SIRT1, the mammalian ortholog of Sir2, is a rather unique NAD+-dependent enzyme and the most studied of the seven members of the mammalian sirtuin family whose primary function is deacetylation of post-translationally modified lysine residues in a variety of protein substrates (1, 2). SIRT3, SIRT4, and SIRT5 are limited to the mitochondrial matrix, while SIRT1 acts upon cellular targets in the cytosol and nucleus (3–6). SIRT1 affects an array of nuclear and cytoplasmic targets that regulate cell apoptosis, genomic stability, and inflammatory networks at the transcriptional level and the post-translational level (3).
SIRT1 activity is upregulated when energy needs are high and caloric availability is limited (7, 8).
SIRT1 uses NAD+ as a cosubstrate when the NAD+/NADH ratio is high during periods of cell energy stress. Important cellular and nuclear targets for SIRT1 include, but are not limited to: transcriptional factors (p65 of NFκB, p53, p300); epigenomic regulation by acetylated histones; stress-related, regulatory enzyme pathways such as heat shock factor protein 1, hypoxia-inducible factor 1α, and DNA repair enzymes; central metabolic enzymes including peroxisome proliferator-activated receptor (PPAR)γ coactivator-1α (PGC-1α), PPARγ-mediated lipid oxidation, and forkhead box subgroup O, along with circadian clock regulatory enzymes (1, 3, 9–13). Genetic deletion or overexpression of SIRT1 can markedly alter the health and longevity of cells in tissue culture or in genetically altered mice (1, 14).
SIRT1 can be allosterically activated by small molecule activating compounds called STACs (15, 16). The original STAC described is resveratrol, a polyphenol compound found in red wines (17), although its low affinity and lack of specificity limits its utility to explore SIRT1-dependent pharmacology (17). However, a number of more specific STACs have been identified and extensively characterized for direct activation of SIRT1 (15, 16).
The first synthetic STACs to enter the clinic were SRT2104 and SRT3025. SRT2104 (18) was the first STAC to show an increase in lifespan in mice fed a normal diet and also to improve overall health of mice most notably muscle and bone health (19). In the clinic, SRT2104 was able to show benefit in psoriasis patients (20) and to reduce cholesterol and triglycerides levels in elderly subjects (21). In preclinical studies, SRT3025 has also been shown to have positive effects in preventing bone loss (22) and to prevent atherosclerosis in ApoE −/− mice fed a high cholesterol diet (23).
In the current studies, the therapeutic utility of SRT3025 was examined in well characterized animal models of acute, generalized inflammation induced by severe bacterial infection. Using the murine CLP model of intra-abdominal sepsis and the murine pneumococcal pneumonia model, we show substantial survival benefits following the addition of SRT3025 along with standard antimicrobial agents. The targeting of SIRT1 by SRT3025 in this context was demonstrated in the lack of efficacy of SRT3025 in SIRT1 conditional (adult-inducible) knockout mice. The impact of SRT3025 on bacterial clearance, cytokine generation, and the transcriptional profiles from various target organs in these sepsis models were analyzed in both the bacterial pneumonia and peritonitis models. The data show wide-ranging effects of SIRT1 activation and indicate that these activating compounds might represent an entirely novel treatment for severe infectious disease.
MATERIALS AND METHODS
Reagents and chemicals
All chemicals and reagents used in these experiments were purchased for Sigma (St. Louis, MO) unless otherwise stated. SRT3025 was synthesized by Sirtris, A GSK Company (Cambridge, MA).
Animal care and use
Eight to twelve week old, specific pathogen-free, female C57BL/6 wild-type mice (Charles River Laboratories, Wilmington, MA) weighing 20 g to 25 g were used in these experiments. The animals were housed in an IACUC-approved facility under BSL-2 safety. The experimental protocols were approved by the institutional animal care committee before any experiments were undertaken. We followed the ARRIVE guidelines for the care of animals in biomedical research in performing these experiments (24). Mice were allowed to adjust to laboratory conditions for at least 7 days before undergoing any experimental procedures. Mice were randomly assigned to the each research group and the investigators were blinded to the treatment assignment and genotype of the mice until the studies were completed and analyzed.
Standard breeding procedures were then used to generate study populations of mice homozygous for the Sirt1-floxed allele (25) and for carrying the R26MCM transgene. The latter transgene expresses the MerCreMer cDNA from the Rosa26 locus and enables tamoxifen-inducible, ubiquitous Cre recombinase-mediated deletion of the floxed region of conditional alleles in mice. The resulting cre/not knockouts and not/not controls were induced with a seven daily doses of 200 mg/kg Tamoxifen (in corn oil). The mice were rested for at least 6 weeks prior to any experimental studies. The knockout was confirmed by RT-PCR using an exon 4 specific probe.
The cecal ligation and puncture model
The cecal ligation and puncture model of sepsis in C57BL/6 mice results in a polymicrobial infection with peritonitis. The procedure has been described in detail previously (26). Briefly, animals were anesthetized using Isoflurane (Baxter, Deerborne, IL), and the cecum was exteriorized through a midline incision. The cecum was then ligated with a 5-0 nylon monofilament suture at 90% of its length and punctured twice with a 21-gauge needle along the antimesenteric side of the cecum. The cecum was then returned to the abdominal cavity, the fascia and skin were then closed and treated with topical anesthetic and bacitracin (IVAX, Miami, FL). The animals then received a single 20 mg/kg subcutaneous dose of moxifloxacin (Schering, Kenilworth, NJ) and a 1-mL subcutaneous bolus of normal saline. The animals were then rewarmed until fully conscious. Animals were followed daily for 7 days for clinical signs of sepsis. Overtly ill animals (hypothermia and loss of righting response) were euthanized by Isoflurane-induced respiratory arrest and scored as lethally infected animals.
Animals were given the SRT3025 or vehicle control at 100 mg/kg given by oral gavage −48, −24, 0, 24, and 48 h from the CLP. Animals were monitored for 7 days S/P CLP and the survival time was assessed along with sham surgery mice (laparotomy and identification of the cecum but without ligation or puncture).
In a separate experiment timed sacrifice of animals was conducted at 24 and 48 h post CLP. Mice (n = 10 per group) were randomly assigned to receive SRT3025 at 100 mg/kg given by oral gavage −48, −24, 0, 24, and 48 h from CLP or vehicle control and were then euthanized 24 or 48 h S/P CLP. Plasma and peritoneal fluid were tested for endotoxin (Associates of Cape Cod, Woods Hole, MA) and a multiplex cytokine assay (Quansys, Logan, UT). Lipopolysaccharide levels were determined by quantitative Limulus amebocyte assay. Lung, liver, and spleen tissue were excised and analyzed histopathologically by a pathologist unaware of the treatment assignment. Five animals in each group had liver, spleen, lung, and small intestinal tissues snap frozen in RNA later and stored at −70°C for transcriptomic analysis.
The pneumococcal pneumonia model
Animals were challenged by S. pneumoniae (ATCC strain 6303) by intratracheal (IT) installation. The C57BL/6 mice were lightly anaesthetized and 50 μL of S. pneumoniae grown in mid-log phase and washed in PBS at 106 CFU was instilled directly onto the glottis by the incisor suspension method.
We used a respiratory fluoroquinolone (moxifloxacin 10 mg/kg given once daily subcutaneously for 3 days) starting 6 h after IT challenge and then at 24 and 48 h later. SRT3025 was given orally 100 mg/kg by oro-gastric feeding versus vehicle control at the same volume 2 h before and 24 and 48 h after IT challenge. Animals were randomized in a blinded fashion to four treatment groups: PBS-phosphate buffered saline + vehicle control (placebo, n = 10 mice); PBS + SRT3205 (n = 9); moxifloxacin + vehicle control (n = 10); and moxifloxacin + SRT3025 (n = 10). The primary endpoint of this study was 7-day mortality rate. Overtly ill animals (hypothermia and loss of righting response) were euthanized by Isoflurane-induced respiratory arrest and scored as lethally infected animals. All mice randomized at the beginning of the experiments were included in the final statistical analyses.
An additional set of experiments were conducted using the same microbial challenge strain and treatment regimen, except that animals were euthanized at 48 h (by Isoflurane-induced respiratory arrest) for a detailed quantitative microbiology, lung tissue histopathology, and cytokine levels found in the blood and lung tissue. The challenge dose of S. pneumoniae used in this experiment was 10-fold lower at 105 cfu per mouse. Ten animals were randomized to each of these four treatment groups. The cytokines were measured with a multiplex ELISA system. Lung tissue was removed aseptically cultured by serial 10-fold dilution. The data are presented as colony forming counts (cfu)/gram tissue. Five animals in each group had spleen, liver, lung, and small intestine excised and placed in RNA later, snap frozen and stored at −70°. Transcriptional profiling was performed by GeneLogic Inc (Gaithersburg, MD) using Illumina WG-6 v2.0 chipset. Data were analyzed using Limma package (Bioconductor, Seattle, WA).
Survival outcomes were compared by Kaplan–Meier survival plots using log-rank statistics and analyzed using a non-parametric analysis of variance test (Kruskal–Wallis test for differential survival times). Differences between two groups were analyzed by using a Mann–Whitney U test for quantitative microbiology in tissue samples, histopathology, cytokine levels, and lipopolysaccharide measurements. Numeric data are presented as the mean ± standard error unless otherwise stated. A two-tailed P value of <0.05 was considered to be statistically significant.
RNA was hybridized to Illumina MouseWG6v2 arrays and preprocessed using the manufacturer's BeadStudio pipeline. Log2 scale processed expression data were then further analyzed using the R/BioConductor (27). To determine the treatment effect, a linear model was defined with disease status (CLP and pneumonia, respectively, or sham), treatment (compound or vehicle), and tissue type as experimental factors. Model estimates were computed using the limma R package which implements an empirical Bayes method to robustly estimate variance after a linear model fit (28) followed by P value adjustment to compute the false discovery rate (FDR) (29). Genes were defined as differentially expressed if they were 1.5-fold up- or down-regulated and had an adjusted P value (FDR) ≤0.1.
SIRTI activating compounds improve survival in CLP
SRT3025 (100 mg/kg) was tested in the mouse CLP model. Oral absorption of SRT3025 was confirmed by measurement of SRT3025 in the plasma 1 h after the last oral dose of 100 mg/kg given once daily for 3 days at 430 ± 98.9 ng/mL (mean ± standard error).
The effect of SRT3025 on survival in C57/BL6 wild-type mice with severe sepsis from CLP is illustrated in Fig. 1. Animals that received SRT3025 orally with antibiotics at the time of CLP had a survival rate of 55% (16/30) at 169 h compared with 5% (1/16) in the vehicle control group (P < 0.01).
The peritoneal fluid bacterial colony counts at 48 h post CLP in the group receiving SRT3025 was significantly lower (2817 ± 974 cfu/mL) compared with control group (11,210 ± 4674; P < 0.05). Blood cultures also showed significantly reduced levels of both gram-negative (1 ± 6 vs. 70 ± 19 cfu) and gram-positive (402 ± 177 vs. 6168 ± 3263 cfu/mL) bacteria in the SRT3025-treated group compared with the vehicle control groups (P < 0.05 for both). Peritoneal LPS levels were also significantly lower in SRT3025-treated animals than in the vehicle-treated animals (1.7 ± 0.9 vs. 10.9 ± 6.5 ng/mL; P < 0.05). No significant histopathological differences were noted when comparing the SRT3025 group samples with the vehicle control group. There were no consistent differences between the SRT3025 and control groups in regard to levels of blood and peritoneal cytokines and chemokines (data not shown).
KO animals with CLP
In a separate experiment, we examined the effect of SRT3025 in SIRT-1 conditional knockout mice (25). We confirmed that the SIRT1 successfully deleted by screening mRNA using probes specific for exon 4 of the Sir1 gene, which was absent in the SIRT-1-deficient animals. Treatment with SRT3025 increased survival in the control mice (5/9) (P = 0.07) but no longer increased survival in the SIRT1 conditional knockouts (2/14) when compared with placebo controls (1/12) (Fig. 2), indicating the SIRT1-dependent nature of the increased survival produced by SRT3025.
STACs in combination with antibiotics improve outcomes in the pneumococcal pneumonia model
The effect of SRT3025 in prolonging survival in C57/BL6 wild-type mice with severe sepsis from an intratracheal challenge with S. pneumoniae (106 cfu/animal) is illustrated in Figure 3 (n = 10 per group). Oral dosing of SRT3025 in combination with moxifloxacin resulted in a 60% survival rate at 169 h compared with 0% in the group receiving placebo with moxifloxacin (P < 0.0005). When given without antibiotics, the survival rate with SRT3025 dropped to 10%. Animals treated with moxifloxacin alone showed no statistically significant mortality benefit compared with vehicle-treated mice.
The pneumococcal colony counts in lung and spleen samples at 48 h postintratracheal challenge with a lower dose of S. pneumoniae 105 cfu is demonstrated in Figure 4. Colony counts were significantly lower in groups receiving SRT3025 in comparison to controls in splenic tissue (P < 0.01). Co-administration with moxifloxacin showed an added benefit in lowering colony counts across all groups. The lowest colony counts were found in the group receiving both SRT3025 and moxifloxacin.
The major findings in the cytokine and chemokine levels in lung tissue in the pneumonia model are depicted in table one. Lung cytokines did not significantly differ by the addition of SRT3025 in the absence of concomitant antimicrobial therapy with moxifloxacin. However, highly consistent reductions in cytokine and chemokine levels were observed by the addition of SRT3025 to groups receiving moxifloxacin when compared with the vehicle control group. These differences reached statistical significance with 12 of the 16 cytokines and chemokines measured including interleukin (IL)-1 alpha, IL-1 beta, IL-2, IL-6, IL-12, IL-17, tumor necrosis factor alpha, macrophage inflammatory protein I (Table 1), but not with interferon gamma, IL-3, MCP 1, or RANTES.
Transcriptomics-common pathways, organ-specific effects of SRT3025, similarities from the lung and peritonitis models
To better understand the mechanisms underlying the protective action of SRT3025 in CLP and pneumonia models, we undertook gene expression analysis using Illumina bead arrays. Since gene expression is affected in multiple tissues, in the CLP model we examined gene expression in ileum, lung, spleen, and liver from sham, vehicle, or SRT3025-treated animals. Tissues were collected 48 h post-CLP or 48 h postintratracheal exposure to S. pneumoniae. The bead array produced expression data corresponding to about 21,000 genes in each treatment group.
A comparison of sham versus vehicle-treated CLP animals revealed a large number of differentially expressed genes (DEG, defined as both FDR <10% and absolute fold change≥1.5 between the two groups) in liver (1169 DEG), ileum (567 DEG), lung (494 DEG), and spleen (496 DEG) (Fig. 5A). Gene expression changes associated with STAC treatment overlapped with the changes in gene expression associated with CLP (Fig. 5A), an effect in the liver. SRT3025 treatment reversed gene expression changes associated with CLP (Fig. 5, A and B), consistent with the protective effects of SRT3025 observed in CLP mice.
The tissues from mice treated with S. pneumoniae also showed significant changes in gene expression relative to sham group across all tissues examined, but the effects were most apparent in lung (749 DEG) and liver (829 DEG) (Fig. 5C). Spleen (119 DEG) and ileum (463 DEG) showed fewer gene expression changes. SRT3025 treatment again produced a number of gene expression changes overlapping with the S. pneumoniae altered genes (Fig. 5C). The effect of SRT3025 was especially obvious in liver and lung (Fig. 5C). Effectively, SRT3025 treatment reversed the gene expression program associated with pneumococcal sepsis (Fig. 5D), consistent with the improved survival and reduced bacterial load observed in SRT3025-treated mice.
We further compared the gene expression program associated with CLP and pneumococcal challenge in liver, lung, ileum, and spleen. Remarkably, both challenges elicit a highly overlapping gene expression program in liver and spleen (Fig. 6). The gene expression programs in the lung and ileum revealed greater amounts of organ-specific expressed genes near the primary site of infection but remarkable levels of overlap between CLP and pneumococcal challenges suggesting that the signaling mechanisms leading to mortality in these models are generally similar, at least at the 48-h time point.
We next looked at the pathways perturbed in tissues from CLP and pneumococcal challenged animals versus sham controls, and the pathways affected by SRT3025 in tissues from CLP and pneumococcal treated mice (Fig. 7). Interestingly, the overlap at pathway level was even greater between CLP and pneumococcal induced sepsis. In liver (see Supplemental Tables 1, 2, Supplemental Digital Content 1, http://links.lww.com/SHK/A344), translational pathways were generally upregulated while a number of pathways associated with oxidoreductase activity were downregulated. Analysis of the spleen pathways indicated downregulation of immune cell activation processes (see Supplemental Tables 3, 4, Supplemental Digital Content 1, at http://links.lww.com/SHK/A344). In the lung, CLP or pneumococcal challenge increased pathways associated with immune and inflammatory processes while suppressing chemotactic and cell motility associated pathways (see Supplemental Tables 5, 6, Supplemental Digital Content 1, at http://links.lww.com/SHK/A344). Ileum responses were much greater in the CLP model and were generally indicative of upregulation of lipid metabolism and inflammatory pathways (see Supplemental Tables 7, 8, Supplemental Digital Content 1, at http://links.lww.com/SHK/A344).
The pathophysiology of sepsis is yet to be fully understood. Eradicating the invasive microbial pathogen is necessary but not always sufficient to resolve sepsis and reverse its sequelae. Therefore, modulation of the host immune response has emerged as a primary therapeutic strategy in combination with source control and antimicrobial agents.
SIRT-1 activator compounds (STACs) are small molecular weight compounds that allosterically activate SIRT1. SIRT1 is an NAD+-dependent deacetylase which modulates the activity and function of an array of nuclear targets that regulate cell survival and metabolism. SIRT1 activity is upregulated during periods of cellular stress particularly when energy needs are high and caloric availability is limited (3, 9, 13).
Our study looked at the effect of the direct SIRT1 activator SRT3205 on survival and immune response in two distinct models of sepsis in mice. When administered in the setting of sepsis in C57BL/6 wild-type mice models, SRT3025 treatment showed significant improvement in survival outcome when compared with placebo with or without antibiotics. Treatment with SRT3025 was accompanied by striking changes in the transcription profiles in multiple inflammatory and metabolic pathways in numerous tissues.
Remarkably, these organ-specific changes in the transcriptome analyses were similar following CLP or pneumonia despite different sets of pathogens and disparate sites of infection, consistent with a general host immunomodulatory mechanism of Sirtuin-1 activation (Figs. 5 and 6). However, important disparities in differentially expressed genes are evident in different organs and, while similarities are marked with down-regulated genes, disparities are notable with up-regulated genes following CLP or pneumonia (Fig. 7). These differences are future explored in heat maps provided in the supplemental files (see Figure, Supplemental Digital Content 2, at http://links.lww.com/SHK/A345). Further research will be required to interrogate these differences and dissect the relevance of specific signaling events affected by SIRT 1 activators to direct the development of novel therapeutics.
SRT3025 promoted bacterial clearance and reduced inflammatory cytokines from the tissues of animals challenged with S. pneumoniae. This effect was most prominently observed in lung. As expected, the antimicrobial agent moxifloxacin also significantly reduced colony counts of pneumococci in tissues and appeared to be additive to the improved microbial clearance provided by the SIRT1 activator. The impact of the antimicrobial might have been greater if larger doses were administered. The dosing strategy used here attempted to employ clinically relevant doses (10 mg/kg in mice rather than the usual dose in patients which is 5 mg/kg as a single dose daily). The ability of SRT3025 to improve survival in CLP mice was lost in SIRT1 conditional KO mice strongly suggesting that the beneficial effects of SRT3025 require SIRT1 catalytic activity.
Our findings are supported by a recent paper which showed that the SIRT1 inhibitor EX-527 worsened survival in a CLP model when administered at the time of insult (30). Interestingly, the same compound, EX-527, was protective when it was administered during the hypoinflammatory phase following CLP-induced intra-abdominal sepsis (30). In these studies with EX-527, the therapy was administered in the absence of concomitant antibiotics against enteric microorganisms. The epigenetic effects of SIRT1 deacetylase inhibition on histone proteins in late sepsis are likely substantially different than the effects of SIRT1 activators given before the infectious insult. Additionally, the adjuvant use of antibiotics in both the CLP and pneumonia models used in the experiments described herein alters the host-pathogen balance favoring attenuation of systemic inflammation by SIRT1 activators in concert with increased bacterial clearance afforded by antimicrobial therapy. In preliminary studies we tested the ability of SRT3025 to rescue mice already ill following CLP and found little evidence for activity. In order for SIRT1 activators to work in sepsis, they will likely need to be given in the early phase of the host inflammatory response to systemic infection.
Three recent reports support our findings that SIRT-1 activation can be protective in the early phases of experimental sepsis. Gao et al. (31) noted that Sirt-1-deficient mice were more likely to succumb from sepsis following CLP and that this was mediated by excess activation of lung inflammasome. Hansen et al. (32) demonstrated that another Sirtuin 1 activator protects mitochondrial function and limits multiorgan injury following intestinal ischemia-reperfusion injury in mice. Yoo et al. (33) found interferon beta-mediated SIRT-1 upregulation limited pro-inflammatory cytokine production and protected against murine endotoxic shock. STACs are most likely to be beneficial clinically if administered early with appropriate antibiotics at the onset of severe infection such as community-acquired pneumonia or the early phases of peritonitis. Plans to develop STACs for clinical use will focus on early intervention before sepsis-induced immune suppression is manifest.
These findings indicate that SIRT1 activation can protect against an array of microbial pathogens by modulating the initial acute phase and innate immune response to numerous pathogens and could represent a novel treatment strategy for severe infection. SIRT1 activators might be particularly useful as an adjuvant therapy with antimicrobial agents in the management of pneumococcal pneumonia. This infection continues to exact an annual toll of over one million deaths of young children and older adults each year worldwide, despite the availability of vaccines and antibiotics active against this common respiratory pathogen (34).
1. Guarente L. Sirtuins
, aging, and medicine. N Engl J Med
2. Feldman JL, Dittenhafer-Reed KE, Denu JM. Sirtuin catalysis and regulation. J Biol Chem
3. Hagis MC, Sinclair DA. Mammalian sirtuins
: biological insights and disease relevance. Annu Rev Pathol
4. Huang JY, Hirschey MD, Shimazu T, Ho L, Verdin E. Mitochondrial sirtuins
. Biochim Biophys Acta
2010; 1804 8:1645–1665.
5. Sol EM, Wagner SA, Weinert BT, Kumar A, Kim HS, Deng CX, Choudhary C. Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3. PLoS One
2012; 7 12:e50545.
6. Chen Y, Zhao W, Yang JS, Cheng Z, Luo H, Lu Z, Tan M, Gu W, Zhao Y. Quantitative acetylome analysis reveals the roles of SIRT1
in regulating diverse substrates and cellular pathways. Mol Cell Proteomics Oct
2012; 11 10:1048–1062.
7. Smith JJ, Kenney RD, Gagne DJ, Frushour BP, Ladd W, Galonek HL, Israelian K, Song J, Razvadauskaite G, Lynch AV, et al. Small molecule activators of SIRT1
replicate signaling pathways triggered by caloric restriction in vivo. BMJ Syst Biol
8. Sauve AA, Wolberger C, Schramm VL, Boeke JD. The biochemistry of sirtuins
. Annu Rev Biochem
9. Denu JM, Gottesfeld JM. Minireview Series on Sirtuins
: from biochemistry to health and disease. J Biol Chem
2012; 287 51:42417–42418.
10. Imai S, Armstrong CM, Kaeberlein M, Guarente L. Transcriptional silencing and longevity protein SIRT1
is NAD-dependent histone deacetylase. Nature
11. Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM, Puigserver P. Nutrient control of glucose homeostasis through a complex of PGC-1α in SIRT-1. Nature
12. Motta MC, Divecha N, Lemieux M, Kamel C, Chen D, Gu W, Bultsma Y, McBurney M, Guerente L. Mammalian SIRT1
represses forkhead transcription factors. Cell
13. Yeung F, Holberg JE, Ramsey CS, Keller MD, Jones DR, Frye RA, Mayo MW. Modulation of NF-kB-dependent transcription and cell survival by the SIRT1
deacetylase. EMBO J
14. Guarente L. Mitochondria: a nexus of aging caloric restriction and sirtuins
15. Dai H, Kustigian L, Carney D, Case A, Considine T, Hubbard BP, Perni RB, Riera TV, Szczepankiewicz B, Vlasuk GP, et al. SIRT1 activation
by small molecules: kinetic and biophysical evidence for direct interaction of enzyme and activator. J Biol Chem
16. Hubbard BP, Gomes AP, Dai H, Li J, Case AW, Considine T, Riera TV, Lee JE, E SY, Lamming DW, et al. Evidence for a common mechanism of SIRT1
regulation by allosteric activators. Science
2013; 339 6124:1216–1219.
17. Baur JA, Sinclair DA. Therapeutic potential of resveratrol: the in vivo evidence. Nat Rev Drug Discov
18. Ng PY, Bemis JE, Disch JS, Vu CB, Oalmann CJ, Lynch AV. The identification of the SIRT1
activator SRT 2014 as a clinical candidate. Lett Drug Design Discov
19. Mercken EM, Mitchell SJ, Martin-Montalvo A, Minor RK, Almeida M, Gomes AP, Scheibye-Knudsen M, Palacios HH, Licata JJ, Zhang Y, et al. SRT2104 extends survival of male mice on a standard diet and preserves bone and muscle mass. Aging Cell
20. Jacobson EW, Suárez-Fariñas M, Cueto I, Katcherian A, Matheson R, Parish LC, Shortino D, Gupta A, Vlasuk GP, Krueger JG. 12 weeks of treatment with an oral SIRT1
activator, SRT2104, leads to clinical improvement and skin microarray modification in patients with psoriasis. J Invest Derm
21. Libri V, Brown AP, Gambarota G, Haddad J, Shields GS, Dawes H, Pinato DJ, Hoffman E, Elliot PJ, Vlasuk GP, et al. A pilot randomized, placebo controlled, double blind phase I trial of the novel SIRT1
activator SRT2104 in elderly volunteers. PLoS One
2012; 7 12:e15395.
22. Artsi H, Cohen-Kfir E, Gurt I, Shahar R, Bajayo A, Kalish N, Bellido TM, Gabet Y, Dresner-Pollak R. The sirtuin1 activator SRT3025 down-regulates sclerostin and rescues ovariectomy-induced bone loss and biomechanical deterioration in female mice. Endocrinology
23. Miranda MX, van Tits LJ, Lohmann C, Arsiwala T, Winnik S, Talleux A, Stein S, Gomes AP, Suri V, Ellis JL, et al. The SIRT1
activator SRT3025 provides atheroprotection in Apoe -/- mice by reducing hepatic PCSK9 secretion and enhancing Ldlr expression. Euro Heart J
24. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol
2010; 8 6:e1000412.
25. Price NL, Gomes AP, Ling AJY, Duarte FU, Martin-Montalvo A, North B, Agarwal B, Ye L, Ramadori G, Teodoro JS, et al. SIRT1
is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function. Cell Metab
26. Hubbard WJ, Choudhry MA, Schwacha MG, Kerby JD, Rue LW, Bland KI, Chaudry IH. Cecal ligation and puncture. Shock
2005; 24 (suppl 1):52–57.
27. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol
2004; 5 10:R80.
28. Smyth GK. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol
2004; 3: Article 3.
29. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Statist Soc Series B
30. Vachharajani VT, Liu TF, Brown CM, Wang X, Buechler NL, Wells JD, Yoza BK, McCall CE. SIRT1
inhibition during the hypoinflammatory phenotype of sepsis
enhances immunity and improves outcome. J Leuk Bio
2014; 96 5:785–796.
31. Gao R, Ma Z, Hu Y, Chen J, Shetty S, Fu J. Sirt1
restrains lung inflammasome activation in a murine model of sepsis
. Am J Physio Lung Mol Physio
2015; 308 8:L847–L853.
32. Hansen LW, Khader A, Yang WL, Prince JM, Nicastro JM, Coppa GF, Wang P. Sirtuin 1 activator SRT1720 protects against organ injury induced by intestinal ischemia-reperfusion. Shock
2016; 45 4:359–366.
33. Yoo C-H, Yeom J-H, Heo J-J, Song E-K, Lee S-I, Han M-K. Interferon beta protects against lethal endotoxic and septic shock
through SITR1 upregulation. Sci Rep
34. Van der Poll T, Opal SM. The molecular pathogenesis of pneumococcal pneumonia