Xin Cheng1, Zhengcai Liu2, Baoling Liu1, Ting Zhao1, Yongqing Li1, Hasan B Alam3. 1. Department of Surgery, University of Michigan Hospital, Ann Arbor, Michigan. 2. Department of Hepatobiliary Surgery, Xijing Hospital, The Fourth Military Medical University, Xi'an, China. 3. Department of Surgery, University of Michigan Hospital, Ann Arbor, Michigan. Electronic address: alamh@med.umich.edu.
Abstract
BACKGROUND: Hemorrhagic shock (HS) followed by a subsequent insult ("second hit") often initiates an exaggerated systemic inflammatory response and multiple organ failure. We have previously demonstrated that valproic acid, a pan histone deacetylase inhibitor, could improve survival in a rodent "two-hit" model. In the present study, our goal was to determine whether selective inhibition of histone deacetylase 6 with Tubastatin A (Tub-A) could prolong survival in a two-hit model where HS was followed by sepsis from cecal ligation and puncture (CLP). METHODS: C57Bl/6J mice were subjected to sublethal HS (30% blood loss) and then randomly divided into two groups (n = 13 per group) such as Tub-A group (treatment) and vehicle (VEH) group (control). The Tub-A group was given an intraperitoneal injection of Tub-A (70 mg/kg) dissolved in dimethyl sulfoxide (DMSO). The VEH group was injected with DMSO (1 μl/g body weight). After 24 h, all mice were subjected CLP followed immediately by another dose of Tub-A or DMSO. Survival was monitored for 10 d. In a parallel study, peritoneal irrigation fluid and liver tissue from Tub-A- or DMSO-treated mice were collected 3 h after CLP. Enzyme-linked immunosorbent assay was performed to quantify activity of the myeloperoxidase and concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) in the peritoneal irrigation fluid. RNA was isolated from the liver tissue, and real-time polymerase chain reaction was performed to measure relative messenger RNA levels of TNF-α and IL-6. RESULTS: Treatment with Tub-A significantly improved survival compared with that of the control (69.2% versus 15.4%). In addition, Tub-A significantly suppressed myeloperoxidase activity (169.9 ± 8.4 ng/mL versus 70.4 ± 17.4 ng/mL; P < 0.01) and reduced levels of cytokines TNF-α and IL-6 in the peritoneal fluid (TNF-α: 105.7 ± 4.7 versus 7.4 ± 2.4 pg/mL; IL-6: 907.4 ± 2.3 versus 483.6 ± 1.6 pg/mL; P < 0.01) compared with those in the VEH control. Gene expression measured by real-time polymerase chain reaction confirmed that Tub-A inhibits transcription of TNF-α and IL-6. CONCLUSIONS: Tub-A treatment significantly improves survival, attenuates inflammation, and downregulates TNF-α and IL-6 gene expression in a rodent two-hit model.
BACKGROUND:Hemorrhagic shock (HS) followed by a subsequent insult ("second hit") often initiates an exaggerated systemic inflammatory response and multiple organ failure. We have previously demonstrated that valproic acid, a pan histone deacetylase inhibitor, could improve survival in a rodent "two-hit" model. In the present study, our goal was to determine whether selective inhibition of histone deacetylase 6 with Tubastatin A (Tub-A) could prolong survival in a two-hit model where HS was followed by sepsis from cecal ligation and puncture (CLP). METHODS: C57Bl/6J mice were subjected to sublethal HS (30% blood loss) and then randomly divided into two groups (n = 13 per group) such as Tub-A group (treatment) and vehicle (VEH) group (control). The Tub-A group was given an intraperitoneal injection of Tub-A (70 mg/kg) dissolved in dimethyl sulfoxide (DMSO). The VEH group was injected with DMSO (1 μl/g body weight). After 24 h, all mice were subjected CLP followed immediately by another dose of Tub-A or DMSO. Survival was monitored for 10 d. In a parallel study, peritoneal irrigation fluid and liver tissue from Tub-A- or DMSO-treated mice were collected 3 h after CLP. Enzyme-linked immunosorbent assay was performed to quantify activity of the myeloperoxidase and concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) in the peritoneal irrigation fluid. RNA was isolated from the liver tissue, and real-time polymerase chain reaction was performed to measure relative messenger RNA levels of TNF-α and IL-6. RESULTS: Treatment with Tub-A significantly improved survival compared with that of the control (69.2% versus 15.4%). In addition, Tub-A significantly suppressed myeloperoxidase activity (169.9 ± 8.4 ng/mL versus 70.4 ± 17.4 ng/mL; P < 0.01) and reduced levels of cytokines TNF-α and IL-6 in the peritoneal fluid (TNF-α: 105.7 ± 4.7 versus 7.4 ± 2.4 pg/mL; IL-6: 907.4 ± 2.3 versus 483.6 ± 1.6 pg/mL; P < 0.01) compared with those in the VEH control. Gene expression measured by real-time polymerase chain reaction confirmed that Tub-A inhibits transcription of TNF-α and IL-6. CONCLUSIONS:Tub-A treatment significantly improves survival, attenuates inflammation, and downregulates TNF-α and IL-6 gene expression in a rodent two-hit model.
Authors: Hasan B Alam; Kathleen Stanton; Elena Koustova; David Burris; Norman Rich; Peter Rhee Journal: Resuscitation Date: 2004-01 Impact factor: 5.262
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