Figure 1
From: Prevention of neonatal oxygen-induced brain damage by reduction of intrinsic apoptosis
Dose–response plots for inhibition of caspase-2 and -3 with TRP601 and its active metabolite. TRP601 (a) and Δ2Me-TRP601 (b) were added to recombinant caspase-2 (gray curves) and caspase-3 (black curves) to determine initial enzyme velocity and IC50 values in chromogenic microplate assay. TRP601 and Δ2Me-TRP601 were added simultaneously to substrates (plain curves; IC50/TRP601/Casp3=25.58±3.1 nM; IC50/Δ2Me-TRP601/Casp3=0.39±0.11 nM; IC50/TRP601/Casp2(a)=471.8±91.3 nM; IC50/Δ2Me-TRP601/Casp2(a)=7.4±3.18 nM) or alternatively 45 min before substrates (dotted curves; IC50/TRP601/Casp2(b)=115.2±39.12 nM; IC50/Δ2Me-TRP601/Casp2(b)=2.67±1.46 nM). (c) Six-day-old Wistar rat pups were either injected with the caspase inhibitor TRP601 (1 mg/kg bodyweight, i.p.) or vehicle control before exposure to 80% O2. After 12 or 24 h, animals were killed, transcardially perfused with PBS, and brain samples were collected in order to perform a fluorometric caspase-3 activity assay. Measurements of hydrolysis of Ac-DEVD-AMC at 460 nm resulted in a highly significant upregulation of caspase-3 activity under hyperoxic conditions, whereas single treatment with TRP601 significantly decreased caspase-3 activity to control levels after 12 and 24 h. Bars represent mean±S.E.M. (thalamus, n=6/group, normalized to control animals (21% O2) ***P<0.001, #P<0.05, ###P<0.001, two-way ANOVA). (d) Inhibition of caspase-2 by TRP601 in vivo (1 mg/kg bodyweight i.p.) lead to decreased protein expression of caspase-2 in thalamus from treated animals, whereas under normoxic conditions TRP601 had no effect. Data are normalized to levels of rat pups exposed to normoxia (control=100%). Representative western blot images of caspase-2 and β-actin are shown for thalamus. Bars represent mean ±S.E.M. (n=8/group, ***P<0.001, ###P<0.001, one-way ANOVA). (e) Six-day-old Wistar rat pups were either injected with the caspase inhibitor TRP601 (1 mg/kg bodyweight, i.p.) or vehicle control before exposure to 80% O2. After 12 or 24 h, animals were killed, transcardially perfused with PBS, and brain samples were collected in order to perform a fluorometric caspase-2 activity assay. Measurements of hydrolysis of VDVAD-AFC at 505 nm resulted in a highly significant upregulation of caspase-2 activity under hyperoxic conditions, whereas single treatment with TRP601 significantly decreased caspase-2 activity to control levels after 12 and 24 h. Bars represent mean ±S.E.M. (thalamus, n=6/group, normalized to control animals (21% O2) ***P<0.001, ###P<0.001, two-way ANOVA)
