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Vignesh Krishnamoorthy, Shaoqing He, Renuka Chaphalkar, Bindu Kodati, Raghu R Krishnamoorthy; Neuroprotective effects of SB203580 (p38 MAP kinase inhibitor) against Endothelin-1-induced retinal ganglion cell death. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4960.
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A growing body of evidence suggests that endothelins (ETs), a family of 21-amino-acid vasoactive peptides, are contributors to neurodegeneration in glaucoma, however, the underlying signaling mechanisms are not well understood. The aim of this study is to assess the neuroprotective properties of SB203580, a p38 MAP kinase inhibitor, against ET-1-induced cell death in primary rat RGCs.
Primary RGCs were isolated from postnatal day 4-6 rats by panning with Thy-1 antibody. RGCs were maintained for 7 days and then treated in trophic factor-free medium with 100nM ET-1 for different time points. After the treatments, immunocytochemistry was used to detect phosphorylated p38/JNK, ATF-1, and ATF-3. In separate experiments, RGCs were treated with SB203580 or a combination of SB203580 and 100nM ET-1 for 24 hours. After the treatments, a Live/Dead assay was used to determine the number of living and dead cells. Images were captured at 10x magnification using the Cytation 5 Imaging system. The experiment was repeated three times and the number of surviving and dead cells were quantitated. Caspase 3 activity was also determined in ET-1-treated RGCs with/without SB203580.
An increase of phospho-p38 staining was detected in RGCs treated with ET-1 for 15 and 30 mins. No appreciable changes in the staining of phospho-JNK was detected in these time points. ET-1 treatment didn’t alter the expression of ATF-1 and ATF-3, which are downstream transcription factors of p38. ET-1 induced RGC death for 24 hour treatment (as seen by an increase in the dead/live ratio from 0.446 to 0.621), compared to untreated controls. Interestingly, compared to the untreated controls, an appreciable decrease in cell death was detected in cells treated with SB203580 alone (a decrease in the dead/live ratio from 0.446 to 0.243). Following treatment with a combination of ET-1 and SB203580, cell counts showed a decrease in cell death when compared with cells treated with ET-1 alone (a change in the dead/live ratio from 0.621 to 0.333).
These results suggest that SB203580 may play a neuroprotective role against ET-1-induced cell death. Elucidation of endothelin-mediated signaling pathways will help understand some mechanisms by which endothelins promote neurodegeneration in glaucoma.
This is a 2020 ARVO Annual Meeting abstract.
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