T. Zhao1, Y. Li1, B. Liu1, R. T. Bronson2, H. B. Alam1 2Harvard School Of Medicine,Brookline, MA, USA 1University Of Michigan,General Surgery,Ann Arbor, MI, USA
Introduction: Six isoforms of histone deacetylase (HDAC) Class III have been reported- Sirtuin (SIRT) 1, 2, 3, 4, 5, and 6. We recently demonstrated that EX-527, an inhibitor of SIRT1, improves survival in a lethal cecal ligation and puncture (CLP) model. The aim of this study was to determine if selective inhibition of SIRT2 with AGK2 could also improve survival in a lethal septic model, and attenuate the inflammatory response.
Methods: Experiment I: C57BL/6J mice were intraperitoneally injected with either AGK2 (82 mg/kg) dissolved in dimethyl sulfoxide (DMSO) or DMSO only, and 2 h later subjected to CLP (n=9/group). Survival was monitored for 10 days. Experiment II: animals, treated the same way as Experiment I, were grouped into (i) DMSO vehicle, and (ii) AGK2 (n=10-14/group), with sham-operated animals serving as controls. Peritoneal fluid and blood samples were collected for measurement of cytokines at 24 or 48 h. Blood at 48 h was also used to assess the coagulation status using Thrombelastography (TEG). In addition, long bones (femurs and tibias) were harvested (n=5-6/group) at 48 h to determine morphological changes in the bone marrow by H&E staining. Bone marrow atrophy was quantified by a blinded pathologist. Experiment III: Normal primary splenocytes were cultured, and treated with lipopolysaccharide (LPS) in the presence or absence of AGK2 (10 μM) for 6 h to assess cytokine production (n=4/group).
Results: AGK2 significantly improved survival (figure), and attenuated the levels of cytokines in the circulation (TNF-α: 298.3±24.6 vs. 26.8±2.8 pg/ml, p=0.003; IL-6: 633.4±82.8 vs. 232.6±133 pg/ml, p=0.034) and peritoneal fluid (IL-6: 704.8±67.7 vs. 391.4±98.5 pg/ml, p=0.033) compared to the vehicle control. It also decreased the TNF-α and IL-6 production by the splenocytes in-vitro (TNF-α: 68.1±6.4 vs. 23.9±2.8 pg/ml, p=0.001; IL-6: 73.1±4.2 vs. 49.6±3.0 pg/ml; p=0.005). The TEG data showed that animals subjected to CLP displayed markers of coagulopathy: prolonged fibrin formation and fibrin cross-linkage time, slower clot formation, decreased platelet function and clot rigidity. Inhibition of SIRT2 was associated with dramatic improvements in fibrin cross-linkage, platelet function and clot rigidity, but without a significant impact on the clot initiation parameters. Meanwhile, inhibition of SIRT2 significantly attenuated the bone marrow atrophy (58.3±6.5 vs. 30.0±8.2%, p=0.026).
Conclusions: Selective inhibition of SIRT2 significantly improves survival, attenuates “cytokine storm” and sepsis-associated coagulopathy, and decreases bone marrow atrophy in a lethal septic model.
