The neuroprotective effect of melatonin in glutamate excitotoxicity of R28 cells and mouse retinal ganglion cells.

Glaucoma is the leading cause of irreversible blindness. The progressive degeneration of retinal ganglion cells (RGCs) is the major characteristic of glaucoma. Even though the control of intraocular pressure could delay the loss of RGCs, current clinical treatments cannot protect them directly. The overactivation of N-methyl-D-aspartic acid (NMDA) receptors by excess glutamate (Glu) is among the important mechanisms of RGC death in glaucoma progression. Melatonin (MT) is an indole neuroendocrine hormone mainly secreted by the pineal gland. This study aimed to investigate the therapeutic effect of MT on glutamate excitotoxicity of mouse RGCs and R28 cells. The Glu-induced R28 cell excitotoxicity model and NMDA-induced retinal injury model were established. MT was applied to R28 cells and the vitreous cavity of mice by intravitreal injection. Cell counting kit-8 assay and propidium iodide/Hoechst were performed to evaluate cell viability. Reactive oxygen species and glutathione synthesis assays were used to detect the oxidative stress state of R28 cells. Retina immunofluorescence and hematoxylin and eosin staining were applied to assess RGC counts and retinal structure. Flash visual-evoked potential was performed to evaluate visual function in mice. RNA sequencing of the retina was performed to explore the underlying mechanisms of MT protection. Our results found that MT treatment could successfully protect R28 cells from Glu excitotoxicity and decrease reactive oxygen species. Also, MT rescued RGCs from NMDA-induced injury and protected visual function in mice. This study enriches the indications of MT in the treatment of glaucoma, providing practical research ideas for its comprehensive prevention and treatment.

Nuclear factor erythroid 2-related factor 2 agonist protects retinal ganglion cells in glutamate excitotoxicity retinas.

OBJECTIVE: To investigate whether tert-Butylhydroquinone (TBHQ) can ameliorate oxidative stress and inflammation induced by glutamate excitotoxicity, and mediate retinal ganglion cell (RGC) damage by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and inhibiting the nuclear factor kappa B (NF-κB) signaling pathway. MATERIALS AND METHODS: TBHQ was used to treat a glutamate excitotoxicity model of retinal cell line 28 and C57 mice. Damage to RGCs and visual function were assessed using flash visual evoked potential (FVEP), immunofluorescence, propidium iodide staining, and hematoxylin and eosin staining. Knockdown of Nrf2 used Nrf2 shRNA. The expression levels of related proteins were detected using western blot and immunofluorescence. RESULTS: Glutamate excitotoxicity down-regulated Nrf2 expression in vitro and in vivo. Nuclear factor erythroid2-related factor 2 activation by TBHQ reduced the damage to retinal ganglion cells, reduced the thinning of the whole retina and the ganglion cell complex, and shortened the latency of the FVEP forward wave after injury. In addition, the levels of NAD(P)H quinone dehydrogenase 1 (NQO1), heme oxygenase 1 (HO-1), and Nrf2 increased significantly, and those of cyclooxygenase-2 (COX2) and NF-κB decreased significantly, after TBHQ treatment. Compared with TBHQ treatment group, the expression level of p-p65 in shRNA transfected group was increased, but still lower than that in Glu group. CONCLUSION: The protective effect of TBHQ on RGC loss under glutamate excitotoxicity might be related to the activation of the Nrf2 signaling pathway, anti-oxidative stress, inhibition of NF-κB activation, and inhibition of retinal inflammation. Thus, TBHQ might be used to treat glutamate excitotoxicity -related retinopathy.