Original article
Synergistic protection of RGCs by olfactory ensheathing cells and alpha-crystallin through regulation of the Akt/BAD PathwayProtection synergique des CGR par des sphingocytes et l’alpha-cristalline par régulation de la voie AKT/BAD

https://doi.org/10.1016/j.jfo.2020.02.003Get rights and content

Summary

Background

Our recent in vivo studies have shown that olfactory ensheathing cells (OECs) and α-crystallin can promote retinal ganglion cell (RGC) survival and axonal regeneration synergistically after optic nerve injury. However, the mechanism is still unknown.

Objectives

Here, we studied the synergistic effect and mechanism of OECs and α-crystallin on RGC survival after H2O2-induced oxidative damage and a crushing injury to the optic nerve in an adult rat model.

Methods

After H2O2-induced oxidative damage, RGC-5 cells were treated with OECs, α-crystallin or a combination of OECs and α-crystallin. Apoptosis of RGC-5 cells was assessed by flow cytometry. Phosphorylated Akt, BAD, and cleaved-caspase3 were detected by Western blot after optic nerve injury in vivo and H2O2-induced RGC-5 oxidative damage in vitro.

Results

The results showed that OECs and α-crystallin could both independently inhibit RGC-5 apoptosis (P < 0.01), increase the phosphorylation of both Akt and BAD, and decrease the activation of caspase-3 (P < 0.01). However, the effect of the combination of both was more significant than either alone.

Conclusion

These findings indicate that inhibition of superoxide damage to RGCs through regulation of the Akt/BAD pathway is one of the mechanisms by which OECs and α-crystallin promote optic nerve recovery after injury.

Résumé

Généralités

Nos dernières études biologiques ont montré que les cellules de sphingocytes (OECS) et l’alpha-cristalline facilitent la survie et la régénération axiale des cellules ganglionnaires rétiniennes après une lésion du nerf optique. Toutefois, son mécanisme n’est toujours pas clair.

Objectif

Les effets de l’OECS et de l’alpha-cristalline sur la survie des CGR et leur mécanisme ont été étudiés sur des rats adultes avec des lésions dues au peroxyde d’hydrogène et un modèle de d'écrasement du nerf optique.

Méthode

Le H2O2 induit des lésions d’oxydation des cellules rgc-5 et les cellules rgc-5 sont traitées en association avec l’OECS, la protéine α-cristalline et l’OECS et l’alpha-cristalline. La cytométrie en flux a détecté l’apoptose des cellules rgc-5. La phosphorylation et le clivage de la caspase3, d’Akt et de Bad ont été détectés par Western Blot.

Résultats

Les résultats montrent que les OECS et l’alpha-cristalline inhibent l’apoptose des cellules rgc-5 (p < 0,01), augmentent la phosphorylation d’Akt et de Bad et réduisent l’activité de la caspase3 (p < 0,01). Toutefois, les combinaisons de médicaments sont les plus efficaces.

Conclusion

L’inhibition des lésions de stress oxydant de rgcs par la voie Akt/BAD est l’un des mécanismes de l’OECS et de alpha-cristalline pour faciliter la récupération des lésions du nerf optique.

Introduction

Retinal ganglion cells (RGCs) fail to spontaneously regenerate their axons after optic nerve injury. Excepting glial scar, and inhibitory microenviroment, apoptosis of RGCs is the major reasons of inhibiting axonal regeneration after optic nerve injury [1], [2]. After transection or crush-axotomy of the optic nerve, about 60% and 90% of the RGCs die within 7 and 14 days [1], [3]. Thus, protecting the survival of RGCs is a crucial factor in promoting axonal regeneration after optic nerve injury.

Olfactory enshesathing cells (OECs) have been trialed in cell transplant therapies to assist in the repair of central nervous system injuries by phagocytosing cellular debris, modulating neuroinflammation, promoting angiogenesis, and secreting extracellular matrix [4]. Furthermore, OECs can secrete variety neurotrophic factors including nerve growth factor(NGF), neurotrophin-3, brain derived neurotrophic factor(BDNF), glial-derived neurotrophic factor(GDNF), ciliary neurotrophic factor(CNTF), and fibroblast growth factor(FGF) [5]. In addition, transplantation of OECs can assist photoreceptors survival of RCS rat [6], spiral ganglion cells survival after T10 spinal level laminectomy [7], and attenuates hippocampus neurons damages induced by acute carbon monoxide poisoning [8]. In optic nerve injury, OEC transplantation delayed the death of axotomized RGCs at 7 days, but the mechanism remains unclear [3].

α-Crystallin, as the small heat shock protein, play a beneficial role in preventing stress-induced cell death, including lens epithelial cells [9], dopaminergic olfactory bulb neurons [10], retinal pigment epithelial cells [11], photoreceptors [12], and astrocyte [13]. The mechanisms has been showed to inhibit cytochrome c release, downregulate of Bcl-2, interact with procaspase-3, Bcl-X(S), Bax and suppress maturation of caspase-3 [14]. After optic nerve axotomy, the RGCs protective effect ofα-crystallin overexpression was approximately 40% at day 7, and 75% at 14 days [15]. However, the authors did not observe more long time.

Our recent studies showed that OECs and α-crystallin could protect RGCs survival respectively after optic nerve injury for 1 month. Moreover, the synergistic effect of OECs and α-crystallin was more effectively. It was 2.72 times of control, 1.45 times of α-crystallin, and 1.23 times of OECs [16]. However, the mechanism is still unknown. In the present work, we studied the synergistic effect and mechanism of OECs and α-crystallin on RGCs survival by H2O2-induced oxidative damage and optic nerve crushed injury of adult rat.

Section snippets

Animals

Adult (200–250 g) Long Evans rats were used for the experiments. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Third Military Medical University. And all studies were conducted according to the Declaration of the NIH Statement for the Use of Animals in Ophthalmic and Vision Research

OECs culture and identification

Primary cultures of OECs/ONFs were prepared as previously described [16]. Briefly, adult rats were killed by decapitation. The outer nerve and glomerular layers of

H2O2 treatment causes RGC-5 injury

First, we detected the injury of RGC-5 after treated with different concentrations of H2O2 (200 μM, 300 μM, 400 μM) for 24 h. The viability of RGC-5 reduced to 0.77% treated with 200 μM H2O2, 0.65% with 300 μM H2O2, and 0.34% with 400 μM H2O2(Fig. 1A; P < 0.01). Compared with the control group, percentage of RGC-5 apoptosis was 21.32 ± 1.64% in 200 μM group, 33.37 ± 0.86% in 300 μM group, 63.83 ± 0.85% in 400 μM group(Fig. 1B, C; P < 0.01). And cleaved-Caspase3 was up-regulated in three H2O2 group (Fig. 2; P < 

Discussion

Recently, our studies showed that OECs and α-crystallin could protect RGCs survival respectively after optic nerve injury. And the synergistic effect of them was more effectively [16]. However, the mechanism is unknown. Here, we studied the synergistic effect and mechanism of OECs and α-crystallin on RGCs survival by H2O2-inudced RGC-5 oxidative injury and optic nerve crushed. These findings indicated that inhibiting RGCs apoptosis through regulation of Akt/Bad pathway is one of the mechanism

Disclosure of interest

The authors declare that they have no competing interest.

Statement of ethics

There is no risk affecting the health and safety of human or laboratory animal.

Acknowledgements

The study is supported by grants from National Natural Science Foundation of China (No. 31100706), China Postdoctoral Science Foundation (No. 2012M521872).

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