OBJECTIVE: To investigate the effect of targeting the silent information regulator 2 hemolog 2 (SIRT2) expression on the apoptosis of drug-resistant AML cell line HL-60/A and its mechanism. METHODS: The expression of SIRT2 and antophagy-related protein LCT, P62 in HL-60/A and HL-60 was detected by Western blot, the effect of cytorabine on the apoptosis of HL-60/A cells was detected by using Annexin V/PI double staining after targeting inhibition of SIRT2 expression resulting from transfecting HL-60/A cells with SiRNA. The Western blot and transmission electray microscopy were used to detect the cell autophagy. To further clarify the role of autophragy in the regulatory effect of SIRT2 on the drug-resistance of HL-60/A cells, the autophagy-specific agonist, repamycin, was added into the cell culture medium after SIRT2-siRNA transfection. Then, the autophagy and apoptosis of HL-60/A were detected, respectively. RESULTS: The SIRT2 protein expression obviously increased in HL-60/A cells than that in HL-60 cells. Moreover, the expression rate of LC3II/I was higher, but P62 expression was lower in HL-60/A cells. After siRNA successfully transfecting into HL-60/A cells, quantitative PCR and Western blot should that the expression of SIRT2 significantly decreased. Meawhile, Western blot showed that the expression of LC3 II/I decreased, but P62 increased. Meanwhile, By TEM found that the number of autophagosome also decreased, suggesting the autophagy was inhibited after down-regulation of SIRT2. In addition, the drug senstivity of HL-60/A cells to cytarabine in siRNA-transfection group increased, and the apoptotic rate detected by Annexin V/PI double staining significantly increased. However, after co-culture with rapamycin, the suppressed autophagy in siRNA-trasfect HL-60/A cells was activated, leading to the reappearance of drug resistance of cells to cytarabine, and more significantly decrease of apoptotic rate. CONCLUSION The high expression of SIRT2 in HL-60/A cells activates the protective autophagy mechanism, which closely related with drug resistance.
Apoptosis ; Autophagy ; Drug Resistance, Neoplasm ; HL-60 Cells ; Humans ; Sirtuin 2 ; metabolism

We investigated the effects of the sirtuin-2 (SIRT2) inhibitor AK-7 on novel object memory, cell proliferation, and neuroblast differentiation in the dentate gyrus. In addition, we also observed the relationships with sodium butyrate, a histone deacetylase inhibitor, on the hippocampal functions. To investigate the effects of AK-7 on hippocampal functions, 10-week-old C57BL/6 mice were daily injected intraperitoneally with 20 mg/kg AK-7 alone or in combination with subcutaneous administration of 300 mg/kg sodium butyrate, a histone deacetylase inhibitor, for 21 days. A novel object recognition test was conducted on days 20 (training) and 21 (testing) of treatment. Thereafter, the animals were sacrificed for immunohistochemistry for Ki67 (cell proliferation) and doublecortin (DCX, neuroblast differentiation). AK-7 administration significantly reduced the time spent exploring new objects, while treatment in combination with sodium butyrate significantly alleviated this reduction. Additionally, AK-7 administration significantly reduced the number of Ki67-positive cells and DCX-immunoreactive neuroblasts in the dentate gyrus, while the treatment in combination with sodium butyrate ameliorated these changes. This result suggests that the reduction of SIRT2 may be closely related to age-related phenotypes including novel object memory, as well as cell proliferation and neuroblast differentiation in the dentate gyrus. In addition, sodium butyrate reverses SIRT2-related age phenotypes.
Animals ; Butyric Acid* ; Cell Proliferation* ; Dentate Gyrus ; Histone Deacetylase Inhibitors ; Immunohistochemistry ; Memory* ; Mice ; Neurogenesis ; Phenotype ; Sirtuin 2 ; Sodium*

Exposure to particulate matter 2.5 (PM2.5) potentially triggers airway inflammation by activating nuclear factor-κB (NF-κB). Sirtuin 2 (SIRT2) is a key modulator in inflammation. However, the function and specific mechanisms of SIRT2 in PM2.5-induced airway inflammation are largely understudied. Therefore, this work investigated the mechanisms of SIRT2 in regulating the phosphorylation and acetylation of p65 influenced by PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Results revealed that PM2.5 exposure lowered the expression and activity of SIRT2 in bronchial tissues. Subsequently, SIRT2 impairment promoted the phosphorylation and acetylation of p65 and activated the NF-κB signaling pathway. The activation of p65 triggered airway inflammation, increment of mucus secretion by goblet cells, and acceleration of tracheal stenosis. Meanwhile, p65 phosphorylation and acetylation, airway inflammation, and bronchial hyperresponsiveness were deteriorated in SIRT2 knockout mice exposed to PM2.5. Triptolide (a specific p65 inhibitor) reversed p65 activation and ameliorated PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Our findings provide novel insights into the molecular mechanisms underlying the toxicity of PM2.5 exposure. Triptolide inhibition of p65 phosphorylation and acetylation could be an effective therapeutic approach in averting PM2.5-induced airway inflammation and bronchial hyperresponsiveness.
Animals ; Inflammation ; Mice ; NF-kappa B/metabolism* ; Particulate Matter/toxicity* ; Signal Transduction ; Sirtuin 2/metabolism* ; Transcription Factor RelA/metabolism*

OBJECTIVETo observe the effect of SIRT1 on intestinal barrier function of epithelial Caco-2 cells under hypoxia and investigate its mechanism.

METHODSCaco-2 cells were randomly divided into three groups: normoxia group (Nx), hypoxia group (Hx,1%O2 for 6 h) and hypoxia plus 40 μmol/L Resveratrol (agonist of SIRT1) group (Hx+Res). Transepithelial electrical resistance (TER) was determined. mRNA and protein expressions of SIRT1 and tight junctions (ZO-1, Occludin, Claudin-1) were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

RESULTSBoth mRNA and protein expressions of SIRT1 were significantly reduced in Hx group as compared with Nx group (0.40±0.02 vs. 0.70±0.07, P=0.001; 0.37±0.03 vs. 0.76±0.03, P=0.001). The mRNA and protein expressions of SIRT1 were significantly increased in Hx+Res group as compared with Hx group(0.50±0.02 vs. 0.40±0.02, P=0.026; 0.54±0.02 vs. 0.37±0.03, P=0.011). The expression levels of ZO-1, Occludin and Claudin-1 in Hx group were lower than those in Nx group (P<0.05), however, pretreatment with Resveratrol could attenuate the decreased expression of above 3 molecules under hypoxia(P<0.05). TERs of Nx group, Hx group and Hx+Res group were (142±7) Ohm/cm(2), (94±3) Ohm/cm(2) and (119±7) Ohm/cm(2) respectively. Compare with the Nx group, the TER of Hx group was significantly decreased(P<0.05). TER of Hx+Res group was significantly increased compare with Hx group, but it was still significantly lower than that in Nx group(P<0.05).

CONCLUSIONSExpression of SIRT1 is significantly reduced under hypoxia. Activation of SIRT1 can maintain the epithelial barrier function through regulating the expression of tight junctions under hypoxia.

Caco-2 Cells ; Cell Hypoxia ; Claudin-1 ; metabolism ; Epithelial Cells ; metabolism ; Humans ; Intestinal Mucosa ; cytology ; Occludin ; metabolism ; Sirtuin 1 ; metabolism ; Zonula Occludens-1 Protein ; metabolism

BACKGROUND/AIMS: Renal aging-related changes are characterized by oxidative stress. SIRT1 regulates cellular conditions by activating Nrf2. The present study investigated the processes of renal changes by antioxidant enzymes and the relationship between SIRT1 and Nrf2. METHODS: We used male 2-, 12-, and 24-month-old C57BL/6 mice. We measured renal function, histological changes, oxidative stress, and expression of SIRT1–Nrf2 signaling in the kidneys. RESULTS: 24-month-old mice exhibited increased albuminuria and serum creatinine. Creatinine clearance was decreased in 24-month-old mice compared with 12-month-old mice. There were increases in mesangial volume and tubulointerstitial fibrosis in 24-month-old mice. Moreover, oxidative stress marker, 3-Nitrotyrosine, expression and apoptosis were increased in 24-month-old mice. The 24 h urinary 8-isoprostane and 8-hydroxy-deoxyguanosine excretion increased with aging. The levels of expression of SIRT1 and nuclear Nrf2 were decreased in 24-month-old mice. The antioxidant enzymes HO-1 and NQO-1 were down-regulated in 24-month-old mice. Another antioxidant enzyme, SOD2, was decreased in 24-month-old mice. CONCLUSIONS: Our results demonstrated that SIRT1 was down-regulated with aging, and this may be related to changes in the expression of target molecules including Nrf2. As a result, oxidative stress was induced. The pharmacological targeting of these signaling molecules may reduce the pathological changes associated with aging in the kidney.
Aging ; Albuminuria ; Animals ; Apoptosis ; Child, Preschool ; Creatinine ; Fibrosis ; Humans ; Infant ; Kidney* ; Male ; Mice ; NF-E2-Related Factor 2 ; Oxidative Stress ; Sirtuin 1

OBJECTIVETo evaluate the feasibility of integrating cancer gene therapy with therapeutic effect evaluation using siRNA-loaded nano-scale microbubbles (siRNA-NBs).

METHODSsiRNA-NBs were prepared by hetero-assembly of polymeric siRNA micelles and liposomal microbubbles, and the particle sizes and surface potentials were examined with dynamic light scattering. The distributions of cy3-labled siRNA in the tumor tissues were evaluated using confocal laser scanning microscopy. A siRNA targeting the anti-apoptosis gene SIRT2 was designed and its gene silencing effects was tested in vivo using siRNA-NBs with ultrasound exposure. The therapeutic effect of the loaded siRNA-NBs was evaluated by contrast-enhanced ultrasonography.

RESULTSThe siRNA-NBs had a mean diameter of 400.7 ± 30.5 nm with a weak positive charge of +8.8 ± 0.8 mV. With ultrasound exposure, siRNA-NBs effectively delivered cy3-siRNA into the cytoplasm of cancer cells and caused SIRT2 suppression and cell apoptosis in tumor tissues, resulting in significantly suppressed tumor growth. In addition, contrast-enhanced ultrasonography of siRNA-NBs provided good imaging quality to allow real-time observation of blood supply during gene therapy.

CONCLUSIONSAs a novel ultrasound contrast agent, siRNA-NBs make possible the integration of tumor gene therapy and therapeutic effect evaluation for cancer.

Apoptosis ; Contrast Media ; Gene Silencing ; Genetic Therapy ; Humans ; Liposomes ; Micelles ; Microbubbles ; Neoplasms ; therapy ; Particle Size ; Polymers ; RNA, Small Interfering ; Sirtuin 2 ; genetics ; Ultrasonics

OBJECTIVE: To investigate the underlying molecular mechanisms by which silence information regulator (SIRT) 2 and glutaminase (GLS) in the amygdala regulate social behaviors in autistic rats. METHODS: Rat models of autism were established by maternal sodium valproic acid (VPA) exposure in wild-type rats and SIRT2-knockout ( SIRT2 ^-/-) rats. Glutamate (Glu) content, brain weight, and expression levels of SIRT2, GLS proteins and apoptosis-associated proteins in rat amygdala at different developmental stages were examined, and the social behaviors of VPA rats were assessed by a three-chamber test. Then, lentiviral overexpression or interference vectors of GLS were injected into the amygdala of VPA rats. Brain weight, Glu content and expression level of GLS protein were measured, and the social behaviors assessed. RESULTS: Brain weight, amygdala Glu content and the levels of SIRT2, GLS protein and pro-apoptotic protein caspase-3 in the amygdala were increased in VPA rats, while the level of anti-apoptotic protein Bcl-2 was decreased (all P<0.01). Compared with the wild-type rats, SIRT2 ^-/- rats displayed decreased expression of SIRT2 and GLS proteins in the amygdala, reduced Glu content, and improved social dysfunction (all P<0.01). Overexpression of GLS increased brain weight and Glu content, and aggravated social dysfunction in VPA rats (all P<0.01). Knockdown of GLS decreased brain weight and Glu content, and improved social dysfunction in VPA rats (all P<0.01). CONCLUSIONS The glutamate circulatory system in the amygdala of VPA induced autistic rats is abnormal. This is associated with the upregulation of SIRT2 expression and its induced increase of GLS production; knocking out SIRT2 gene or inhibiting the expression of GLS is helpful in maintaining the balanced glutamate cycle and in improving the social behavior disorder of rats.
Animals ; Rats ; Amygdala/metabolism* ; Autistic Disorder/metabolism* ; Behavior, Animal ; Disease Models, Animal ; Glutamates/metabolism* ; Glutaminase/metabolism* ; Sirtuin 2/metabolism* ; Social Behavior

OBJECTIVETo study the regulatory rolesof SIRT1 on EZH2 expression and the further effects on EZH 2’ s repression of target gene expression.

METHODSThe stable SIRT1 RNAi and Control RNAi HeLa cells were established by infection with retroviruses expressing shSIRT1 and shLuc respectively followed by puromycin selection. EZH2 protein level was detected by Western blot in either whole cell lysate or the fractional cell extract. Reverse transcription-polymerase chain reaction was performed to detect the mRNA level of EZH2. Cycloheximide was used to treat SIRT1 RNAi and Control RNAi cells for protein stability assay. Chromatin immunoprecipitation(ChIP) assay was applied to measure enrichment of SIRT1, EZH2, and trimethylated H3K27 (H3K27me3) at SATB1 promoter in SIRT1 RNAi and Control RNAi cells.

RESULTSWestern blot results showed that EZH2 protein level increased upon SIRT1 depletion. Fractional extraction results showed unchanged cytoplasmic fraction and increased chromatin fraction of EZH2 protein in SIRT1 RNAi cells. The mRNA level of EZH2 was not affected by knockdown of SIRT1. SIRT1 recruitment was not detected at the promoter regionof EZH2 gene locus. The protein stability assay showed that the protein stability of EZH2 increases upon SIRT1 knockdown. Upon SIRT1 depletion, EZH2 and H3K27me3 recruitment at SATB1 promoter increases and the mRNA level of SATB1 decreases.

CONCLUSIONSDepletion of SIRT1 increases the protein stability of EZH2. The regulation of EZH2 protein level by SIRT1 affects the repressive effects of EZH2 on the target gene expression.

DNA-Binding Proteins ; analysis ; chemistry ; physiology ; Enhancer of Zeste Homolog 2 Protein ; Gene Expression Regulation ; HeLa Cells ; Humans ; Polycomb Repressive Complex 2 ; Repressor Proteins ; physiology ; Sirtuin 1 ; antagonists & inhibitors ; physiology ; Transcription Factors ; analysis ; chemistry ; physiology

OBJECTIVETo observe the expression of SIRT1 and mitochondrial uncoupling protein 2 (UCP2) in the liver of rats with type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver (NAFLD) and explore the possible pathogenesis of T2DM and NAFLD.

METHODSTwenty-four male SD rat were randomized equally into control group and T2DM and NAFLD group (MC group), fed with standard diet and high-fat and high-sugar diet, respectively. At 12 weeks, the rats in MC group received a single dose of STZ (30 mg/kg) injected into the abdominal cavity for pancreatic islet destruction, and those in the control group received an equivalent volume of citric acid buffer. At 14 weeks, the body weight, FBG, hepatic function, blood lipid levels, FFAs, FINs and HOMA-IR of the rats were measured, and the liver pathology was examined with HE staining. The expression of SIRT1 and UCP2 in the rat liver was detected by immunohistochemistry and real-time quantitative PCR.

RESULTSAt 14 weeks, FBG, ALT, AST, TC, TG, LDL-C, VLDL, FFAs, FINs and HOMA-IR were significantly higher and HDL-C was significantly lower in MC group than in the control group (P<0.05). Pathological examination showed good structural integrity of the liver in the control group, and the liver cells were closely arranged with rich cytoplasm and round cell nuclei; in MC group, moderate to severe fatty liver was detected, and the liver cells showed severe ballooning degeneration and contained lipid vacuoles in the cytoplasm. The expression of SIRT1 was significantly lower and UCP2 significantly higher in MC group than in the control group (P<0.05).

CONCLUSIONThe expression of SIRT1 is significantly lowered and UCP2 increased in the liver of rats with T2DM and NAFLD.

Animals ; Diabetes Mellitus, Type 2 ; complications ; metabolism ; Fatty Liver ; complications ; metabolism ; Ion Channels ; metabolism ; Liver ; metabolism ; Male ; Mitochondrial Proteins ; metabolism ; Non-alcoholic Fatty Liver Disease ; Rats ; Rats, Sprague-Dawley ; Sirtuin 1 ; metabolism ; Uncoupling Protein 2

The prevalence of type 2 diabetes mellitus (T2DM) has been increasing worldwide. Therefore, a novel therapeutic strategy by which to prevent T2DM is urgently required. Calorie restriction (CR) can retard the aging processes, and delay the onset of numerous age-related diseases including diabetes. Metabolic CR mimetics may be therefore included as novel therapeutic targets for T2DM. Sirtuin 1 (SIRT1), a NAD+-dependent histone deacetylase that is induced by CR, is closely associated with lifespan elongation under CR. SIRT1 regulates glucose/lipid metabolism through its deacetylase activity on many substrates. SIRT1 in pancreatic beta-cells positively regulates insulin secretion and protects cells from oxidative stress and inflammation, and has positive roles in the metabolic pathway via the modulation in insulin signaling. SIRT1 also regulates adiponectin secretion, inflammation, glucose production, oxidative stress, mitochondrial function, and circadian rhythms. Several SIRT1 activators, including resveratrol have been demonstrated to have beneficial effects on glucose homeostasis and insulin sensitivity in animal models of insulin resistance. Therefore, SIRT1 may be a novel therapeutic target for the prevention of T2DM, implicating with CR. In this review, we summarize current understanding of the biological functions of SIRT1 and discuss its potential as a promising therapeutic target for T2DM.
Adiponectin ; Aging ; Caloric Restriction ; Circadian Rhythm ; Diabetes Mellitus, Type 2 ; Glucose ; Histone Deacetylases ; Homeostasis ; Inflammation ; Insulin ; Insulin Resistance ; Metabolic Networks and Pathways ; Models, Animal ; Oxidative Stress ; Prevalence ; Sirtuin 1 ; Stilbenes