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Observational Study
. 2017 Oct;23(10):1282-1293.
doi: 10.1002/lt.24821.

Sirtuin 1 attenuates inflammation and hepatocellular damage in liver transplant ischemia/Reperfusion: From mouse to human

Kojiro Nakamura  1 , Shoichi Kageyama  1 , Bibo Ke  1 , Takehiro Fujii  1 , Rebecca A Sosa  2 , Elaine F Reed  2 , Nakul Datta  1 , Ali Zarrinpar  1 , Ronald W Busuttil  1 , Jerzy W Kupiec-Weglinski  1
Affiliations
Observational Study

Sirtuin 1 attenuates inflammation and hepatocellular damage in liver transplant ischemia/Reperfusion: From mouse to human

V体育ios版 - Kojiro Nakamura et al. Liver Transpl. 2017 Oct.

"V体育2025版" Abstract

Hepatic ischemia/reperfusion injury (IRI), an inevitable antigen-independent inflammation response in cadaveric liver transplantation, correlates with poor early graft function, rejection episodes, and contributes to donor organ shortage. Sirtuin 1 (SIRT1) is a histone deacetylase that may regulate inflammatory cell activity and manage liver function in IRI, though its functional role and clinical relevance remains to be elucidated. We investigated the efficacy of SIRT1 activation in a murine liver IRI model and verified the concept of putative SIRT1-mediated hepatoprotection in clinical liver transplantation. In the experimental arm, mice were subjected to 90 minutes of liver partial warm ischemia followed by 6 hours of reperfusion with or without adjunctive SIRT1 activation in vivo (resveratrol [Res]). In parallel, bone marrow-derived macrophage (BMDM) or spleen lymphocyte cultures were treated with Res. In the clinical arm, liver biopsies from 21 adult primary liver transplant patients (2 hours after reperfusion) were divided into "low" (n = 11) versus "high" (n = 10) SIRT1 expression groups, assessed by Western blots. Treatment with Res attenuated murine liver IRI while up-regulating SIRT1, suppressing leukocyte infiltration, and decreasing proinflammatory cytokine programs. SIRT1 silencing (small interfering RNA) in BMDM cultures enhanced inflammatory cytokine programs, whereas addition of Res decreased proinflammatory response in a SIRT1-dependent manner. In addition, Res decreased interferon γ production in liver-infiltrating and spleen lymphocyte cultures. Human liver transplants with high SIRT1 levels showed improved hepatocellular function and superior survival (P = 0 VSports手机版. 04), accompanied by lower proinflammatory cytokine profile. In conclusion, our translational study is the first to identify SIRT1 as a regulator of hepatocellular function in human liver transplant recipients under ischemia/reperfusion stress. By targeting innate and adaptive immune activation, manipulation of SIRT1 signaling should be considered as a novel means to combat inflammation in liver transplantation. Liver Transplantation 23 1282-1293 2017 AASLD. .

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Conflict of interest statement (VSports app下载)

Potential conflict of interest: Nothing to report.

Figures (V体育官网入口)

FIG. 1
FIG. 1
Res attenuates hepatic IRI. Groups of mice (C57/BL6) were subjected to 90 minutes of partial liver warm ischemia followed by 6 hours of reperfusion. Some animals were pretreated with Res (25 mg/kg IP) or VHC (15% ethanol) at 1 hour of the ischemia insult. (A) Representative liver histology (original magnification, ×100). (B) Suzuki’s histological grading of liver IRI. (C) Hepatocellular function evaluated by serum ALT and AST levels (IU/L); n = 5–6/group; data are presented as mean ± SD. *P < 0.05 versus IR+Res (1-way ANOVA).
FIG. 2
FIG. 2
Res mitigates apoptosis/inflammatory cell trafficking in IR-stressed liver. (A) Representative immunohistochemical staining of TUNEL (apoptosis), Ly6-G (neutrophil), and CD68 (macrophage; original magnification, ×400). (B) Quantification of positive cells/HPF; n = 4/group; data are presented as mean ± SD. *P < 0.05 versus IR+Res (1-way ANOVA).
FIG. 3
FIG. 3
Res up-regulates SIRT1 and suppresses inflammatory cytokine production in IR-stressed liver. (A) Protein levels of SIRT1, COX-2, iNOS, Tbet, and cleaved caspase 3 were evaluated by Western blotting. The intensity of the bands was quantified with ImageJ and normalized by dividing the target band intensity by that of housekeeping β-actin. The values under the bands represent the relative ratio of normalized intensity as compared with that of IR. Representations of 3 experiments are shown. (B) Quantitative RT-PCR–assisted detection of mRNA coding for SIRT1, IL1β, MCP1, TNF-α, CXCL10, and IFNγ. Expression levels were normalized to HPRT expression, then normalized to expression of IR group (*P < 0.05 versus IR+Res; n = 4; 1-way ANOVA). Data are presented as mean ± SD. (C) Liver samples without IR stress were harvested from wild-type naïve mice 8 hours after treatment with Res (25 mg/kg IP) or VHC. The values under the bands represent the relative ratio of normalized intensity compared with that of untreated mice. Representations of 3 experiments are shown.
FIG. 4
FIG. 4
SIRT1 suppresses macrophage activation in vitro. BMDM cultures were pretreated with SIRT1-siRNA or Res (100 μM, 12 hours), and then stimulated with LPS (100 ng/mL, 6 hours). (A) Western blot–assisted detection of SIRT1, COX-2, iNOS, p-IκBα, IκBα, and p-Stat1. The intensity of the bands was normalized by dividing the target band intensity by that of β-actin. The values under the bands represent the relative ratio of normalized intensity compared with that of cells+LPS. Representatives of at least 3 experiments are shown. (B) Quantitative RT-PCR–assisted detection of mRNA coding for SIRT1, IL1β, MCP1, CXCL10, and CCL5. Expression levels were normalized to HPRT expression, then normalized to expression of cells+LPS (*P < 0.05 versus cells+LPS; n = 4; 1-way ANOVA). Data are presented as mean ± SD.
FIG. 5
FIG. 5
Res decreases the number of liver-infiltrating IFNγ+ T cells. Groups of mice (C57/BL6) were injected with Res (25 mg/kg IP) or VHC (15% ethanol). Eight hours later, lymphocytes isolated from liver samples were stained with anti-CD3 Ab and anti-IFNγ Ab and analyzed by FACS. Representations of 2 experiments are shown.
FIG. 6
FIG. 6
Res increases SIRT1 and mitigates Th1 cytokine production in vitro. Spleen lymphocytes were stimulated with ConA (5 μg/mL) with or without Res (100 μM, 12 hours). (A) Western blot–assisted detection of SIRT1, p-Stat1, and Tbet. The intensity of the bands was normalized by dividing the target band intensity by that of β-actin. The values under the bands represent the relative ratio of normalized intensity compared with that of cells+ConA. Representations of 3 experiments are shown. (B) Quantitative RT-PCR–assisted detection of mRNA coding for SIRT1, IFNγ, and IL2. Expression levels were normalized to HPRT and normalized to the expression of cells+ConA (*P < 0.05 versus cells+ConA; n = 4; 1-way ANOVA). Data are presented as mean ± SD.
FIG. 7
FIG. 7
Posttransplant hepatic SIRT1 levels and OLT patient outcomes. (A) Twenty-one posttransplant human liver bxs were divided, based on the relative SIRT1 expression level in OLT, ie, “low” (n = 11) versus “high” (n = 10), as measured by Western blots. (B) Serum ALT and AST levels at POD 1–14. The solid line indicates the high SIRT1 patient groups while the dotted line indicates the low SIRT1 patient group. (C) The cumulative probability of survival after liver transplantation, analyzed by Kaplan-Meier method. Solid line indicates high-SIRT1, and dotted line indicates low-SIRT1 patient groups. #P < 0.05 versus low-SIRT1 bx group (log-rank test). (D) Representative Western blot–assisted expression of SIRT1, COX-2, Tbet, and β-actin in bx samples (A/B: low-SIRT1; P/Q: high-SIRT1). (E) Western blot–assisted protein quantification of COX-2 and Tbet. Relative expressions were calculated by dividing the target band intensity by that of β-actin, then normalized to expression of low-SIRT1 bx. Mean ± SEM are shown. #P < 0.05 low (n = 11) versus high (n = 10) SIRT1 bx group (Mann-Whitney U test). (F) Quantitative RT-PCR–assisted detection of mRNA coding for IL1β, MCP1, TNF-α, CXCL10, and IFNγ. Expression levels were first normalized to GAPDH and then normalized to the expression of low-SIRT1 bx group. Mean ± SEM are shown. #P < 0.05 low (n = 11) versus high (n = 10) SIRT1 bx group (Mann-Whitney U test).
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