Inhibitory Effect of Paeonol on Vascular Smooth Muscle Cell Senescence by Reducing SIRT6/PARP1-mediated DNA Damage / 中国实验方剂学杂志

ObjectiveTo investigate whether the effects of paeonol （Pae） on angiotensin Ⅱ （AngⅡ）-induced senescence in vascular smooth muscle cells （VSMCs） were related to angiotensinogen of silencing regulatory information factor 6 （SIRT6）/adenosine diphosphate ribose polymerase 1 （PARP1） signaling pathway in VSMCs. MethodThe model of VSMC-stress aging induced by AngⅡ （100 nmol·L-1） was established. The rats were divided into normal group， model group， low， medium， and high-concentration Pae groups （30， 60， 120 μmol·L-1）. The positive rate of cell senescence was detected by SA-β-Gal staining， the ability of cell proliferation was detected by the cell counting kit-8 （CCK-8） method， the expression of SIRT6， PARP1， p16， p21， p53， proliferating cell nuclear antigen （PCNA）， deoxyribonucleic acid （DNA）-damaged protein γ-H2AX was detected by Western blot， and VSMC proliferation was detected by EdU staining. The silenced VSMCs were prepared by siRNA-SIRT6 transfection， and the protein expressions of SIRT6， PARP1， p16， and γ-H2AX in VSMCs silenced by SIRT6 were observed. ResultThe results of SA-β-Gal staining showed that the senescence positive rate of SA-β-Gal staining in the model group was higher than that in the normal group （P<0.01）， and the positive rate of SA-β-Gal staining in the Pae group was significantly lower than that in the model group （P<0.05， P<0.01）. The results of Western blot showed that as compared with the normal group， the expression of PCNA， SIRT6， and PARP1 in the model group was down-regulated， and the expression of aging-related proteins p16， p21， p53， and γ-H2AX was up-regulated in the model group （P<0.05， P<0.01）. Compared with the model group， Pae promoted the protein expression of PCNA， SIRT6， and PARP1 and inhibited the protein expression of p16， p21， p53， and γ-H2AX in a dose-dependent manner （P<0.05， P<0.01）. The results of EdU staining showed that the number of EdU positive cells in the model group was lower than that in the normal group （P<0.01）， and the number of EdU positive cells in Pae groups was significantly higher than that in the model group （P<0.05， P<0.01）. After SIRT6 silencing， the effects of Pae on promoting SIRT6 and PARP1 and inhibiting P16 were reversed （P<0.05， P<0.01）. In addition， the addition of SIRT6 inhibitor （IN-1） promoted the occurrence of cell senescence induced by AngⅡ （P<0.05， P<0.01）. ConclusionPae can effectively inhibit the aging of VSMCs， and its mechanism may be related to the regulation of SIRT6/PARP1 signal pathway.

Overexpressed SIRT6 ameliorates doxorubicin-induced cardiotoxicity and potentiates the therapeutic efficacy through metabolic remodeling

Since the utilization of anthracyclines in cancer therapy, severe cardiotoxicity has become a major obstacle. The major challenge in treating cancer patients with anthracyclines is minimizing cardiotoxicity without compromising antitumor efficacy. Herein, histone deacetylase SIRT6 expression was reduced in plasma of patients treated with anthracyclines-based chemotherapy regimens. Furthermore, overexpression of SIRT6 alleviated doxorubicin-induced cytotoxicity in cardiomyocytes, and potentiated cytotoxicity of doxorubicin in multiple cancer cell lines. Moreover, SIRT6 overexpression ameliorated doxorubicin-induced cardiotoxicity and potentiated antitumor efficacy of doxorubicin in mice, suggesting that SIRT6 overexpression could be an adjunctive therapeutic strategy during doxorubicin treatment. Mechanistically, doxorubicin-impaired mitochondria led to decreased mitochondrial respiration and ATP production. And SIRT6 enhanced mitochondrial biogenesis and mitophagy by deacetylating and inhibiting Sgk1. Thus, SIRT6 overexpression coordinated metabolic remodeling from glycolysis to mitochondrial respiration during doxorubicin treatment, which was more conducive to cardiomyocyte metabolism, thus protecting cardiomyocytes but not cancer cells against doxorubicin-induced energy deficiency. In addition, ellagic acid, a natural compound that activates SIRT6, alleviated doxorubicin-induced cardiotoxicity and enhanced doxorubicin-mediated tumor regression in tumor-bearing mice. These findings provide a preclinical rationale for preventing cardiotoxicity by activating SIRT6 in cancer patients undergoing chemotherapy, but also advancing the understanding of the crucial role of SIRT6 in mitochondrial homeostasis.

Immunofluorescent distribution of SIRT1, SIRT2, SIRT6 protein in the skin of SKH1 mouse after ultraviolet B irradiation for 10 Weeks / Distribución inmunofluorescente de la proteína SIRT1, SIRT2, SIRT6 en la piel del ratón SKH1 después de la irradiación ultravioleta B durante 10 semanas

SUMMARY: The skin, located on the outermost part of the body, is always exposed to external stimuli such as sunlight. The exposure of skin to ultraviolet B (UVB) radiation from sunlight is known to be a major environmental factor in inducing photoaging. After exposure to UVB, an increase in reactive oxygen species can affect the expression and activity of many critical proteins depending on the duration and dose of the UVB radiation. Mammalian sirtuins (SIRTs), which are nicotinamide dinucleotide-dependent protein deacetylases, are well known for playing a role in cellular longevity. However, little is known about SIRT protein alterations in keratinocytes upon UVB irradiation according to SIRT subtypes. Therefore, in this study, the distribution of non-mitochondrial SIRT1, SIRT2, and SIRT6 proteins was investigated by immunofluorescence (IF) staining of the skin of SKH-1 mice (n=12) after UVB irradiation for 10 weeks. After UVB irradiation for 10 weeks, the IF of both SIRT1 and SIRT6 was significantly increased in the UVB-irradiated mice group (UG), but the difference in SIRT2 IF was not statistically significant between the control group (CG) and the UG. The translocation of both SIRT1 and SIRT6 IF from the nucleus to the cytoplasm of keratinocytes was observed in the upper epidermis of the UG, whereas SIRT2 IF was localized in the cytoplasm of keratinocytes in the epidermis in both the CG and the UG. The translocation of SIRT1 and SIRT6 IF from the nucleus to the cytoplasm of keratinocytes may account for the physiologically protective action of keratinocytes against UVB irradiation. However, the exact role of SIRT1 and SIRT6 translocation in keratinocytes, where SIRT1 and SIRT6 shuttle from the nucleus to the cytoplasm, is not well known. Therefore, further studies are needed to understand the molecular mechanisms of SIRT1 and SIRT6 translocation in keratinocytes upon UVB irradiation.

La piel, situada en la parte más externa del cuerpo, está siempre expuesta a estímulos externos como la luz solar. Se sabe que la exposición de la piel a la radiación ultravioleta B (UVB) de la luz solar es un factor ambiental importante en la inducción del fotoenvejecimiento. Después de la exposición a los rayos UVB, un aumento en las especies reactivas de oxígeno puede afectar la expresión y la actividad de muchas proteínas críticas según la duración y la dosis de la radiación UVB. Las sirtuinas de mamíferos (SIRT), que son proteínas desacetilasas dependientes de dinucleótidos de nicotinamida, son bien conocidas por desempeñar un papel en la longevidad celular. Sin embargo, se sabe poco sobre las alteraciones de la proteína SIRT en los queratinocitos tras la irradiación UVB según los subtipos de SIRT. Por lo tanto, en este estudio, se investigó la distribución de las proteínas SIRT1, SIRT2 y SIRT6 no mitocondriales mediante tinción de inmunofluorescencia (IF) de la piel de ratones SKH-1 (n = 12), después de la irradiación con UVB durante 10 semanas. Posterior a la irradiación, el IF de SIRT1 y SIRT6 aumentaron significativamente en el grupo de ratones irradiados con UVB (UG), pero la diferencia en SIRT2 IF no fue estadísticamente significativa entre el grupo control (CG) y el UG. La translocación de SIRT1 y SIRT6 IF desde el núcleo al citoplasma de los queratinocitos se observó en la epidermis superior de la UG, mientras que SIRT2 IF se localizó en el citoplasma de los queratinocitos en la epidermis, tanto en el GC, como en la UG. La translocación de SIRT1 y SIRT6 IF del núcleo al citoplasma de los queratinocitos puede explicar la acción protectora fisiológica de estos contra la radiación UVB. Sin embargo, el papel exacto de la translocación de SIRT1 y SIRT6 en los queratinocitos, donde SIRT1 y SIRT6 se trasladan desde el núcleo al citoplasma, no se conoce bien. Por lo tanto, se necesitan más estudios para comprender los mecanismos moleculares de la translocación SIRT1 y SIRT6 en los queratinocitos tras la irradiación UVB.

A novel SIRT6 activator ameliorates neuroinflammation and ischemic brain injury

Ischemic stroke is the second leading cause of death worldwide with limited medications and neuroinflammation was recognized as a critical player in the progression of stroke, but how to control the overactive neuroinflammation is still a long-standing challenge. Here, we designed a novel SIRT6 activator MDL-811 which remarkably inhibited inflammatory response in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and primary mouse microglia, which were abolished by silencing SIRT6. RNA-seq screening identified the forkhead box C1 (

miR-92a-3p promotes ox-LDL induced-apoptosis in HUVECs via targeting SIRT6 and activating MAPK signaling pathway

Atherosclerosis could be induced by multiple factors, including hypertension, hyperlipidemia, and smoking, and its pathogenesis has not been fully elucidated. MicroRNAs have been shown to possess great anti-atherosclerotic potential, but the precise function of miR-92a-3p in atherosclerosis and its potential molecular mechanism have not been well clarified. Flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay were performed to evaluate effects of oxidized low-density lipoprotein (ox-LDL) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs), respectively. Malondialdehyde and superoxide dismutase levels in cell lysate were assessed with biochemical kits. The expression levels of miR-92a-3p and Sirtuin6 (SIRT6) in HUVECs exposed to ox-LDL were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the protein levels of SIRT6, c-Jun N-terminal kinase (JNK), phosphorylation JNK (p-JNK), p38 mitogen activated protein kinase (p38 MAPK), and phosphorylation p38 MAPK (p-p38 MAPK) were measured by western blot assays. The relationship between miR-92a-3p and SIRT6 was confirmed by dual-luciferase reporter assay. Ox-LDL induced apoptosis and oxidative stress in HUVECs in concentration- and time-dependent manners. Conversely, miR-92a-3p silencing inhibited apoptosis and SIRT6 expression in HUVECs. The overexpression of miR-92a-3p enhanced apoptosis and phosphorylation levels of JNK and p38 MAPK as well as inhibited proliferation in ox-LDL-induced HUVECs. In addition, SIRT6 was a target of miR-92a-3p. miR-92a-3p negatively regulated SIRT6 expression in ox-LDL-induced HUVECs to activate MAPK signaling pathway in vitro. In summary, miR-92a-3p promoted HUVECs apoptosis and suppressed proliferation in ox-LDL-induced HUVECs by targeting SIRT6 expression and activating MAPK signaling pathway.

Effect of SIRT6 on migratory and proliferative activity of skin fibroblasts in the elderly and their mechanisms / 中华皮肤科杂志

Objective To assess the effect of silent mating type information regulation 2 homolog 6 (SIRT6) on migratory and proliferative activity of skin fibroblasts in the elderly,and to explore their mechanisms.Methods Circumcised foreskins were obtained from patients of different ages in Department of Urologic Surgery,Second Hospital of Shanxi Medical University,including 8 elderly patients and 8 young patients.Human skin fibroblasts were isolated from the foreskin tissues by using a collagenase digestion method.Western blot analysis was performed to determine the expression of SIRT6 and phosphorylated p65 (p-p65) in human skin fibroblasts in different age groups,scratch assay to evaluate cell migratory activity,and cell counting kit-8 (CCK8) assay to assess cellular proliferative activity.Skin fibroblasts in the elderly group were divided into 2 groups:SIRT6 group transfected with a lentiviral vector overexpressing SIRT6,and control group transfected with an empty lentiviral vector.Then,the cell migratory and proliferative activity as well as p-p65 expression were measured by the above methods,and the mRNA expression of type Ⅰ and Ⅲ collagens,and integrin subunits α3,α5 and β1 was determined by real-time PCR in the SIRT6 group and control group.Statistical analysis was carried out with GraphPad Prism 5 software by using t test for comparison between 2 groups.Results Compared with the young group,the elderly group showed significantly decreased SIRT6 expression in skin fibroblasts (0.434 ±0.179 vs.1.000 ± 0.067,t =3.040,P =0.012),migration rate (43.81％ ± 18.84％ vs.94.63％ ± 12.32％,t =5.903,P =0.003)and cellular proliferative activity at 24 and 48 hours (both P ＜ 0.05),but significantly increased p-p65 expression (1.694 ± 0.148 vs.1.000 ± 0.093,t =2.949,P =0.015).Compared with the control group,the SIRT6 group showed significantly decreased p-p65 expression (P ＜ 0.05),but significantly increased migratory and proliferative activity (both P ＜ 0.05),and elevated mRNA expression of type Ⅲ collagen and integrin subunits oα3,α5 and β1 (all P ＜ 0.05).Conclusion SIRT6 can improve the migratory and proliferative activity of human fibroblasts in the elderly,possibly by inhibiting the nuclear factor-κB pathway.

lncRNA XIST/miR-34a-5p/SIRT6 axis regulates proliferation and metastasis of oral squamous cell carcinoma cells / 中国肿瘤生物治疗杂志

@# Objective: To investigate the molecular mechanisms of lncRNA XIST/miR-34a-5p/SIRT6 axis regulating the proliferation and metastasis of oral squamous cell carcinoma (OSCC) cells. Methods: Specimens of tumor tissues and paracancer tissues of 47 patients with OSCC, who visited the Qingdao Stomatological Hospital from March 2013 to March 2018, were enrolled in this study. qPCR was performed to measure the mRNA expressions of lncRNA XIST, miR-34a-5p and SIRT6 in OSCC tissues and cell lines. WB was performed to measure the protein levels of SIRT6, Ki67, pcDNA, cleaved-caspase3, cleaved-caspase8, E-cadherin and vimentin in OSCC tissues and cell lines. CCK-8 assay was performed to observe the effect of lncRNA XIST knockdown on proliferation of Cal-27 and Tca-8113 cells; Tanswell assay was performed to detect migration and invasion of Cal-27 and Tca-8113 cells; flow cytometry was performed to detect the apoptosis of Cal-27 and Tca-8113 cells; and dual luciferase reporter assay was performed to verify the relationship between lncRNAXIST and miR-34a, or miR-34a and SIRT6. Results: Expressions of lncRNAXIST and SIRT6 were up-regulated in OSCC tissues and cell lines (all P<0.05), reversely, miR-34a-5p was down-regulated in OSCC tissues and cell lines (P<0.01). lncRNA XIST knockdown significantly suppressed OSCC cells proliferation, migration and invasion, and induced apoptosis of OSCC cells (all P<0.01); however, simultaneous transfection with miR-34a-5p inhibitor or pcDNA-SIRT6 vector exerted opposite effect. lncRNA XIST knockdown significantly increased cell proliferation and metastasis related protein expression in OSCC cells (all P< 0.01), and simultaneous transfection with miR-34a-5p inhibitor or pcDNA-SIRT6 vector exerted opposite effect. In addition, lncRNA XIST directly targeted miR-34a-5p, and miR-34a-5p targeted SIRT6, lncRNA XIST upregulated SIRT6 expression via inhibiting miR34a-5p (P<0.01). Conclusion: Taken together, lncRNA XIST/miR-34a-5p/SIRT6 axis regulates the proliferation and metastasis of OSCC cells and provides potential therapeutic targets for OSCC.

Effects of saikosaponin b_2 on inflammation and energy metabolism in mice with acute liver injury induced by LPS/GalN / 中国中药杂志

To study the effects of saikosaponin b2( SS-b2) on inflammatory factors and energy metabolism against lipopolysaccharide/galactosamine( LPS/Gal N) induced acute liver injury in mice. Mice were randomly divided into normal group( equal amount of normal saline),model group( 100 g·kg~(-1) LPS and 400 mg·kg~(-1) Gal N),low,medium,high dose group of SS-b2( SS-b25,10,20 mg·kg~(-1)·d-1) and positive control group( dexamethasone,10 mg·kg~(-1)). All of the groups except for the normal group were treated with LPS/Gal N though intraperitoneally injection to establish the acute liver injury model. The organ indexes were calculated. The levels of serum transaminases( ALT and AST) and the activities of ATPase( Na+-K+-ATPase,Ca2+-Mg2+-ATPase) in liver were detected. The activity of tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β) and interleukin-6( IL-6) were determined by the enzyme-linked immunosorbent assay( ELISA). The contents of lactate dehydrogenase( LDH) in liver were determined by micro-enzyme method. HE staining was used to observe the histopathological changes of the liver. Histochemical method was used to investigate the protein expression of liver lactate dehydrogenase-A( LDH-A). The protein expressions of Sirt-6 and NF-κB in the liver were detected by Western blot. According to the results,compared with the model group,there were significant changes in organ indexes in the high-dose group of SS-b2( P<0. 05). The level of ALT,AST,TNF-α,IL-1β,IL-6 and the activities of LDH in serum of mice with liver injury were significantly reduced in the medium and high dose groups of SS-b2( P<0. 01). With the increase of the concentration of SS-b2,the range of hepatic lesions and the damage in mice decreased. The activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in liver of mice were significantly enhanced in each dose group( P<0. 01). The expression of NF-κB in liver tissues was significantly down-regulated in the medium and high dose group( P<0. 01). Meanwhile,the expression of Sirt-6 protein in the liver of mice with acute liver injury was significantly increased in each dose group( P<0. 01).In summary,SS-b2 has a significant protective effect on LPS/Gal N-induced acute liver injury in mice,which may be related to the down-regulation of NF-κB protein expression and up-regulation of Sirt-6 protein expression to improve inflammatory injury and energy metabolism.

SIRT6 regulating IGF2 though recruiting mRNA binding protein IMP1 in 293T cells / 基础医学与临床

Objective To investigate the post-transcriptional regulation mechanism of insulin-like growth factor 2 (IGF2),which is an important tumor regulator,by SIRT6. Methods SIRT6 was overexpressed in 293T cells and IP was used to enrich SIRT6 and mass spectrometry(MS)was used to detect the protein that interacted with SIRT6. Western blot was used to validate the interacted protein and its acetylation/phosphorylation modification status. Results SIRT6 did not change the acetylation modification status of IMP1,but the phosphorylation status of IMP1 was elevated in the presence of SIRT6. Conclusions SIRT6 may regulate IGF2 though promoting the phosphoryla-tion of IMP1 in 293T cells.

Research progress of SIRT6 in cardiovascular diseases / 中国药理学通报

Cardiovascular disease is a serious disease harmful to human health with high morbidity and high mortality.SIRT6 plays an important role in cardiovascular diseases such as atherosclerosis,myocardial infarction and ischemia/reperfusion injury,pathological cardiac hypertrophy and heart failure,as an important member of the histone deacetylase family.In this paper,the role of SIRT6 in cardiovascular diseases is reviewed,and the development of SIRT6 as a target agonist and inhibitor is summarized.

Effect of ginsenoside Rg1 on delaying radiation-induced senescence of hematopoietic stem cell and progenitor cell based on SIRT6/NF-κB signal pathway / 中草药

Objective To investigate the effect of SIRT6/NF-κB signal axis on delaying radiation-induced senescence of hematopoietic stem cell and progenitor cell with ginsenoside Rg1. Methods C57BL/6 mice were randomly divided into control group, model group and ginsenoside Rg1 group. The mice in model and ginsenoside Rg1 groups were exposed to total body irradiation with 6.5 Gy 60Coγ, the mice in ginsenoside Rg1 group were intraperitoneal injected with ginsenoside Rg1 (20 mg/kg) for 7 d before and after radiation. On day after taking medicine, the indicators in peripheral blood were observed to definite the information of hematopoietic reconstruction and the effect of ginsenoside Rg1 on it. Sca-1+ HSCs/HPCs were isolated and purified by magnetic activated cell sorting (MACS). The effect of ginsenoside Rg1 on delaying radiation-induced senescence of hematopoietic stem cell and progenitor cell was evaluated by mixed hematopoietic progenitor cell culture, cell cycle assay and senescence-associated β-galactosidase (SA-β-gal) staining. The expression of senescence associated SIRT6 and NF-κB mRNA and protein was examined by realtime fluorescence quantitative PCR (qRT-PCR) and Western blotting. Results After radiation, the indicator in peripheral blood of model group was decreased compared with control group. Sca-1+ HSC/HPC appeared aging character: The number of cells entered G0/G1 phase, the percentage of SA-β-gal positive cells was increased, the expressing of SIRT6 mRNA and protein were down regulated and NF-κB mRNA and protein were up regulated, and the number of CFU-Mix was decreased. Compared with the model group, the number of white blood cells (RBC), red blood cells (RBC), and platelet (PLT) in mice were increased, the number of cells entered G0/G1 phase and the percentage of SA-β-gal positive cells were decreased, and the number of CFU-Mix was increased in Sca-1+ HSC/HPC from the ginsenoside Rg1 group. Moreover, the expression of SIRT6 mRNA and protein were up regulated, and NF-κB mRNA and protein were down regulated in the ginsenoside Rg1 group. Conclusion SIRT6/NF-κB signal axis may play a key role in the antiaging effect of ginsenoside Rg1 to Sca-1+ HSC/HPC senescence induced by radiation.

Autophagy induction by SIRT6 is involved in oxidative stress-induced neuronal damage

SIRT6 is a NAD(+)-dependent histone deacetylase and has been implicated in the regulation of genomic stability, DNA repair, metabolic homeostasis and several diseases. The effect of SIRT6 in cerebral ischemia and oxygen/glucose deprivation (OGD) has been reported, however the role of SIRT6 in oxidative stress damage remains unclear. Here we used SH-SY5Y neuronal cells and found that overexpression of SIRT6 led to decreased cell viability and increased necrotic cell death and reactive oxygen species (ROS) production under oxidative stress. Mechanistic study revealed that SIRT6 induced autophagy via attenuation of AKT signaling and treatment with autophagy inhibitor 3-MA or knockdown of autophagy-related protein Atg5 rescued H2O2-induced neuronal injury. Conversely, SIRT6 inhibition suppressed autophagy and reduced oxidative stress-induced neuronal damage. These results suggest that SIRT6 might be a potential therapeutic target for neuroprotection.

Function of SIRT6 in tumor initiation and progression / 生物工程学报

As a member of the sirtuins family, also called Class III histone deacetylases (HDACs), SIRT6 has many catalytic enzyme activities and plays a pivotal role in biological processes including anti-aging, chromatin regulation, transcriptional control, glucose and lipid metabolism, and DNA damage repair. Recently, increasing evidences indicated that SIRT6 was related to initiation and development of tumors, such as hepatic cancer, lung cancer, breast cancer and genital system tumors. However, SIRT6 might play a dual role in tumorigenesis and progression. SIRT6 often acted as a tumor suppressor, but might play an oncogenic role. Based on our current study, we depicted the essential roles of SIRT6 in the initiation and progression of various tumors, and summarized its mode of actions, which might provide clues for cancer therapy.