小鼠心脏过表达SIRT1显性失活突变体导致心肌细胞凋亡和早发性心衰

本研究旨在探究SIRT1的去乙酰化活性在调节心脏功能方面的重要作用.与成年小鼠心脏相比,在出生后早期小鼠的心脏中SIRT1去乙酰化活性较高.为了进一步研究SIRT1酶活性在出生后心脏中的功能,本研究构建了心脏特异性表达人源SIRT1显性失活突变体(SIRT1 H363Y)的转基因小鼠,SIRT1 H363Y能够抑制内源性SIRT1的去乙酰化活性.这种转基因小鼠表现为心室与心房腔扩张,并且出生后早夭.在病理学方面,超声心动图与分子表型证实转基因小鼠罹患扩张型心肌病.同时,本研究通过TdT介导的dUTP缺口末端标记技术检测到转基因小鼠的心脏心肌凋亡更为严重.进一步研究发现,SIRT1活性的抑制造成的心肌细胞凋亡至少一部分原因归结为p53乙酰化水平的升高与Bax表达的上调.以上结果表明,小鼠心脏特异性地过表达SIRT1的显性失活突变体(SIRT1 H363Y)能够导致心肌细胞凋亡以及心衰早发,这提示SIRT1在出生后早期维持心脏正常功能方面发挥着非常关键的作用.     

SIRT1功能及其对卵泡发育和卵母细胞成熟的调控作用 *

沉默信息调节因子1(silent information regulator1, SIRT1)是NAD⁺ 依赖的去乙酰化酶,通过使底物发生去乙酰化而参与细胞众多生理功能的调节,在糖脂代谢、衰老、细胞凋亡、氧化应激等过程中发挥了重要作用。另外,众多研究表明,SIRT1是调控动物卵巢老化、卵泡发育和卵母细胞成熟的重要因子,SIRT1 表达下降或活性改变将导致卵母细胞老化,降低动物的繁殖力。为了充分理解SIRT1功能,并通过调控SIRT1活性而延缓卵巢和卵母细胞老化,从而提高动物繁殖力,简述了SIRT1的激活及其参与细胞内调控的生物过程,并从能量代谢、抗氧化胁迫、染色质重塑的角度讨论了SIRT1的主要功能,重点阐述了SIRT1对动物卵泡发育和卵母细胞成熟的调控作用。     

SIRT1在内皮细胞中抑制PMA和ionomycin诱导的ICAM-1的表达

白细胞在内皮中的富集能够引起炎症并触发动脉粥样硬化,intercellular adhesion molecule-1(ICAM-1)在该过程中发挥了重要作用.本实验室先前研究显示,内皮特异过表达Ⅲ类组蛋白去乙酰化酶SIRT1能够抑制动脉粥样硬化.因此,提出这样的假设:SIRT1能够抑制内皮细胞中ICAM-1的表达.实验发现,PMA和ionomycin(PMA/Io)能够在人脐静脉内皮细胞(HUVECs)中明显诱导SIRT1和ICAM-1的表达.而且,腺病毒介导的SIRT1过表达在HUVECs中能显著抑制PMA/Io诱导的ICAM-1的表达,而敲低SIRT1的表达则导致ICAM-1表达上调.双荧光素酶报告基因分析表明,过表达SIRT1抑制基础水平和PMA/Io诱导下的ICAM-1的启动子活性.进一步通过染色质免疫共沉淀(ChIP)实验发现,SIRT1参与转录复合物结合在ICAM-1启动子区,而且SIRT1的干扰能够提高NF-κB的亚基p65结合到ICAM-1启动子区的能力.总之,这些数据提示,SIRT1在内皮细胞中抑制ICAM-1表达的作用可能有助于其对抗动脉粥样硬化的发生.     

沉默信息调节因子1通过诱导miR-221/222表达促进细胞自噬

微小RNA（microRNAs, miRNAs,）是一类强大的基因表达调控子,可在转录及转录后水平负调控靶基因的表达来参与生物学过程。沉默信息调节因子1 (silent information regulator1, SIRT1)底物众多,可通过去乙酰化作用参与多种细胞生命活动进程。尽管如此,SIRT1与非编码RNA如miRNA的表达调控关系仍有待深入研究。本文利用荧光定量PCR 检测发现,SIRT1与miR-221和miR-222的表达呈正相关：干扰SIRT1后,miR-221/222呈低水平表达;而过表达SIRT1则促进miR-221/222的表达。将miR-221/222基因簇启动子区序列插入pEZX-GA01构建双荧光素酶报告载体,与SIRT1过表达质粒或干扰序列共转至细胞。结果显示,SIRT1可显著提高miR-221/222启动子区活性,提示SIRT1可在转录水平调节miR-221/222的表达。进一步运用Western 印迹研究发现,在HEK293细胞中过表达miR-221/222可促进细胞的自噬能力,而抑制miR-221/222的表达可减弱自噬。此外,过表达SIRT1的同时抑制miR-221/222 的表达可减弱SIRT1的自噬诱导作用。综上所述,SIRT1可通过诱导miR-221/222的表达促进细胞自噬,其具体作用机制有待进一步探讨。     

大蒜素激活SIRT1通路抑制过氧化氢诱导人脐静脉内皮细胞的衰老

Sirtuin1(SIRT1)活性的异常与血管内皮细胞的衰老密切相关。大蒜素作为一种生物活性分子具有抗氧化、抗炎及调脂作用,然而目前尚未见关于大蒜素与SIRT1的活性调节的报道。本研究旨在阐明大蒜素对过氧化氢（H2O2）诱导人脐静脉内皮细胞（HUVECs）衰老的影响,以及Sirtuin1(SIRT1)在其中的作用。SA-β-gal染色及活性氧检测提示,与对照组相比,大蒜素明显减少H2O2诱导半乳糖苷酶阳性细胞数及活性氧的产生。用Western印迹、MTT、RT-PCR及SIRT1活化检测对SIRT1、p-SIRT1、PAI-1的蛋白质、SIRT1mRNA表达及细胞活力进行检测,结果显示,大蒜素可以逆转H2O2诱导的PAI-1表达的升高、SIRT1磷酸化及活性的降低,并且上调细胞的活力。当采用SIRT1抑制剂NAM处理后,大蒜素的这些作用均被阻断。以上结果表明,大蒜素通过激活SIRT1抑制H2O2诱导的HUVECs ROS的产生、活力的下降及细胞的衰老。     

SIRT6在非酒精性脂肪性肝炎中的作用

SIRT6是沉默信息调节蛋白家族中的一员,具有烟酰胺腺嘌呤二核苷酸依赖的组蛋白去乙酰化酶活性和单ADP-核糖基转移酶活性。SIRT6在机体关键的生理和病理过程中起着重要作用,包括脂代谢、炎症、氧化应激以及纤维化等,被认为是代谢综合征的潜在治疗靶点。SIRT6敲除小鼠表现出严重的脂肪肝,非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)小鼠模型的肝组织中SIRT6的表达量明显低于正常小鼠,而过表达SIRT6能显著减轻NASH所引起的肝损伤,提示SIRT6可能具有保护NASH动物模型的作用。本文将重点综述SIRT6在NASH发生、发展过程中的重要调节作用。     

姜黄素后处理通过SIRT1/FOXO1信号通路拮抗小鼠脑缺血再灌注损伤

目的:探究姜黄素后处理是否通过激活SIRT1/FOXO1信号通路抵抗小鼠脑缺血再灌注损伤。方法:小鼠脑缺血30 min,再灌注24 h建立脑缺血再灌注模型。手术前脑室内注射SIRT1特异性抑制剂EX527。再灌注后腹腔注射姜黄素。小鼠随机分为以下6组:假手术组;单纯姜黄素后处理组;缺血再灌注组;缺血再灌注+姜黄素后处理组;EX527预处理+缺血再灌注+姜黄素后处理组;EX527预处理+脑缺血再灌注组。再灌注24 h检测脑梗体积、Complex I活性、ROS含量以及SIRT1、Ac-FOXO1、Bax、Bcl-2、Caspase-3蛋白表达情况。结果:与手术组相比,姜黄素后处理组梗死区脑组织SIRT1的表达量及活性明显增加,脑梗体积降低,ROS含量降低而Complex I活性增高,Bcl-2的表达增高而Bax和Caspase-3的表达量降低(均P0.05)。阻断SIRT1信号通路后上述姜黄素脑保护作用均减弱(P0.05)。结论:我们的研究首次证实姜黄素后处理通过激活SIRT1/FOXO1信号通路,进而降低氧化应激与凋亡,最终减轻脑缺血再灌注损伤。     

Sirtuin在热量限制延长寿命机制中的研究进展

热量限制(caloric restriction, CR)可以引起细胞、生物体寿命延长和降低衰老相关疾病的发生,其中Sirtuin起着关键作用．Sirtuin将机体能量代谢和基因表达调控相偶联,通过赖氨酸去乙酰化改变蛋白质的活性和稳定性,从而调节衰老进程．酵母中度CR影响其复制寿命和时序寿命,主要依赖于激活Sir2,增加细胞内NAD+/NADH的比例和调节尼克酰胺浓度来实现．类似的机制也存在于秀丽线虫和果蝇中．哺乳动物在CR条件下SIRT1蛋白表达应答性上升,细胞中NAM磷酸基转移酶能够直接影响NAM和NAD+浓度,并影响SIRT1活性．NO表达增加能导致SIRT1上调和线粒体合成增加．SIRT1可能通过改变组蛋白、p53、NES1、FOXO等底物蛋白的乙酰化影响到细胞和个体的衰老．表明不同生物体中的Sirtuin及其同源类似物在CR条件下对衰老进程和寿命都起着非常重要的作用．     

lncRNA SGO1-AS1通过调节SIRT1去乙酰化酶活性抑制细胞凋亡

沉默信息调节因子1(silent information regulator 1, SIRT1)是NAD~+依赖的组蛋白去乙酰化酶,可以通过去乙酰化底物调节多种生物学功能。长链非编码RNA(long non-coding RNA, lncRNA)是一类新兴的基因表达调控子,可以影响多种生命进程。尽管如此,SIRT1与lncRNA之间的调控关系以及lncRNA在SIRT1介导的生物学功能中的作用还有待进一步阐明。因此,本研究旨在探讨SIRT1相关lncRNA SGO1-AS1在SIRT1介导的细胞凋亡中的作用及其分子机制。荧光定量PCR检测发现,过表达SIRT1可显著促进lncRNA SGO1-AS1的表达(P0.05),反之沉默SIRT1则抑制SGO1-AS1的表达(P0.001)。进一步利用Western印迹、胱天蛋白酶3/7活性检测和TUNEL实验发现,沉默SGO1-AS1可显著促进细胞凋亡(P0.05),但并不明显影响DNA损伤修复。此外,Western印迹结果显示,SGO1-AS1还可显著促进SIRT1蛋白的去乙酰化酶活性。综上所述,lncRNA SGO1-AS1可以抑制细胞凋亡,且长链非编码RNA SGO1-AS1有可能与SIRT1形成正反馈调节环路,从而调控细胞凋亡。尽管如此,SGO1-AS1调节细胞凋亡分子机制依然有待深入研究。     

SIRT1 regulates inflammation response of macrophages in sepsis mediated by long noncoding RNA

Molecular mechanisms for macrophage immune responses modulated by SIRT1 during sepsis remain unclear. Here, we show that SIRT1 expression is down-regulated in macrophages from mouse sepsis model or LPS stimulation. SIRT1 expression in macrophages correlates with low levels of a long noncoding RNA (lncRNA)-NONMMUT003701 [named as lncRNA-CCL2]. SIRT1 inhibits lncRNA-CCL2 expression via sustaining a repressive chromatin state in the lncRNA-CCL2 locus. The inflammation cytokines expression is downregulated by knockdown of lncRNA-CCL2. Such inhibition can be reversed partly by decreased SIRT1 activity. Thus, this work uncovers previously unidentified mechanisms in which SIRT1 associates with lncRNA and lncRNA regulates macrophage inflammatory response.     

SIRT3, a Mitochondrial NAD+-Dependent Deacetylase,Is Involved in the Regulation of Myoblast Differentiation

Sirtuin 3 (SIRT3), one of the seven mammalian sirtuins, is a mitochondrial NAD⁺-dependent deacetylase known to control key metabolic pathways. SIRT3 deacetylases and activates a large number of mitochondrial enzymes involved in the respiratory chain, in ATP production, and in both the citric acid and urea cycles. We have previously shown that the regulation of myoblast differentiation is tightly linked to mitochondrial activity. Since SIRT3 modulates mitochondrial activity, we decide to address its role during myoblast differentiation. For this purpose, we first investigated the expression of endogenous SIRT3 during C2C12 myoblast differentiation. We further studied the impact of SIRT3 silencing on both the myogenic potential and the mitochondrial activity of C2C12 cells. We showed that SIRT3 protein expression peaked at the onset of myoblast differentiation. The inhibition of SIRT3 expression mediated by the stable integration of SIRT3 short inhibitory RNA (SIRT3shRNA) in C2C12 myoblasts, resulted in: 1) abrogation of terminal differentiation - as evidenced by a marked decrease in the myoblast fusion index and a significant reduction of Myogenin, MyoD, Sirtuin 1 and Troponin T protein expression - restored upon MyoD overexpression; 2) a decrease in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and citrate synthase protein expression reflecting an alteration of mitochondrial density; and 3) an increased production of reactive oxygen species (ROS) mirrored by the decreased activity of manganese superoxide dismutase (MnSOD). Altogether our data demonstrate that SIRT3 mainly regulates myoblast differentiation via its influence on mitochondrial activity.     

Overexpression of a dominant-negative mutant of SIRT1 in mouse heart causes cardiomyocyte apoptosis and early-onset heart failure

SIRT1,a mammalian ortholog of yeast silent information regulator 2(Sir2),is an NAD+-dependent protein deacetylase that plays a critical role in the regulation of vascular function.The current study aims to investigate the functional significance of deacetylase activity of SIRT1 in heart.Here we show that the early postnatal hearts expressed the highest level of SIRT1deacetylase activity compared to adult and aged hearts.We generated transgenic mice with cardiac-specific expression of a dominant-negative form of the human SIRT1(SIRT1H363Y),which represses endogenous SIRT1 activity.The transgenic mice displayed dilated atrial and ventricular chambers,and died early in the postnatal period.Pathological,echocardiographic and molecular phenotype confirmed the presence of dilated cardiomyopathy.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling analysis revealed a greater abundance of apoptotic nuclei in the hearts of transgenic mice.Furthermore,we show that cardiomyocyte apoptosis caused by suppression of SIRT1 activity is,at least in part,due to increased p53acetylation and upregulated Bax expression.These results indicate that dominant negative form of SIRT1(SIRT1H363Y)overexpression in mouse hearts causes cardiomyocyte apoptosis and early-onset heart failure,suggesting a critical role of SIRT1 in preserving normal cardiac development during the early postnatal period.     

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.     

SIRT1/FoxO3 axis alteration leads to aberrant immune responses in bronchial epithelial cells

Inflammation and ageing are intertwined in chronic obstructive pulmonary disease (COPD). The histone deacetylase SIRT1 and the related activation of FoxO3 protect from ageing and regulate inflammation. The role of SIRT1/FoxO3 in COPD is largely unknown. This study evaluated whether cigarette smoke, by modulating the SIRT1/FoxO3 axis, affects airway epithelial pro‐inflammatory responses. Human bronchial epithelial cells (16HBE) and primary bronchial epithelial cells (PBECs) from COPD patients and controls were treated with/without cigarette smoke extract (CSE), Sirtinol or FoxO3 siRNA. SIRT1, FoxO3 and NF‐κB nuclear accumulation, SIRT1 deacetylase activity, IL‐8 and CCL20 expression/release and the release of 12 cytokines, neutrophil and lymphocyte chemotaxis were assessed. In PBECs, the constitutive FoxO3 expression was lower in patients with COPD than in controls. Furthermore, CSE reduced FoxO3 expression only in PBECs from controls. In 16HBE, CSE decreased SIRT1 activity and nuclear expression, enhanced NF‐κB binding to the IL‐8 gene promoter thus increasing IL‐8 expression, decreased CCL20 expression, increased the neutrophil chemotaxis and decreased lymphocyte chemotaxis. Similarly, SIRT1 inhibition reduced FoxO3 expression and increased nuclear NF‐κB. FoxO3 siRNA treatment increased IL‐8 and decreased CCL20 expression in 16HBE. In conclusion, CSE impairs the function of SIRT1/FoxO3 axis in bronchial epithelium, dysregulating NF‐κB activity and inducing pro‐inflammatory responses.