美洲大蠊提取物CⅡ-3调控SIRT1/mTOR信号通路诱导白血病K562细胞衰老  

Periplaneta americana extract CⅡ-3 induces senescence of leukemia K562 cells via SIRT1/mTOR signaling pathway

作　　者：何思悦 张成桂 刘衡 周玥 唐紫云 毕子莹 田露 李敏瑞 HE Si-yue;ZHANG Cheng-gui;LIU Heng;ZHOU Yue;TANG Zi-yun;BI Zi-ying;TIAN Lu;LI Min-rui(Department of Histology and Embryology,Dali University,Dali 671000,China;Yunnan Provincial Key Laboratory of Insect Biomedicine Research and Development,Dali 671000,China)

机构地区：[1]大理大学组织学与胚胎学教研室,云南大理671000 [2]云南省昆虫生物医药研发重点实验室,云南大理671000

出　　处：《中国中药杂志》2023年第11期3039-3045,共7页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81860038,81660731,81873103)。

摘　　要：该研究旨在探讨沉默信息调节因子2相关酶1(slient mating-type information regulation 2 homolog 1,SIRT1)/结节性硬化症复合物Ⅱ(tuberous sclerosis complex 2,TSC2)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路在美洲大蠊提取物CⅡ-3诱导人白血病细胞K562细胞衰老中的作用。体外培养K562细胞,设对照组(0μg·mL^(-1))及美洲大蠊提取物CⅡ-3不同剂量组(5、10、20、40、80、160μg·mL^(-1)),细胞计数试剂盒(cell counting kit-8,CCK-8)检测CⅡ-3对K562细胞的增殖抑制作用;流式细胞术检测CⅡ-3对K562细胞周期分布的影响;β-半乳糖苷酶染色试剂盒(senescence-associatedβ-galactosidase stain kit,SA-β-gal)检测衰老细胞染色阳性百分率;流式细胞术检测线粒体膜电位的改变;荧光定量PCR检测端粒酶逆转录酶(telomerase reverse transcriptase,TERT)的表达;荧光定量PCR和免疫印迹法(Western blot)检测SIRT1、TSC2和mTOR mRNA及蛋白的表达。结果显示,经美洲大蠊提取物CⅡ-3诱导作用后,CⅡ-3组细胞增殖抑制率高于对照组,当CⅡ-3质量浓度为80μg·mL^(-1),作用时间为72 h时,CⅡ-3对K562细胞的增殖抑制率最高,故选为实验标准进行后续研究。与对照组相比,CⅡ-3组阻滞在G0/G1的细胞比例升高,S期细胞比例降低;细胞SA-β-Gal衰老染色阳性率升高;线粒体膜电位降低;端粒酶TERT mRNA表达下调;SIRT1和TSC2 mRNA的表达下调,mTOR mRNA的表达上调;SIRT1和p-TSC2蛋白的表达下调,p-mTOR蛋白的表达上调。以上研究结果表明,美洲大蠊提取物CⅡ-3可通过SIRT1/mTOR信号通路诱导K562细胞衰老。This study aims to investigate the role of slient mating-type information regulation 2 homolog 1(SIRT1)/tuberous sclerosis complex 2(TSC2)/mammalian target of rapamycin(mTOR)signaling pathways in the Periplaneta americana extract CⅡ-3-induced senescence of human leukemia K562 cells.K562 cells were cultured in vitro and treated with 0(control),5,10,20,40,80,and 160μg·mL^(-1) of P.americana extract CⅡ-3.Cell counting kit-8(CCK-8)and flow cytometry were employed to examine the proliferation and cell cycle of the K562 cells.Senescence-associatedβ-galactosidase stain kit(SA-β-gal)was used to detect the positive rate of senescent cells.Mitochondrial membrane potential was detected by flow cytometry.The relative mRNA level of telomerase reverse transcriptase(TERT)was determined by fluorescence quantitative PCR.The mRNA and protein levels of SIRT1,TSC2,and mTOR were determined by fluorescence quantitative PCR and Western blot,respectively.The results showed that CⅡ-3 significantly inhibited the proliferation of K562 cells and the treatment with 80μg·mL^(-1) CⅡ-3 for 72 h had the highest inhibition rate.Therefore,80μg·mL^(-1) CⅡ-3 treatment for 72 h was selected as the standard for subsequent experiments.Compared with the control group,CII-3 increased the proportion of cells arrested in G0/G1 phase,decreased the proportion of cells in S phase,increased the positive rate of SA-β-Gal staining,elevated the mitochondrial membrane potential and down-regulated the mRNA expression of TERT.Furthermore,the mRNA expression of SIRT1 and TSC2 was down-regulated,while the mRNA expression of mTOR was up-regulated.The protein expression of SIRT1 and p-TSC2 was down-regulated,while the protein expression of p-mTOR was up-regulated.The results indicated that P.americana extract CⅡ-3 induced the senescence of K562 cells via the SIRT1/mTOR signaling pathway.

关 键 词：美洲大蠊提取物CⅡ-3 K562细胞 衰老 SIRT1 mTOR 

分 类 号：R285[医药卫生—中药学]