SIRT1 Activator Resveratrol Research Articles

Involvement of TACE in colon inflammation: A novel mechanism of regulation via SIRT-1 activation

TNF-α converting enzyme (TACE) processes the membrane TNF-α to release the bioactive soluble TNF-α. Several evidences suggest the involvement of TNF-α and TACE in inflammatory bowel disease (IBD). Tissue inhibitor of metalloproteinase (TIMP)-3, an endogenous inhibitor of TACE, is positively associated with silent information regulator (SIRT)-1. We aimed to study the expression of TACE, TIMP-3 and SIRT-1 at different stages of colitis and how TACE is regulated in response to SIRT-1 activation. Acute colitis was induced by 3.5% dextran sulfate sodium (DSS) in drinking water for 5days and levels of cytokines and mRNA expression of TACE, TIMP-3 and SIRT-1 were measured in colon at different time intervals. Next, the effect of SIRT-1 activator (resveratrol) or a selective TACE inhibitor (compound 11p) treatment was evaluated. Elevated levels of TNF-α, interleukin (IL)-6, IL-1β, interferon (IFN)-γ and IL-17 were observed during DSS exposure phase which restored to the normal level after DSS removal. A significant increase in TACE and suppression in TIMP-3 and SIRT-1 mRNA level was observed during DSS exposure phase which reverts back to normal towards the remission phase. Treatment with resveratrol significantly elevated SIRT-1 and TIMP-3 and suppressed TACE mRNA expression and was associated with amelioration of disease. Furthermore, treatment with selective TACE inhibitor significantly suppressed body weight loss, disease activity index, colonic myeloperoxidase activity and the elevated levels of cytokines after DSS challenge. These results strongly emphasize the involvement of TACE in colon inflammation and inhibition of TACE directly or indirectly via SIRT-1 activation ameliorates colitis.

Vitamin C protected human retinal pigmented epithelium from oxidant injury depending on regulating SIRT1.

The purpose was to investigate the protective effects of Vitamin C (Vit C) and the regulatory mechanism between Vit C and sirtuin 1 (SIRT1) in PREs during oxidative stress as Vit C and SIRT1 exerted famous effects as antioxidants. We found that moderate Vit C (100 µM) prevented ARPE-19 cells from damages induced by H2O2, including increasing viability, reducing apoptosis, and attenuating intracellular ROS levels. But lower and higher concentration of Vit C had no effects. Further results indicated that Vit C caused the dysregulation of some stress responses factors (SIRT1, p53 and FOXO3) in ARPE-19 cells response to H2O2. Moreover we found that SIRT1 activator resveratrol (SRV) stimulated significantly the protective effects of moderate Vit C, provided the property of antioxidative stress for the lower and higher concentration of Vit C in ARPE-19 cells as well. Consistently, nicotinamide (NA) relieved the protective functions of moderate Vit C. Interestingly, data also revealed the dysregulation of p53 and FOXO3 was dependent on the regulation of SIRT1 rather than Vit C. Summarily, the protective effect of Vit C against oxidative stress was involved in regulation of SIRT1. It suggested that combined application of Vit C and RSV might be a promising therapeutic method for AMD.

Enhancement in efferocytosis of oxidized low-density lipoprotein-induced apoptotic RAW264.7 cells through Sirt1-mediated autophagy

Macrophages play a key role in atherosclerotic plaque formation and rupture. These phagocytic cells are important in the scavenging of modified lipoproteins, unwanted or dead cells and cellular debris through efferocytosis. Sirtuin1 (Sirt1), a member of the conserved sirtuin family and a key regulator in the progression of atherosclerosis exerts protective effects by regulating autophagy, a well-known survival mechanism. Inhibition of autophagy may also result in defective efferocytosis. This study aimed to investigate the effect of Sirt1 on the efferocytosis of oxidized low-density lipoprotein (ox-LDL)-induced apoptotic RAW264.7 cells through upregulation of autophagy. The apoptotic cells were incubated with high and low concentrations of Sirt1 activator resveratrol (RSV) and Sirt1 inhibitor nicotinamide (NAM) as well as autophagy inhibitor 3-methyladenine (3-MA) + low concentration RSV. Apoptosis was determined by flow cytometry (FCM) of annexin-V/propidium iodide (AV/PI) dual staining. Total proteins were extracted and protein levels were detected through western blot analysis. The ox-LDL uptake and efferocytosis of apoptotic RAW264.7 cells were detected by oil red O staining and calculation of the phagocytic index of apoptotic RAW264.7 cells. The expression of Sirt1 and autophagy marker proteins was simultaneously increased with the stimulation of low concentration RSV (all P<0.05) and decreased in low and high NAM groups (all P<0.05), compared with the control group. Efferocytosis was highest in the low concentration RSV group (P<0.001) and relatively lower in the low and high concentration NAM groups (both P<0.05) compared with the control group, which was similar to the change in the expression of Sirt1 and autophagy marker proteins. The results showed that the efferocytosis of apoptotic RAW264.7 cells was significantly improved with the upregulation of Sirt1-mediated autophagy. Therefore, Sirt1 may serve as a novel therapeutic target for the treatment of atherosclerosis.

Dysfunction of Endothelial Progenitor Cells from Smokers and Chronic Obstructive Pulmonary Disease Patients Due to Increased DNA Damage and Senescence

Cardiovascular disease (CVD) is a major cause of death in smokers, particularly in those with chronic obstructive pulmonary disease (COPD). Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis, and their dysfunction contributes to CVD. To investigate EPC dysfunction in smokers, we isolated and expanded blood outgrowth endothelial cells (BOEC) from peripheral blood samples from healthy nonsmokers, healthy smokers, and COPD patients. BOEC from smokers and COPD patients showed increased DNA double-strand breaks and senescence compared to nonsmokers. Senescence negatively correlated with the expression and activity of sirtuin-1 (SIRT1), a protein deacetylase that protects against DNA damage and cellular senescence. Inhibition of DNA damage response by silencing of ataxia telangiectasia mutated (ATM) kinase resulted in upregulation of SIRT1 expression and decreased senescence. Treatment of BOEC from COPD patients with the SIRT1 activator resveratrol or an ATM inhibitor (KU-55933) also rescued the senescent phenotype. Using an in vivo mouse model of angiogenesis, we demonstrated that senescent BOEC from COPD patients are dysfunctional, displaying impaired angiogenic ability and increased apoptosis compared to cells from healthy nonsmokers. Therefore, this study identifies epigenetic regulation of DNA damage and senescence as pathogenetic mechanisms linked to endothelial progenitors' dysfunction in smokers and COPD patients. These defects may contribute to vascular disease and cardiovascular events in smokers and could therefore constitute therapeutic targets for intervention.

Resveratrol Attenuates CoCl2-Induced Cochlear Hair Cell Damage through Upregulation of Sirtuin1 and NF-κB Deacetylation

The goals of this study were to investigate the effects of hypoxia on cochlear hair cell damage, and to explore the role of sirtuin1 in hypoxia-induced hair cell damage. Cochlear organotypic cultures from postnatal day 4 rats were used in this study. Hypoxia was induced by treating cochlear explants with CoCl2. Cochlear cultures were treated with CoCl2 alone or in combination with the sirtuin1 activator resveratrol and the sirtuin1 inhibitor sirtinol. Hair cell damage was identified by phalloidin staining and imaged using scanning electron microscopy. RT-PCR and Western blot analyses were used to detect the expression of sirtuin1 and acetylated nuclear factor-κB (NF-κB). Low concentrations of CoCl2 (100–200 μM) did not cause an obvious change in the number and morphology of hair cells, whereas higher concentrations of CoCl2 (300–400 μM) induced swelling of hair cells, accompanied by cell loss. Increased sirtuin1 expression was induced by CoCl2 at 100 to 200 μM, but not at 400 μM. NF-κB acetylation was significantly increased in explants treated with 400 μM CoCl2. Pretreatment with resveratrol prevented CoCl2-induced hair cell loss and acetylation of NF-κB. The protective effect of resveratrol was significantly reduced by sirtinol. CoCl2 induces hair cell damage in organotypic cochleae cultures. Resveratrol attenuates CoCl2-induced cochlear hair cell damage possibly via activation of sirtuin1, which deacetylates NF-κB.

T5 Circulating endothelial progenitor cells in smokers and patients with COPD are dysfunctional due to increased DNA damage and senescence

IntroductionCardiovascular disease (CVD) is a major cause of death in smokers, particularly in patients with chronic obstructive pulmonary disease (COPD). Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis,...

Crystal Structures of Sirt3 Complexes with 4′-Bromo-Resveratrol Reveal Binding Sites and Inhibition Mechanism

Sirtuins are protein deacetylases regulating aging processes and various physiological functions. Resveratrol, a polyphenol found in red wine, activates human Sirt1 and inhibits Sirt3, and it can mimic calorie restriction effects, such as lifespan extension in lower organisms. The mechanism of Sirtuin modulation by resveratrol is not well understood. We used 4'-bromo-resveratrol (5-(2-(4-hydroxyphenyl)vinyl)-1,3-benzenediol) to study Sirt1 and Sirt3 modulation. Despite its similarity to the Sirt1 activator resveratrol, the compound potently inhibited both, Sirt1 and Sirt3. Crystal structures of Sirt3 in complex with a fluorophore-labeled and with a native substrate peptide, respectively, in presence of 4'-bromo-resveratrol reveal two compound binding sites. Biochemical studies identify the internal site and substrate competition as the mechanism for inhibition, providing a drug target site, and homology modeling suggests that the second, allosteric site might indicate the site for Sirt1 activation.

168 CIRCULATING ENDOTHELIAL PROGENITOR CELLS IN SMOKERS ARE DYSFUNCTIONAL DUE TO INCREASED DNA DAMAGE AND SENESCENCE

Introduction Cardiovascular disease (CVD) is a major cause of death in smokers, particularly in patients with chronic obstructive pulmonary disease (COPD). Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis, and their dysfunction contributes to CVD. DNA damage has also been recognized as an important contributor to CVD. Our aim was to investigate whether EPC from smokers and COPD patients are dysfunctional, and to investigate the role of DNA damage pathways in mediating endothelial dysfunction in these patients. Methods To investigate EPC dysfunction in smokers, we isolated and expanded blood outgrowth endothelial cells (BOEC) from peripheral blood samples of healthy non-smokers, healthy smokers and COPD patients. The mononuclear fraction was placed in culture in the presence of endothelial growth factors and BOEC colonies appeared between days 7 and 24. BOEC colonies were expanded and used at passages 4 to 6 for all experiments. Endothelial senescence was measured by senescence-associated β-galactosidase (SA-β-Gal) activity. Expression of sirtuin (SIRT)-1 and of markers of senescence and DNA damage were measured by Western blotting and/or immunofluorescence confocal microscopy. SIRT1 activity was measured using a SIRT1 fluorescent activity assay kit. To investigate in vivo function, BOEC were labelled with Vybrant DiI Cell-Labelling Solution, mixed with Matrigel and injected subcutaneously into the back of NOD.CB17-Prkdcscid/NcrCrl mice. Seven days later, the mice were sacrificed and the plugs were harvested and cryosectioned. Results In vitro, BOEC from smokers and COPD patients showed increased DNA double-strand breaks (measured by γ-H2AX, 53BP1) and senescence (senescence associated-β-galactosidase activity, p16 and p21 levels) compared to non-smokers. Senescence negatively correlated with sirtuin-1 (SIRT1) expression and activity, a protein deacetylase that inhibits DNA damage and cellular senescence. Inhibition of DNA damage response by silencing of ataxia telangiectasia-mutated (ATM) kinase resulted in up-regulation of SIRT1 expression and decreased senescence. Interestingly, treatment of BOEC from COPD patients with the SIRT1 activator resveratrol or a selective ATM inhibitor rescued the senescent phenotype. Using the in vivo Matrigel plug angiogenesis assay, BOEC from COPD patients displayed reduced ability to form capillary-like structures and increased DNA damage, senescence and apoptosis (measured by 53BP1, p16, TUNEL and cleaved-caspase 3 staining) compared to non-smokers. Conclusions BOEC from smokers and COPD patients show reduced angiogenesis in vivo and display increased DNA damage and senescence, associated with reduced SIRT1 expression. These defects may contribute to endothelial dysfunction and cardiovascular events in smokers and COPD patients and could potentially constitute therapeutic targets for intervention.

Endothelial progenitor cells in smokers are dysfunctional because of increased DNA damage and senescence

BackgroundCardiovascular disease (CVD) is a major cause of death in smokers, especially in patients with chronic obstructive pulmonary disease (COPD). The molecular pathways that lead to endothelial dysfunction and CVD due to cigarette smoke remain unclear. DNA damage has been recognised as an important contributor in ageing disorders, including CVD. Circulating endothelial progenitor cells (EPC) are required for endothelial homoeostasis, and their dysfunction contributes to CVD. This study aimed to examine whether circulating EPC (also called blood outgrowth endothelial cells [BOEC]) from smokers and COPD patients are dysfunctional, and to investigate the role of DNA damage pathways in mediating endothelial dysfunction in these patients. MethodsBOEC were isolated from peripheral blood samples received from 16 healthy non-smokers (five men, 11 women; mean age 57 years [SE 2·7]), ten healthy smokers (five men, five women; 57 years [2·6]), and 16 COPD patients (11 men, five women; 67 years [1·6]). Endothelial senescence was measured by senescence-associated β-galactosidase (SA-β-gal) activity. Protein levels of sirtuin 1 (SIRT1) were measured by western blotting, expression of p16, γ-H2AX, and 53BP1 by immunofluorescence, and p21 by western blotting and immunofluorescence. SIRT1 activity was measured with a SIRT1 fluorescent activity assay kit. FindingsBOEC from smokers and COPD patients showed evidence of increased DNA double-strand breaks (increased γ-H2AX, 53BP1) compared with non-smokers. BOEC from healthy smokers and COPD patients displayed increased senescence (measured by SA-β-gal activity, p16, and p21) and decreased SIRT1 expression and activity compared with controls. SIRT1 protein levels and activity negatively correlated with senescence, indicating a regulatory role of SIRT1 on senescence. Interestingly, treatment of BOEC from COPD patients with a SIRT1 activator (resveratrol) rescued the senescent phenotype. InterpretationThe results from our study demonstrate that BOEC from smokers and COPD patients display increased DNA damage and senescence, associated with reduced SIRT1 expression and activity. These defects may contribute to endothelial dysfunction and cardiovascular events in people who smoke and could potentially constitute therapeutic targets for intervention. FundingImperial College London.

Sirt1 Mediates Hyperbaric Oxygen Preconditioning-Induced Ischemic Tolerance in Rat Brain

Our previous studies have shown that hyperbaric oxygen preconditioning (HBO-PC) induces tolerance to cerebral ischemia/reperfusion (I/R). This study aimed to investigate whether SirT1, a class III histone deacetylase, is involved in neuroprotection elicited by HBO-PC in animal and cell culture models of ischemia. Rats were subjected to middle cerebral artery occlusion for 120 minutes after HBO-PC (once a day for 5 days). Primary cultured cortical neurons were exposed to 2 hours of HBO-PC after 2 hours of oxygen–glucose deprivation (OGD). We showed that HBO-PC increased SirT1 protein and mRNA expression, promoted neurobehavioral score, reduced infarct volume, and improved morphology at 24 hours and 7 days after cerebral I/R. Neuroprotection of HBO-PC was attenuated by SirT1 inhibitor EX527 and SirT1 knockdown by short interfering RNA (siRNA), whereas it was mimicked by SirT1 activator resveratrol. Furthermore, HBO-PC enhanced SirT1 expression and cell viability and reduced lactate dehydrogenase release 24 hours after OGD/re-oxygenation. The neuroprotective effect of HBO-PC was emulated through upregulating SirT1 and, reversely, attenuated through downregulating SirT1. The modulation of SirT1 was made by adenovirus infection carrying SirT1 or SirT1 siRNA. Besides, SirT1 increased B-cell lymphoma 2 (Bcl-2) expression and decrease cleaved caspase 3. These results indicate that SirT1 mediates HBO-PC-induced tolerance to cerebral I/R through inhibition of apoptosis.

Sirtuin 1 affects the transcriptional expression of adipose triglyceride lipase in porcine adipocytes1

To investigate whether sirtuin 1 (Sirt1) could affect the transcriptional expression of the adipose triglyceride lipase (ATGL) gene, we treated porcine adipocytes with the general Sirt1 activator resveratrol (RES) with the Sirt1 inhibitor nicotinamide (NAM) or a knockdown of Sirt1 by Sirt1-specific small interfering RNA (siRNA). The RES (50 μM) activated Sirt1 gene expression and increased ATGL gene expression and glycerol release (P < 0.01). The Sirt1 inhibitor NAM or knockdown with Sirt1 siRNA further proved that the ATGL mRNA abundances were decreased (P < 0.05) after inhibition with Sirt1 in adipocytes. Furthermore, we found the opposite Sirt1 regulation pattern for PPARγ to that of ATGL in adipocytes. In summary, Sirt1 regulates the transcriptional expression of ATGL in adipocytes, and PPARγ appears to have an important role in this process. These results add to our understanding of the role of Sirt1 in adipose mobilization.

Measles virus nucleocapsid protein, a key contributor to Paget's disease, increases IL-6 expression via down-regulation of FoxO3/Sirt1 signaling

Measles virus plays an important role as an environmental factor in the pathogenesis of Paget's disease (PD). Previous studies have shown that IL-6 is increased in the bone marrow of Paget's patients and that measles virus nucleocapsid protein (MVNP) induces IL-6 secretion by pagetic osteoclasts. Further, IL-6 plays a critical role in the development of pagetic osteoclasts and bone lesions induced by PD, but the mechanisms regulating IL-6 production by MVNP remain unclear. Our current studies revealed that MVNP expression in osteoclast precursors down-regulated Sirt1 mRNA and protein, a negative regulator of NF-κB activity, which is a key factor for IL-6 expression. MVNP expression in NIH3T3 cells also elevated Il-6 transcription and impaired the expression of Sirt1 mRNA both under basal conditions and upon activation of the Sirt1 upstream regulator FoxO3 by LY294002 (a PI3K/AKT inhibitor). Luciferase activity assays showed that constitutively active FoxO3 abolished the repressive effect of MVNP on reporters driven by either FoxO3 response elements or the Sirt1 promoter. Further, protein stability assays revealed that FoxO3 was degraded more rapidly in MVNP-expressing cells than in control cells following the addition of cycloheximide. Similarly, co-transfection of MVNP and FoxO3 into HEK293 cells demonstrated that MVNP decreased the protein levels of over-expressed FoxO3 in a dose-dependent manner. Treatment with the proteasome inhibitor, MG132, blocked the MVNP-triggered decrease of FoxO3, and the treatment with the serine/threonine phosphatase inhibitor, calyculin A, revealed that MVNP increased phosphorylation of FoxO3. Further, over-expression of Sirt1 or treatment with the Sirt1 activator resveratrol blocked the increase in Il-6 transcription by MVNP. Finally, resveratrol reduced the numbers of TRAP positive multi-nuclear cells in bone marrow cultures from TRAP-MVNP transgenic mice to wild type levels. These results indicate that MVNP decreases FoxO3/Sirt1 signaling to enhance the levels of IL-6, which in part mediate MVNP's contribution to the development of Paget's disease.

Mitochondria targeted therapeutic approaches in Parkinson's and Huntington's diseases

Substantial evidence from both genetic and toxin induced animal and cellular models and postmortem human brain tissue indicates that mitochondrial dysfunction plays a central role in pathophysiology of the neurodegenerative disorders including Parkinson's disease (PD), and Huntington's disease (HD). This review discusses the emerging understanding of the role of mitochondrial dysfunction including bioenergetics defects, mitochondrial DNA mutations, familial nuclear DNA mutations, altered mitochondrial fusion/fission and morphology, mitochondrial transport/trafficking, altered transcription and increased interaction of pathogenic proteins with mitochondria in the pathogenesis of PD and HD. This review recapitulates some of the key therapeutic strategies applied to surmount mitochondrial dysfunction in these debilitating disorders. We discuss the therapeutic role of mitochondrial bioenergetic agents such as creatine, Coenzyme-Q10, mitochondrial targeted antioxidants and peptides, the SIRT1 activator resveratrol, and the pan-PPAR agonist bezafibrate in toxin and genetic cellular and animal models of PD and HD. We also summarize the phase II-III clinical trials conducted using some of these agents. Lastly, we discuss PGC-1α, TORC and Sirtuins as potential therapeutic targets for mitochondrial dysfunction in neurodegenerative disorders. This article is part of a Special Issue entitled 'Mitochondrial function and dysfunction in neurodegeneration'.

Resveratrol Rescues SIRT1-Dependent Adult Stem Cell Decline and Alleviates Progeroid Features in Laminopathy-Based Progeria

Abnormal splicing of LMNA gene or aberrant processing of prelamin A results in progeroid syndrome. Here we show that lamin A interacts with and activates SIRT1. SIRT1 exhibits reduced association with nuclear matrix (NM) and decreased deacetylase activity in the presence of progerin or prelamin A,leading to rapid depletion of adult stem cells (ASCs) in Zmpste24(-/-) mice. Resveratrol enhances the binding between SIRT1 and A-type lamins to increases its deacetylase activity. Resveratrol treatment rescues ASC decline, slows down body weight loss, improves trabecular bone structure and mineral density, and significantly extends the life span in Zmpste24(-/-) mice. Our data demonstrate lamin A as an activator of SIRT1 and provide a mechanistic explanation for the activation of SIRT1 by resveratrol. The link between conserved SIRT1 longevity pathway and progeria suggests a stem cell-based and SIRT1 pathway-dependent therapeutic strategy for progeria.

Regulation of SIRT1 determines initial step of endometrial receptivity by controlling E-cadherin expression

Sirtuin 1 (SIRT1), originally found as a class III histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. We examined the role of SIRT1 in the regulation of uterine receptivity using Ishikawa and RL95-2 endometrial carcinoma cell lines. Exogenous expression of SIRT1 significantly enhanced E-cadherin expression, while small interfering RNA-mediated depletion of endogenous SIRT1 resulted in a significant reduction of E-cadherin expression. A SIRT1 activator resveratrol elevated E-cadherin expression in a dose dependent manner, while SIRT1 repressors nicotinamide and sirtinol exhibited a dose dependent reduction of E-cadherin expression. We also showed that both forced expression of SIRT1 and activation of SIRT1 promote E-cadherin-driven reporter gene constructs, and SIRT1 is localized at E-cadherin promoter containing E-box elements in Ishikawa cells. Using an in vitro model of embryo implantation, we demonstrate that exogenous expression of SIRT1 and stimulation of SIRT1 activity resulted in the Ishikawa cell line becoming receptive to JAR cell spheroid attachment. Furthermore, resveratrol enhanced E-cadherin and Glycodelin protein expression at sites of intercellular contact, suggesting an additive role of resveratrol in promoting implantation. The initial step of human reproduction depends on the capacity of an embryo to attach and implant into the endometrial wall, and these results revealed the novel mechanism that activation and increased expression of SIRT1 play an important role in uterine receptivity.

Sirt-1 Is Required for the Inhibition of Apoptosis and Inflammatory Responses in Human Tenocytes

Tendon overuse injuries and tendinitis are accompanied by catabolic processes and apoptosis of tenocytes. However, the precise molecular mechanisms of the destructive processes in tendon are not fully understood. Sirt-1, a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, has been linked to transcriptional silencing and appears to play a key role in inflammation. The purpose of this study was to examine whether down-regulation of Sirt-1 using antisense oligonucleotides (ASO) affects inflammatory and apoptotic signaling in tenocytes. Transient transfection of tenocytes with ASO against Sirt-1 induced expression of Bax and other proteins involved in apoptosis (cleaved caspase-3 and poly(ADP-ribose)polymerase), acetylation of tumor suppressor p53, and mitochondrial degradation. Interestingly, Sirt-1 was found to interact directly with p53. In contrast, Sirt-1 activator resveratrol inhibited interleukin-1β (IL-1β)- and nicotinamide-induced NF-κB activation and p65 acetylation and suppressed the activation of IκB-α kinase. Resveratrol also reversed the IL-1β- or nicotinamide-induced up-regulation of various gene products that mediate inflammation (cyclooxygenase-2) and matrix degradation (matrix metalloproteinase-9) that are known to be regulated by NF-κB. Knockdown of Sirt-1 by using ASO abolished the inhibitory effects of resveratrol on inflammatory and apoptotic signaling including Akt activation and SCAX suppression. Down-regulation of histone deacetylase Sirt-1 by mRNA interference abrogated the effect of resveratrol on NF-κB suppression, thus highlighting the crucial homeostatic role of this enzyme. Overall, these results suggest for the first time that Sirt-1 can regulate p53 and NF-κB signaling via deacetylation, demonstrating a novel role for resveratrol in the treatment of tendinitis/tendinopathy.

Mitochondrial dysfunction accounts for aldosterone-induced epithelial-to-mesenchymal transition of renal proximal tubular epithelial cells

Epithelial–mesenchymal transition (EMT) plays a pivotal role in the pathogenesis of renal tubulointerstitial fibrosis. We previously demonstrated that aldosterone (Aldo)-induced EMT is dependent on mitochondrial-derived oxidative stress. This study investigated whether mitochondrial dysfunction (MtD) is involved in the pathogenesis of EMT and whether peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a major regulator of oxidative metabolism and mitochondrial function, prevents EMT by improving MtD. Aldo decreased PGC-1α expression while increasing its acetylation and induced MtD, as evidenced by oxidative stress, mitochondrial membrane potential collapse, mitochondrial DNA damage, and mitochondrial complex activity reduction. Aldo time-dependently induced p66Shc phosphorylation and expression. Mineralocorticoid receptor antagonist eplerenone and p66Shc short interfering RNA prevented Aldo-induced MtD and EMT, as evidenced by downregulation of α-smooth muscle actin and upregulation of E-cadherin. Mitochondrial DNA depletion by ethidium bromide or mitochondrial transcription factor A inhibitory RNA (RNAi) induced MtD, further promoting EMT. RNAi-mediated suppression of PGC-1α induced MtD and EMT, whereas overexpression of PGC-1α prevented Aldo-induced MtD and inhibited EMT. Similarly, overexpression of silent mating type information regulation 2 homolog 1 (SIRT1), a gene upstream of PGC-1α, or the SIRT1 activator resveratrol restored Aldo-induced MtD and EMT by upregulating PGC-1α. These findings, which implicate a role for MtD in EMT and suggest that SIRT1 and PGC-1α coordinate to improve mitochondrial function and EMT, may guide us in therapeutic strategies for renal tubulointerstitial fibrosis.

Sirt1 overexpression protects murine osteoblasts against TNF-α-induced injury in vitro by suppressing the NF-κB signaling pathway

Sirtuin 1 (Sirt1) is the class III histone/protein deacetylase that interferes with the NF-κB signaling pathway, thereby has anti-inflammatory function. This study was undertaken to investigate whether Sirt1 could protect osteoblasts against TNF-α-induced injury in vitro. Murine osteoblastic cell line, MC3T3-E1, was used. Overexpress of Sirt1 protein in MC3T3-E1 cells was made by transfection the cells with Sirt1-overexpressing adenovirus. The levels of mRNAs and proteins were determined with qRT-PCR and Western blotting, respectively. The activity of NF-κB was examined using NF-κB luciferase assay. The NO concentration was measured using the Griess method. Treatment of MC3T3-E1 cells with TNF-α (2.5-10 ng/mL) suppressed Sirt1 protein expression in a concentration-dependent manner. TNF-α (5 ng/mL) resulted in an increase in apoptosis and a reduction in ALP activity in the cells. Overexpression of Sirt1 in the cells significantly attenuated TNF-α-induced injury through suppressing apoptosis, increasing ALP activity, and increasing the expression of Runx2 and osteocalcin mRNAs. Furthermore, overexpression of Sirt1 in the cells significantly suppressed TNF-α-induced NF-κB activation, followed by reducing the expression of iNOS and NO formation. Sirt1 activator resveratrol (10 μmol/L) mimicked the protection of the cells by Sirt1 overexpression against TNF-α-induced injury, which was reversed by the Sirt1 inhibitor EX-527 (5 μmol/L). Overexpression of Sirt1 protects MC3T3-E1 osteoblasts aganst TNF-α-induced cell injury in vitro, at least in part, via suppressing NF-κB signaling. Sirt1 may be a novel therapeutic target for treating rheumatoid arthritis-related bone loss.

Hypoxanthine–xanthine oxidase down-regulates GLUT1 transcription via SIRT1 resulting in decreased glucose uptake in human placenta

Appropriate foetal growth and development is dependent on adequate placental glucose uptake. Oxidative stress regulates glucose uptake in various tissues. The effect of oxidative stress on placental glucose transport is not known. Thus, the aim of this study was to determine the effect of oxidative stress on glucose uptake and glucose transporters (GLUTs) in human placenta. Human placenta was incubated in the absence or presence of 0.5 mM hypoxanthine+15 mU/ml xanthine oxidase (HX/XO) for 24 h. Gene and protein expressions of the GLUTs were analysed by quantitative RT-PCR and western blotting respectively. Glucose uptake was measured using radiolabelled ((14)C) glucose. HX/XO significantly decreased GLUT1 gene and protein expression and resultant glucose uptake. There was no effect of the antioxidants N-acetylcysteine, catalase and superoxide dismutase or the NF-κB inhibitor BAY 11-0782 on HX/XO-induced decrease in glucose uptake. However, HX/XO treatment significantly decreased both gene and protein expression of SIRT1. In the presence of the SIRT1 activator resveratrol, the decrease in GLUT1 expression and glucose uptake mediated by HX/XO was abolished. Collectively, the data presented here demonstrate that oxidative stress reduces placental glucose uptake and GLUT1 expression by a SIRT1-dependent mechanism.

SIRT1 activating compounds reduce oxidative stress and prevent cell death in neuronal cells

Activation of SIRT1, an NAD+-dependent deacetylase, prevents retinal ganglion cell (RGC) loss in optic neuritis, an inflammatory demyelinating optic nerve disease. While SIRT1 deacetylates numerous protein targets, downstream mechanisms of SIRT1 activation mediating this neuroprotective effect are unknown. SIRT1 increases mitochondrial function and reduces oxidative stress in muscle and other cells, and oxidative stress occurs in neuronal degeneration. We examined whether SIRT1 activators reduce oxidative stress and promote mitochondrial function in neuronal cells. Oxidative stress, marked by reactive oxygen species (ROS) accumulation, was induced in RGC-5 cells by serum deprivation, or addition of doxorubicin or hydrogen peroxide, and resulted in significant cell loss. SIRT1 activators resveratrol (RSV) and SRTAW04 reduced ROS levels and promoted cell survival in RGC-5 cells as well as primary RGC cultures. Effects were blocked by SIRT1 siRNA. SIRT1 activators also increased expression of succinate dehydrogenase (SDH), a mitochondrial enzyme, and promoted deacetylation of PGC-1α, a co-enzyme involved in mitochondrial function. Results show SIRT1 activators prevent cell loss by reducing oxidative stress and promoting mitochondrial function in a neuronal cell line. Results suggest SIRT1 activators can mediate neuroprotective effects during optic neuritis by these mechanisms, and they have the potential to preserve neurons in other neurodegenerative diseases that involve oxidative stress.