W. He1, P. Zhou1, Z. Chang1, B. Liu1, J. Maxwell1, I. Halaweish1, Y. Li1, H. B. Alam1 1University Of Michigan,General Surgery,Ann Arbor, MI, USA
Introduction: We have recently shown that inhibition of peptidylarginine deiminase (PAD) improves survival in a rodent model of lethal cecal ligation and puncture. However, the role of PAD inhibitor in hemorrhagic shock is largely unknown. The goal of this study was to investigate whether and how YW3-56, a selective PAD4 inhibitor, could improve survival following lethal hemorrhagic shock.
Methods: Anesthetized male Wistar-Kyoto rats (n=10/group) were subjected to 55% blood loss over 40 minutes (35% arterial hemorrhage in 10 minutes and 20% venous hemorrhage in 30 minutes), and randomized into three groups: (1) Dimethyl sulfoxide (DMSO) vehicle, (2) YW3-56 (10 mg/kg) in DMSO, and (3) Sham (no hemorrhage, no treatment). Survival was monitored for 24 h. In a second study, RAW 264.1 mouse macrophages were exposed to hypoxic conditions (0.5% O2, 5% CO2, and 94.5% N2) at 37°C in the presence or absence of YW3-56 (2.5 μM). The cells and cultured medium were harvested at 1, 3, and 6 hours. Sham (no hypoxia, no YW3-56) group served as a control. Cell viability was determined by MTT assay. Enzyme-linked immunosorbent assay was performed to analyze the secreted tumor necrosis factor α (TNF-α) and interleukin (IL)-6 in the culture medium.
Results: Hemorrhage was associated with 100% mortality in the DMSO treated animals within 3 hours, whereas 57% of the YW3-56-treated animals survived over 24 hours (Fig 1A, p< 0.05). YW3-56 treatment also significantly increased the cellular viability in the hypoxic macrophages (Fig 1B, p<0.001). Moreover, hypoxic insult induced TNF-α and IL-6 secretion in the medium, which was significantly attenuated by YW3-56 treatment (p<0.05).
Conclusion: Our results demonstrate for the first time that administration of YW3-56 significantly improves survival in vivo and in vitro, and inhibits hypoxia-induced production of key pro-inflammatory cytokines.
