. LYC promoted the expression of NRF2 in both the neural retina and the RPE choroid in vivo. This evidence revealed the potential of LYC in retinal protection under I/R, uncovering the pharmacological effect of the KEAP1/NRF2/ARE pathway in BRB targeting. The study generates new insights into scientific practices in retinal research.

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.

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    These authors contributed equally to this work.

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