Abstract 17393: Early Reperfusion Phase as the Critical Therapeutic Window for Flavonoids Baicalein's Cardioprotection via Oxidant Scavenging and Nitric Oxide Signaling

Abstract

Background: Baicalein is a natural flavonoid with antioxidant activities protecting against ischemia-reperfusion (IR) injury. Previous studies suggest that oxidative burst occurs early after reperfusion that leads to accelerated cell death thereafter. We therefore investigated the timing of baicalein treatment in relation to its oxidant modulating effects and IR protection in order to sort out the critical therapeutic window. Methods: Using an established cardiomyocyte model of IR, we administered baicalein (100 μ M) at 0, 15, 30, 60 and 120 min after reperfusion and compared the cell viability, reactive oxygen species (ROS) and nitric oxide (NO) profiles with those of IR control. The cell viability was assessed by propidium iodide. The intracellular ROS were monitored by 2’,7’-dichlorofluorescin diacetate (DCFH-DA), while NO was assessed by 4’,5’-diaminofluorescin diacetate (DAF-2 DA). Results: Baicalein administered at 0 min of reperfusion significantly reduced cell death (35.5 ± 2.6% vs. 57.4 ± 1.9% in IR control, P < 0.001). Delay of baicalein for 15 or 30 min led to time-dependent decrease of cytoprotection (cell death 40.6 ± 1.7% and 44.5 ± 3.0%, P < 0.001 and 0.05 vs. IR control). If baicalein was delayed for 60 or 120 min, the protective effect was lost. In IR control, a transient but significant ROS burst was seen within 15 min of reperfusion. Baicalein treated at 0 min significantly decreased the ROS burst ( P < 0.01). If treatment was delayed for 15 min, the ROS burst became unaffected at all. In terms of NO, baicalein treatment at 0 min resulted in a significant increase of NO in the later phase (> 30 min) of reperfusion. Cotreatment of baicalein with NO synthase (NOS) inhibitor L-NAME (200 μ M) not only decreased NO at reperfusion but abrogated the protective effect. If baicalein was delayed for 15 min, not only the NO increase was delayed, the level of NO was also lower. These suggest that NOS activation in the early phase of reperfusion is critical for NO signaling and protection. Conclusion: Baicalein protects cardiomyocytes from IR injury via oxidant scavenging and NOS-mediated NO signaling. The critical therapeutic window is within the first 15 to 30 min of reperfusion, during which reperfusion ROS burst can be timely scavenged and NOS effectively activated.