Abstract
Purpose :
Several preclinical data have highlighted a main role of Muller glia (MG) in sensing hyper-glycemia and acquiring a pro-inflammatory polarization during diabetic retinopathy (DR) onset and progression.
Recently, we have described a novel and atypical NF-kB pathway during the early MG activation by high glucose (HG). Here, we monitored the expression of key NF-kB target genes in the rat Muller glia cell strain (rMC-1) challenged with HG over 24h and in primary retina cultures isolated from Sprague-Dawley rats after 1h of HG stimulation.
Methods :
rMC1 were grown in complete 5 mmol/L glucose medium (LG) 37°C, 5%CO2. The glucose challenge (HG, 25 mmol/L) was performed by delivering LG, HG or mannitol (as hyperosmolar control) for 45 min, 3h, 8h and 24h (n=3 per group).
Primary retinal cultures were isolated from 7-8 weeks aged Sprague-Dawley rats and stimulated in the presence of LG, HG and mannitol for 1h (n=4 per group) . In all cases, cells/tissues were collected, RNA isolated and Real-Time PCR studies performed for: IL-1β; IL-8; IL-2; IL-12; INF-γ; Ccl-11; Ccl-5; CxCl-3 e CxCl-9. One-way ANOVA followed by Tukey post-hoc significance test was applied for statistics. .
Results :
Expression analysis (2-ΔΔCt) indicated a robust increase of IL-1β, IL-8, IL-2 and Ccl5 transcripts after 45 min of HG exposure with rispect to LG and mannitol (hereafter referred to as controls because fully comparable) in rMC1 (p<0.001 for all genes). This increment was maximum for IL-8 (6-fold increase). After 3h of stimulation, IL-8 and IL-2 still increased reaching 8- and 5-fold increase (vs controls) respectively (p<0.0001), whereas IL-1β and Ccl5 did not. After 8h, IL-8 level progresively decreased (with respect to 3h), whilst IL-1β underwent a further increase (with respect to 3h, p<0.001) . Finally, after 24h, the level of all transcripts investigated was comparable to that of controls. Importantly, data obtained in primary rat retina confirmed the IL-8 (10-fold, p<0.0001) and IL-1β (2-fold, p < 0.001) increase with respect to controls.
Conclusions :
These data highlight a key role of IL-8 and IL-1β in the early pro-inflammatory polarization of rMC1 and primary rat retina cultures challenged with HG. These data confirm our previous studies on an early NF-kB pathway regulated by proteasome phosphorylation by CamKII and highlight a possible mecchanism that sustain RD pathogenesis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.