The SIRT1 activator SRT1720 extends lifespan and improves health of mice fed a standard diet

The prevention or delay of the onset of age-related diseases prolongs survival and improves quality of life while reducing the burden on the health care system. Activation of sirtuin 1 (SIRT1), an NAD+ deacetylase, improves metabolism and confers protection against physiological and cognitive disturbances in old age. SRT1720 is a specific SIRT1 activator that has health and lifespan benefits in adult mice fed a highfat diet. We found extension in lifespan, delayed onset of age-related metabolic diseases, and improved general health in mice fed a standard diet after SRT1720 supplementation. Inhibition of pro-inflammatory gene expression both in the liver and muscle of SRT1720-treated animals was noted. SRT1720 lowered phosphorylation of NF-κB pathway regulators in vitro only when SIRT1 was functionally present. Combined with our previous work, the current study further supports the beneficial effects of SRT1720 on health across the lifespan in mice. Groups of 28 week old male C57BL/6J mice were maintained on ad libitum AIN-93G SD diet, or an ad libitum AIN-93G diet supplemented with SRT1720 for the rest of their lives. SRT1720 was added at a dose of 1.33 g drug per kg of chow, formulated to provide daily doses of approximately 100 mg drug per kg bodyweight to the mice. 5 mice from each group were selected and RNA was extracted from both muscle and liver tissue using 1.0mm glass beads in a Precellys 24 Tissue Homogenizer and Qiagen RNeasy Mini Kits for Fibrous Tissue according to manufacturer's specifications. Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. RNA samples were labeled using the Illumina TotalPrep RNA Amplification Kit. In short, 0.5ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP. Arrays were hybridized using a total of 0.75ug of biotin-labeled cRNA at 58 degrees C for 16 hours to Illumina's Sentrix MouseRef-8 v2 Expression BeadChips. Each BeadChip has ~24,000 well-annotated RefSeq transcripts with approximately 30-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3. Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina and the data was extracted using the Illumina GenomeStudio software(v1.6.0). Any spots at or below the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std.预防或延缓衰老相关疾病的发作，可延长生存期、提升生活质量，同时减轻医疗系统负担。激活沉默信息调节因子2相关酶1（sirtuin 1, SIRT1）——一种烟酰胺腺嘌呤二核苷酸（NAD+）依赖性脱乙酰酶——能够改善代谢，并对老年时期的生理与认知功能障碍起到保护作用。SRT1720是一种特异性SIRT1激活剂，在高脂饮食喂养的成年小鼠中可带来健康益处并延长寿命。本研究发现，在标准饮食喂养的小鼠中补充SRT1720后，其寿命得以延长，衰老相关代谢疾病的发病延迟，整体健康状况得到改善。同时观察到，经SRT1720处理的动物的肝脏与肌肉组织中促炎基因的表达受到抑制。体外实验中，仅当SIRT1功能正常存在时，SRT1720才能降低核因子κB（NF-κB）通路调控因子的磷酸化水平。结合我们此前的研究，本研究进一步证实了SRT1720对小鼠全生命周期健康的有益作用。将28周龄的雄性C57BL/6J小鼠分为两组，一组自由采食AIN-93G标准合成饲料（AIN-93G SD diet），另一组在自由采食AIN-93G饲料的基础上终身补充SRT1720。SRT1720的添加剂量为每千克饲料1.33克药物，按此配方可使小鼠每日获得约每千克体重100毫克的药物剂量。从每组中选取5只小鼠，使用Precellys 24组织匀浆器配合1.0mm玻璃珠，并按照制造商说明书使用Qiagen RNeasy纤维组织迷你试剂盒，从肌肉与肝脏组织中提取RNA。采用安捷伦2100生物分析仪搭配RNA 6000 Nano芯片，对总RNA的质量与浓度进行检测。使用Illumina TotalPrep RNA扩增试剂盒对RNA样本进行标记：首先取0.5μg总RNA，以带有T7 RNA聚合酶启动子序列的寡聚dT为引物，通过逆转录酶将其反转录为单链cDNA，随后合成双链cDNA分子。将双链cDNA通过配套柱进行纯化与浓缩，随后用于过夜体外转录反应，通过掺入生物素-16-UTP生成并标记单链RNA（cRNA）。取总计0.75μg生物素标记的cRNA，在58℃下与Illumina Sentrix MouseRef-8 v2表达微珠芯片杂交16小时。每张微珠芯片包含约24000个注释完善的RefSeq转录本，冗余度约为30倍。对芯片进行洗涤、封闭后，采用链霉亲和素-Cy3染色对生物素标记的cRNA进行检测。使用Illumina的Beadstation 500 X以0.8μm分辨率扫描芯片，并通过Illumina GenomeStudio软件（v1.6.0）提取数据。采用Illumina检测p值≥0.02的标准过滤掉背景信号及以下的斑点。对剩余所有信号值取自然对数，计算每张芯片的平均值与标准差，随后计算z分数标准化值并展示如下：z分数=(原始值-平均值)/标准差。