A2-4-2-4 pharmacodynamics. (A) A2-4-2-4 showed the heart failure attenuation activity in a dosage-dependent manner. (B) Time course experiments showed that A2-4-2-4 is required at the early phase of AA-induced AHF. Embryos were treated with AA from 24–72 hpf and A2-4-2-4 added at different times, 24, 30, 36, 42, or 48 hpf. The attenuation of A2-4-2-4 was about 70% when added at 24 hpf but dropped to 35% when added six hours later at 30 hpf (* indicates statistical significance by t-test, p = 0.038; ** between A2-4-2-4 (36) and A2-4-2-4 (30), p = 0.014). A2-4-2-4 lost 90% of the attenuation efficacy when added 24 h later (70% at 24 hpf vs. 7% at 48 hpf). (C) A combinational experiment with sub-dosage of the compounds identified in our lab, 10 μM of NS398, 1:500 dilutions of A2-4-2-4, 5 μM of MEK-I, 2 μM of C25, showed a possible synergistic effect between A2-4-2-4 and NS398, comparing the attenuation of double treatment by NS398; A2-4-2-4 (78.6%) with that of lone NS398 (25.2%) or A2-4-2-4 (34.9%) treatment. (* indicates the statistical significance by t-test, p < 0.05). Similarly, the data suggest a possible synergistic effect between A2-4-2-4 and C25 (* indicates statistical significance by t-test, p < 0.05 between the double versus single treatments) but an additive effect between A2-4-2-4 and MEK-I.
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