Measures of lipid peroxidation and cell death show an increase with age and reduction by both Lip-1 and SIH treatment.
In all panels, vehicle control (0.5% v/v DMSO, Ctl)-treated worms are shown in orange, Lip-1-treated (200 µM Lip-1) are green and SIH-treated (250 µM SIH) are blue. (A) Representative propidium iodide fluorescence (red) overlay of bright field micrograph depicting dead intestinal cells (marked by nuclear signal, yellow triangles) within a live Day 4 adult treated with DEM. Smaller fluorescent puncta were also observed, consistent with neuronal cell nuclei (white unfilled triangles). Untreated Day 4 adult control animals (Ctl) showed no cell death. (B) Proportion of live animals at Day 4 (±95% confidence intervals) of adulthood showing dead cell fluorescence (propidium iodide)±exposure to 10 mM DEM for 24 hr. Cohorts of animals included: co-treatment with a vehicle control (-DEM, Ctl, n = 102; +DEM Ctl n = 117), Lip-1 (-DEM, Lip-1, n = 84; +DEM Ctl n = 106) or SIH (-DEM, Ctl, n = 89; +DEM Ctl n = 129). Lip-1 and SIH both markedly reduced the proportion of animals with dead cells after DEM treatment (z-test: Ctl vs Lip-1 Z = 6.37 ***p<0.001; Ctl vs SIH Z = 7.24, ***p<0.001). (C) Proportion of live animals at 4, 6 and 8 days of adulthood (±95% confidence intervals) showing propidium iodide nuclear fluorescence. Day 6 Ctl adults (n = 160) had a significantly higher proportion of animals with dead cells than Day 4 Ctl (n = 263; z-test: Z = 4.70 ***p<0.001). Similarly, Day 8 Ctl adults (n = 119) had significantly higher prevalence of animals with propidium iodide nuclear fluorescence than Day 4 Ctl (z-test: Z = 6.65 ***p<0.001). No significant difference was observed between Day 6 and Day 8 control populations. Within the Day 6 adult cohorts treatment with Lip-1 (n = 77) or SIH (n = 81) markedly reduced the prevalence of animals with propidium iodide nuclear fluorescence compared to vehicle control (z-test: Ctl vs Lip-1 Z = 2.57 # p<0.001; Ctl vs SIH Z = 2.64, # p<0.001). Within the Day 8 adult cohorts treatment with Lip-1 (n = 234) or SIH (n = 308) markedly reduced the prevalence of animals with propidium iodide nuclear fluorescence compared to vehicle control (Ctl, n = 119; z-test: Ctl vs Lip-1 Z = 5.67 # p<0.001; Ctl vs SIH Z = 6.28, # p<0.001). (D) Levels of the lipid peroxidation end product malondialdehyde (MDA) increases in C. elegans following acute glutathione depletion by 20 mM DEM exposure for 6 hr. MDA levels are shown as values normalized against the mean of untreated Day 4 Adults (Ctl) for independent samples. (Ctl vs +DEM, unpaired 2-tailed t-test ***p<0.001) (E) Malondialdehyde (MDA) increases in aged C. elegans (Day 1 vs Day 8 adults, ANOVA *p<0.05). Treatment with either Lip-1 (Day 8 vs Day 8 +Lip-1 adults, ANOVA ***p<0.001) or SIH (Day 8 vs Day 8 +SIH adults, ANOVA *p<0.05) reduces levels of MDA. Data represent independent samples with values normalized against the mean of untreated Day 1 adults. (F) Representative immunoblot against 4-HNE protein adducts comparing Day 1 and Day 8 control adults and aged adults treated with Lip-1 and SIH with corresponding tubulin blot below (representative of triplicate experiments). The relative intensity of the bands show an age-related increase that is ameliorated by Lip-1 and SIH.