高良姜素对非小细胞肺癌A549细胞SIRT1/mTOR通路及放射敏感性的影响

摘要：目的:探讨高良姜素对非小细胞肺癌（NSCLC）A549细胞沉默信息调节因子1（SIRT1）/哺乳动物雷帕霉素靶蛋白（mTOR）通路及放射敏感性的影响。方法:体外培养NSCLC A549细胞,细胞计数试剂盒（CCK-8）法检测不同浓度(10,20,40,60,80,100μmol·L-1)高良姜素处理的A549细胞的增殖抑制率;将A549细胞经不同剂量（0,1,2,4,6,8 Gy）放射处理,并设置各剂量与35μmol·L-1高良姜素联用组,克隆形成实验观察高良姜素的放射增敏作用;将A549细胞分为高良姜素+放射线组(35μmol·L-1+6 Gy)、高良姜素组(35μmol·L-1)、放射线组（6 Gy）及对照组,流式细胞术检测细胞凋亡率;蛋白免疫印迹法检测增殖蛋白细胞周期蛋白D1（CyclinD1）、细胞增殖核抗原（Ki67）、凋亡蛋白半胱氨酸蛋白酶（Caspase）-3、Caspase-9及STRT1/mTOR通路蛋白SIRT1、腺苷酸活化蛋白激酶（AMPK）、磷酸化AMPK（p-AMPK）、磷酸化mTOR（p-mTOR）、mTOR的表达。结果:与对照组比较,随着高良姜素浓度的增加,A549细胞增殖抑制率逐渐增加（P<0.05）;与各剂量射线单独处理的A549细胞相比,35μmol·L-1高良姜素+各剂量射线处理的A549细胞存活分数降低（P<0.05）,辐射增敏比（SER）=1.522;与对照组比较,高良姜素组、放射线组、高良姜素+放射线组A549细胞凋亡率、Caspase-3、Caspase-9、p-AMPK/AMPK蛋白表达显著升高,CyclinD1、Ki67、SIRT1、p-mTOR/mTOR蛋白表达显著降低（P<0.05）;与高良姜素组、放射线组比较,高良姜素+放射线组A549细胞凋亡率、Caspase-3、Caspase-9、p-AMPK/AMPK蛋白表达显著升高,高良姜素+放射线组CyclinD1、Ki67、SIRT1、p-mTOR/mTOR蛋白表达显著降低（P<0.05）。结论:高良姜素可能通过调控SIRT1/mTOR通路影响A549细胞的增殖凋亡及放射敏感性,可能是治疗NSCLC的潜在药物。     

辛伐他汀联合顺铂对人非小细胞肺癌A549细胞增殖的抑制作用

目的研究辛伐他汀联合顺铂对人非小细胞肺癌A549细胞的抑制作用,并探讨其作用机制。方法采用MTT法检测0.625、1.25、2.5、5、10μmol/L顺铂和3.125、6.25、12.5、25、50μmol/L辛伐他汀对A549细胞增殖抑制率的影响;考察2.5μmol/L顺铂与3.125、6.25、12.5、25、50μmol/L辛伐他汀联用对A549细胞增殖抑制作用的协同效果;Annexin V-FITC/PI双染后流式细胞术检测5μmol/L顺铂与25μmol/L辛伐他汀联合应用对对A549细胞凋亡率的影响;分光光度法检测辛伐他汀(25μmol/L)联合顺铂(2.5μmol/L)对A549细胞caspase-3活性的影响。结果辛伐他汀或顺铂对A549细胞的增殖有显著的抑制作用,随着浓度的增加、时间的延长,呈时间、剂量相关性,其抑制作用增强(P0.01)。顺铂(2.5μmol/L)与不同浓度(3.125、6.25、12.5、25、50μmol/L)辛伐他汀联用对A549细胞的增殖有抑制作用,随着时间的延长,辛伐他汀剂量的增加,两者联用抑制作用呈增强的趋势(P0.01),2.5μmol/L顺铂联用辛伐他汀25μmol/L的抑制作用具有协同效果。25μmol/L辛伐他汀和2.5μmol/L顺铂能有效地诱导A549细胞的凋亡,而且两者联合使用可以显著增加A549细胞的凋亡率。25μmol/L辛伐他汀联合2.5μmol/L顺铂应可以显著增加A549细胞的caspase-3酶活性。结论辛伐他汀能够显著增强顺铂对A549细胞增殖抑制、诱导凋亡的作用,可能通过上调caspase-3活性诱导A549细胞凋亡。     

EX527对胰腺癌细胞 PANC1侵袭迁移能力的影响

目的 应用EX527特异性下调胰腺癌细胞PANC1中SIRT1的去乙酰化活性,观察其对细胞侵袭转移能力的影响。方法 使用EX527处理PANC1细胞,下调SIRT1乙酰化转移酶活性,通过transwell侵袭及划痕实验,观察EX527对PANC1细胞侵袭转移能力的影响,应用Western blot方法观察其对侵袭转移相关因子MMP-2和MMP-9蛋白表达的影响。结果 EX527可以明显降低细胞去乙酰化转移酶活性,但对SIRT1蛋白表达无影响。与对照组相比,经EX527处理后的PANC1细胞的侵袭及转移能力呈现明显抑制作用,且细胞MMP-2和MMP-9的mRNA及蛋白表达水平明显降低,差异具有统计学意义(P<0.01)。结论 EX527可以下调胰腺癌细胞SIRT1去乙酰化活性,明显抑制胰腺癌PANC1细胞的侵袭转移能力,作用机制可能与下调MMP-2和MMP-9表达相关。     

白黎芦醇对心肌细胞去乙酰化酶1表达时序的影响

目的探讨白黎芦醇(RES)在心肌细胞中对去乙酰化酶(SIRT)1表达时序的影响。方法大鼠心肌细胞株H9c2培养48h后,分别以0(对照组)、10(低剂量组)、20(中等剂量组)、50、100μmol/L(高剂量组)的RES处理0、12、24、36、48h,采用噻唑蓝(MTT)比色法测定细胞活力;反转录-聚合酶链反应(RT-PCR)分析SIRT1 mRNA表达情况,Western blot法检测SIRT1蛋白表达水平的变化。结果H9c2心肌细胞经RES处理后,MTT测得的吸光度(A)值在低、中剂量升高,20μmol/L组作用最显著(P<0.05),50μmol/L组开始下降,低于对照组(P>0.05),100μmol/L组降至最低(P<0.05);各组SIRT1 mRNA和蛋白表达A值均高于对照组(P<0.05),与低、高剂量组相比,中等剂量组表达最高(P<0.05);中等剂量组RES处理0、12、24、36、48h后,不同时间点SIRT1 mRNA和蛋白表达A值均高于对照组(P<0.05),以24h时表达最强(P<0.01)。结论20μmol/LRES作用24h时心肌细胞的SIRT1表达最强。     

西达本胺对胃癌细胞株SGC-7901的体外抗增殖作用

目的 研究组蛋白去乙酰化酶(HDACs)抑制剂西达本胺对胃癌细胞株体外抗增殖作用及其可能机制.方法 用西达本胺终浓度为0、16、32、64、128、256μmol/L(分别为B、A1、A2、A3、A4、A5组)处理SGC-7901细胞24、48、72 h.采用MTT法及流式细胞术分别检测细胞增殖及细胞凋亡,反转录PCR(RT-PCR)检测沉默信息调节因子2相关酶1(SIRT1)及HDAC11 mRNA表达,实时荧光定量PCR(RT-qPCR)检测表皮生长因子受体(EGFR) mRNA表达,Western blot检测Notch1蛋白的表达.结果 与B组相比,西达本胺呈浓度依赖性地抑制SCC-7901细胞增殖(P＜0.05),提高SGC-7901细胞凋亡率(P＜0.05),阻断SIRT1、HDAC11、EGFR mRNA以及Notch1蛋白表达(P＜0.05).结论 西达本胺可抑制SGC-7901细胞增殖,促进细胞凋亡;其作用机制可能是降低SIRT1、HDAC11基因的表达或与Notch及EGFR信号通路相关.     

高良姜素调节MCP-1/CCR2信号轴对肺癌细胞恶性生物学行为的影响

目的 研究高良姜素通过调节单核细胞趋化蛋白-1(MCP-1)/趋化因子受体-2(CCR2)信号通路对肺癌细胞的恶性生物学行为产生的影响。方法 将人肺癌细胞系A549分为ctrl组、低浓度高良姜素组(5μmol/L)、高浓度高良姜素组(100μmol/L)、吉非替尼组(10μmol/L)、高浓度高良姜素+MCP-1组(100μmol/L高良姜素+75 ng/mL MCP-1)。CCK-8试剂盒检测细胞活性;流式细胞术检测细胞凋亡;细胞划痕实验检测细胞迁移;transwell检测细胞侵袭能力;western blot检测MCP-1、CCR2、凋亡蛋白半胱氨酸蛋白酶(Caspase-3)、细胞增殖核抗原(Ki67)、基质金属蛋白酶2(MMP-2)蛋白表达。结果 与ctrl组比较,低浓度高良姜素组、高浓度高良姜素组、吉非替尼组肺癌细胞A549的细胞存活率、细胞迁移愈合率、细胞侵袭数、MCP-1、CCR2、Ki67、MMP-2蛋白表达降低,而细胞凋亡率、Caspase-3含量升高(P<0.05);与高浓度高良姜素组比较,高浓度高良姜素+MCP-1组A549细胞存活率、细胞迁移愈合率、细胞侵...     

LncRNA ITGB2-AS1靶向miR-338-3p对肺癌细胞多西他赛耐药性的影响

目的研究lncRNA ITGB2-AS1对肺癌细胞增殖、凋亡及多西他赛耐药性的影响及潜在的分子机制。方法用不同浓度(6.25μg/L、12.5μg/L、25μg/L、50μg/L、100μg/L、200μg/L)多西他赛(DTX)处理肺癌A549和多西他赛耐药细胞A549/DTX细胞,CCK8法测定DTX对A549细胞增殖抑制率和IC 50,qRT-PCR检测A549和A549/DTX细胞中lncRNA ITGB2-AS1和miR-338-3p的水平,Western blot检测细胞中CyclinD1、p21、Bax和Bcl-2表达水平,流式细胞术检测细胞凋亡率,双荧光素酶报告系统验证lncRNA ITGB2-AS1和miR-338-3p的靶向关系。结果多西他赛(DTX)(6.25μg/L、12.5μg/L、25μg/L、50μg/L、100μg/L、200μg/L)对A549/DTX细胞的抑制率显著低于A549细胞,呈浓度依赖性,A549/DTX对DTX的IC 50(256.80±10.22)μg/L显著高于A549细胞(23.57±2.11)μg/L;在A549/DTX中,lncRNA ITGB2-AS1含量显著高于A549细胞(P<0.05),miR-338-3p含量显著低于A549细胞(P<0.05);抑制lncRNA ITGB2-AS1联合12.5μg/L DTX处理可提高A549/DTX细胞凋亡率,增强DTX对A549/DTX细胞的增殖抑制率,上调p21和Bax水平,下调Cyclin D1和Bcl-2蛋白含量;lncRNA ITGB2-AS1靶向负调控miR-338-3p的表达;下调miR-338-3p可逆转抑制lncRNA ITGB2-AS1对A549/DTX细胞增殖、凋亡和多西他赛耐药性的作用。结论LncRNA ITGB2-AS1可靶向miR-338-3p调控A549/DTX细胞的增殖、凋亡及多西他赛耐药性。LncRNA ITGB2-AS1是肺癌潜在的分子靶点。     

SIRT1介导的七氟醚后处理对失血性休克复苏小鼠学习与记忆损伤的保护作用

目的探究沉默信息调节因子(silent information regulation 1,SIRT1)介导的凋亡相关通路在七氟醚后处理对失血性休克复苏小鼠海马神经元损伤中的保护作用。方法建立失血性休克与复苏小鼠模型,雄性C57BL/6J小鼠60只随机分为:假手术组(Sham组)、HSR组(Shock组)、七氟醚处理组(Sevo组)、七氟醚联合SIRT1特异性抑制剂处理组(EX527+Sevo组)以及EX527处理组(EX527组)。通过TTC染色法检测小鼠脑梗死体积,TUNEL染色法检测各组小鼠海马神经细胞的变化,水迷宫实验检测小鼠学习记忆能力,Western blot检测SIRT1和凋亡相关蛋白Bcl-2、Bax、Cleaved caspase-3的表达。结果造模小鼠在水迷宫检测中到达平台的潜伏期延长,在目标象限的运动距离减少,脑梗死体积增大,TUNEL染色阳性细胞数增多,SIRT1、Bcl-2蛋白表达降低,Bax、Cleaved-caspase3蛋白表达增加;七氟醚处理后改善了失血性休克与复苏小鼠的神经损伤情况,七氟醚与SIRT1抑制剂EX527联合处理后,七氟醚对失血性休克与复苏小鼠的神经损伤保护作用减弱。结论七氟醚可能通过SIRT1介导的凋亡相关通路发挥对失血性休克复苏引起的海马神经元损伤的保护作用。     

芍药苷对多囊卵巢综合征大鼠卵巢颗粒细胞增殖和凋亡的影响机制

目的 分析芍药苷调节单磷酸腺苷酸活化蛋白激酶/沉默信息调节因子2相关酶1/过氧化物酶体增殖物激活受体γ辅激活物1-α(AMPK/SIRT1/PGC-1α)通路对多囊卵巢综合征(PCOS)大鼠颗粒细胞增殖和凋亡的影响。方法 10只雌性大鼠挑选5只作为对照组,剩余5只大鼠按照6 mg/100 g皮下注射脱氢表雄酮(DHEA)构造PCOS模型,提取卵巢颗粒细胞,分为对照组、PCOS组、芍药苷低浓度组(200μg/ml)、芍药苷高浓度组(800μg/ml)、芍药苷高浓度+Compound C(AMPK抑制剂)组(800μg/ml+20μmol/L)、芍药苷高浓度+EX527(SIRT1抑制剂)组(800μg/ml+100μmol/L),对照组卵巢颗粒细胞来源于对照组,其余组卵巢颗粒细胞均来自PCOS大鼠。放射免疫法测定细胞培养液雌二醇(E₂)和孕酮(P)含量,CCK8法以及克隆形成实验测定卵巢颗粒细胞增殖能力,流式细胞仪法测定卵巢颗粒细胞凋亡能力,Western blot法测定卵巢颗粒细胞AMPK/SIRT1/PGC-1α通路蛋白表达情况。结果 随着培养时间延长,卵巢颗粒...     

SIRT1通过CREB/BDNF通路调控吗啡诱导大鼠条件位置偏爱的机制研究北大核心CSCD

目的研究腹外侧眶皮层(ventrolateral orbital cortex,VLO)内微量注射sirtuin1(SIRT1)抑制剂EX527对吗啡诱导大鼠条件位置偏爱(conditioned place preference,CPP)的影响,并探讨CREB/BDNF通路在其中的作用。方法应用脑立体定位术在大鼠双侧VLO内留置导管,建立吗啡诱导大鼠CPP模型,d 1为适应,d 2~4为前测,d 5~14为条件性训练,隔日交替腹腔注射吗啡(1 mL·kg^(-1),5 mg·kg^(-1))或生理盐水(1 mL·kg^(-1)),提前30 min通过导管向双侧VLO内微量注射EX527(1μL,5 g·L^(-1))或DMSO(1%,1μL),d 15为测试。CPP测试后立即取脑分离VLO组织,Western blot检测VLO内SIRT1、PSD95、c-fos、p-ERK、CREB、BDNF的表达水平。结果吗啡诱导大鼠CPP模型构建成功,吗啡诱导CPP形成组VLO内SIRT1、PSD95、c-fos、p-ERK、CREB蛋白表达明显增高(P<0.05),BDNF蛋白表达明显降低(P<0.05)。VLO内微量注射EX527抑制了吗啡诱导大鼠CPP形成,且明显降低了VLO内SIRT1、PSD95、c-fos、p-ERK、CREB、BDNF蛋白表达(P<0.05)。结论EX527通过抑制吗啡引起的VLO内SIRT1及其下游相关分子表达的增高,并进一步降低BDNF表达,从而有效阻断了大鼠吗啡成瘾的形成。     

Studies on the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells

OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells(HSCs).METHODS Normal human Chang liver cells and human hepatic stellate cell line,LX-2 cells were treated with SRT1720(10μmol·L~(-1))and AICAR(500μmol·L~(-1))prior to ethanol(50 mmol·L~(-1)) for 24 and 48 h.Cell viability was analyzed by methyl thiazolyl tetrazolium assay.SIRT1,AMPK and p-AMPK m RNA levels for 24 h and 48 h were analyzed by RT-PCR,SIRT1,AMPK and p-AMPK protein expressions in the supernatant at 24 and 48 h was detected by Western blot.RESULTS SRT1720 and AICAR effectively decreased LX-2 cell viabilities and exhibited scarcely little toxicity in human Chang liver cells.SRT1720 and AICAR attenuated collagen-I,α-smooth muscle actin(α-SMA)levels,activated liver kinase B-1(LKB1)and AMPK phosphorylation in ethanol treated LX-2 cells.Meanwhile,SRT1720 and AICAR enhanced SIRT1 expression mediated by ethanol both in Chang liver cells and LX-2 cells.Furthermore,SRT1720 and AICAR suppressed the expression of sterol regulatory element-binding protein-1(SREBP-1)to regulate fatty acid synthesis.CONCLUSION SIRT1 agonist and AMPK agonist blocked the crosstalk between hepatocytes and HSCs via SIRT1/AMPK signaling pathway to modulate hepatocytes accumulation of lipid and HSCs activation.     

Calycosin induces apoptosis in adenocarcinoma HT29 cells by inducing cytotoxic autophagy mediated by SIRT1/AMPK‐induced inhibition of Akt/mTOR

Autophagy promotes cell survival or induces apoptosis in cancer cells. While SIRT1 and AMPK induce autophagy in both normal and cancer cells, Akt and mTOR can inhibit it. Calycosin, a methoxyisoflavone, protects against several types of solid tumours including colorectal cancer. However, the mechanisms behind the antitumour effect of Calycosin remain largely unknown. This study investigates if autophagy mediates the anti‐tumourigenesis effect afforded by Calycosin and examines if this effect involves activation of SIRT1 and/or AMPK. Human colorectal (HT29) carcinoma cells were cultured under normal conditions with Calycosin (50 μmol/L) in the presence or absence of chloroquine (10 μmol/L), EX‐527 (100 nmol/L, SIRT1 inhibitor), or IGF‐1 (100 ng/mL, Akt/mTOR activator) for 48 hours. Calycosin inhibited cell growth, proliferation and invasion and increased protein levels of Beclin‐1 and LC3II, markers of autophagy. It significantly increased protein levels of cleaved caspase‐3, Bax, and SIRT1, and activity of AMPK and reduced those of Bcl‐2. These effects were parallel with concomitant reduction in protein levels p‐src, integrin‐β1 and Cyclin‐D1 and activities of Akt and mTOR. Inhibition of autophagy by CQ reversed all these effects except cell invasion. Interestingly, co‐incubating the cells with either EX‐527 or IGF‐1 completely prevented Calycosin‐induced autophagy and all other associated effects and increased cell invasion. Also, blockade of SIRT‐1 prevented the activation of AMPK, Akt, and mTOR, suggesting it to be an upstream regulator of these markers. In conclusion, Calycosin stimulates CRC cell apoptosis and inhibits their invasion by acting as SIRT1 activator which induces activation of AMPK‐induced inhibition of Akt/mTOR axis.     

SIRT1 alleviates isoniazid-induced hepatocyte injury by reducing histone acetylation in the IL-6 promoter region

Silent information regulator 1 (SIRT1) is a type III histone deacetylase that is related to the inhibition of the inflammatory response. The aim of this study was to investigate the regulation of SIRT1 on isoniazid-induced hepatocyte injury and the possible mechanism of histone modification. We found that compared with the blank control group, expression of SIRT1 was decreased in the isoniazid group and that expression of NF-κB p65 was increased, leading to an increase of the expression of inflammatory cytokines Interleukin-6 (IL-6) and Tumour necrosis factor alpha (TNF-α). The level of histone H3K9 acetylation in the promoter region of IL-6 was increased as well. Addition of a SIRT1 agonist (SRT1720) alleviated the inflammatory reaction caused by isoniazid, while the use of a SIRT1 inhibitor (EX527) aggravated the inflammatory damage to cells. In conclusion, these findings indicated that during the period of isoniazid-induced hepatocyte injury, SIRT1 levels were decreased and inflammatory factor levels were increased. Activation of SIRT1 may reduce hepatocyte injury by reducing the level of histone H3K9 acetylation in the promoter region of the IL-6 gene.     

Overexpression of miR‐138‐5p suppresses MnCl2‐induced autophagy by targeting SIRT1 in SH‐SY5Y cells

The mechanism of manganism caused by manganese (Mn), an important environmental risk factor for Parkinson's disease, is still unclear. Recent evidence suggested that autophagy participated in neurodegenerative diseases, in which microRNA played a crucial role. However, roles of microRNA in the aberrant autophagy that occurs in neurodegenerative diseases remains controversial. In nervous system, miRNA‐138‐5p is highly expressed and plays a key role in regulating memory and axon regeneration. Importantly, we also found that miR‐138‐5p expression decreased significantly after SH‐SY5Y cells exposed to manganese chloride (MnCl₂) in previous study. To explore the role of miR‐138‐5p in Mn‐induced autophagy, autophagy associated indicators were detected. And we found that MnCl₂ could induce autophagic dysregulation and inhibit expression of miR‐138‐5p. While the levels of LC3‐II/LC3‐I, Beclin1, and p62, the number of autophagosome formation significantly decreased after miR‐138‐5p over‐expression, which demonstrated that miR‐138‐5p could clearly retard Mn‐induced autophagy. In additional, we found there were classical and evolutionarily conserved miR‐138‐5p binding sites in 3′‐UTR region of SIRT1, which was inhibited when overexpression of miR‐138‐5p. Therefore, it was speculated that elevated expression of SIRT1 may be resulted from inhibition of miR‐138‐5p after cells exposed to MnCl₂. Finally, we found that SIRT1 inhibitor EX‐527 suppressed Mn‐induced autophagy as well as miR‐138‐5p, while the suppression was reversed by SIRT1‐specific activator SRT1720. These results indicated that overexpression of miR‐138‐5p suppressed Mn‐induced autophagy by targeting SIRT1.     

白藜芦醇通过SIRT1激活ADAM10促进APPα代谢

目的：探讨白藜芦醇可否通过SIRT1激活解整合素金属蛋白酶10（ADAM10）促进APPα代谢抑制Aβ分泌。方法：选取过表达人瑞典突变淀粉样前体蛋白APP695sw的细胞模型,分别设立DMSO空白对照组、SIRT1激活剂白藜芦醇组和SIRT1抑制剂EX527组。给药后,ELISA法分别检测细胞培养上清中sAPPα和Aβ的含量,免疫印记Western Blot法检测细胞SIRT1和ADAM10的蛋白水平。结果：与对照组比较,白藜芦醇组细胞培养上清sAPPα的含量增高,Aβ含量下降,sAPPα/Aβ比值增大,细胞SIRT1和ADAM10蛋白水平增高;而抑制剂EX527组与对照相比,细胞培养上清sAPPα的含量降低,Aβ含量升高,sAPPα/Aβ比值减小,细胞SIRT1和ADAM10蛋白水平降低;抑制剂下调SIRT1后各项指标与激动剂组呈现相反的趋势（P<0.05）。结论：白藜芦醇可通过SIRT1激活ADAM10,促进APP进行α非淀粉样代谢途径,增强sAPPα分泌并抑制Aβ产生。     

不同浓度米诺环素对人胶质瘤细胞增殖、自噬、凋亡的影响及其机制

摘要：目的 探讨不同浓度米诺环素对人类胶质瘤 U87 和 LN229 细胞增殖及凋亡的影响和作用机制。方法 （1）U87和LN229细胞设对照组（DMSO处理），5 μmol/L米诺环素组和10 μmol/L米诺环素组，分组处理72 h 后采用 MTT法检测细胞增殖水平，免疫荧光标记检测细胞自噬蛋白微管相关蛋白l轻链3B亚基（LC3B）表达水平，Western blot法检测细胞自噬和凋亡相关蛋白的表达变化。（2）构建沉默信息调节因子2相关酶1（SIRT1）-shRNA载体，观察 敲低SIRT1表达后，不同浓度米诺环素对U87和LN229细胞自噬的影响。结果 （1）与对照组相比，5 μmol/L和10 μmol/L米诺环素组细胞增殖能力下降，免疫荧光标记显示胞浆内自噬标记蛋白LC3B的表达增多，Western blot结果 显示哺乳动物雷帕霉素靶蛋白（mTOR）水平显著降低，自噬基因相关蛋白5（Atg5）、磷酸化AMP依赖的蛋白激酶α亚 基（p-AMPKα）、SIRT1水平显著增加（P＜0.05）。与对照组相比，10 μmol/L米诺环素组B淋巴细胞瘤-2（Bcl-2）、磷 酸化 p70 核糖体蛋白 S6 激酶（p-p70s6k）水平显著降低，活化形式的 Caspase-3（Cleaved caspase-3）水平显著增加 （P＜0.05），而5 μmol/L米诺环素组除Cleaved Caspase-3表达水平下降外其他凋亡相关蛋白水平未见显著变化（P＞ 0.05）。（2）敲低SIRT1表达后，shSIRT1组mTOR和LC3B表达水平较对照组明显下降；而经过米诺环素处理后，mTOR 的表达出现明显升高，而LC3B表达水平仅部分恢复。结论 5、10 μmol/L的米诺环素均能有效抑制人类胶质瘤U87 和LN229细胞的生长，其机制可能与AMPK/SIRT1通路诱导细胞自噬和p70s6k/Bcl-2通路诱导细胞凋亡有关。     

木犀草素对K562细胞增殖与凋亡的影响及其机制

目的　探讨木犀草素对K562细胞增殖、凋亡的影响及其作用机制。方法　取对数生长期K562细胞分别加入0、10、25、50、100 μmol/L木犀草素培养24 h、48 h、72 h,采用CCK-8法检测细胞增殖抑制率;K562细胞分别加入0、25、50 μmol/L木犀草素培养48 h,采用流式细胞术检测细胞凋亡情况;K562细胞分别加入0、10、50、100 μmol/L木犀草素培养48 h,采用Westem blot检测B细胞淋巴瘤2蛋白（Bcl-2）、Bcl-2相关X蛋白（Bax）、多聚ADP核糖聚合酶（PARP）、Cleaved-PARP、半胱氨酸天冬氨酸蛋白水解酶3（Caspase3）、Cleaved-Caspase3、蛋白激酶B（AKT）、磷酸化AKT（p-AKT）、断裂点簇集区蛋白（BCR）、c-abl癌基因1（c-ABL）BCR-ABL蛋白表达。结果　CCK-8检测结果显示,随木犀草素浓度增加及作用时间的延长,K562细胞增殖抑制率均呈增长趋势（P＜0.05）。流式细胞仪检测结果显示,木犀草素0、25、50 μmol/L组K562细胞的凋亡率依次升高（分别为8.21%±0.55%、23.43%±1.50%和40.47%±2.97%）。Western blot结果显示,Bax、Cleaved-PARP、Cleaved-Caspase3表达水平随木犀草素浓度的增加而升高（P＜0.05）。木犀草素0、10、50 μmol/L组PARP蛋白表达水平依次升高（P＜0.05）,100 μmol/L组与50 μmol/L组差异无统计学意义。100 μmol/L组Caspase3、Bcl-2蛋白表达水平均低于其余组（P＜0.05）。50、100 μmol/L组p-AKT蛋白表达水平低于0、10 μmol/L组,100 μmol/L组低于50 μmol/L组。50 μmol/L组BCR-ABL融合蛋白表达水平高于0 μmol/L组,100 μmol/L组低于0、50 μmol/L组（P＜0.05）。结论　木犀草素可抑制K562细胞增殖,促进细胞凋亡,其机制可能与调控BCR-ABL蛋白表达及PI3K/AKT信号通路有关。     

Role of sirtuin-1 (SIRT1) in hypoxic injury in pancreatic β-cells

Abstract

Islet transplantation (ITx) is being developed as a treatment for type 1 diabetes mellitus, but hypoxic damage to transplanted islet grafts is an important factor affecting successful transplantation. To investigate the role of sirtuin-1 (SIRT1) under hypoxic injury in INS-1 cells, one type of pancreatic β-cell lines, we used SRT1720 and GW4064 for SIRT1 activation. The small interfering RNA SIRT1 (si-SIRT1) was used to suppress SIRT1 gene expression. We measured cell viability, apoptosis, and the levels of inflammatory cytokines, including tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and reactive oxygen species (ROS), under hypoxic conditions. Real-time PCR and Western blot analysis were performed. Cell viability was significantly reduced to 71% and 40% after 4 and 6?h of hypoxic conditions, respectively. Apoptosis increased significantly 2.8-fold and 5.3-fold after 4 and 6?h of hypoxia, respectively. SIRT1 expression was significantly reduced at the mRNA and protein levels during hypoxia. Hypoxic damage significantly increased the TNF-α, IL-6 and ROS levels in INS-1 cells. However, the reduced cell viability and increased inflammatory cytokines from hypoxic damage were ameliorated by SIRT1 activation in INS-1 cells. These results suggest that SIRT1 is a potential target for the protection of pancreatic β-cells against hypoxic damage during ITx.