RISK信号通路在1-磷酸鞘氨醇后适应减轻心肌细胞缺氧/复氧损伤中的作用

目的研究再灌注损伤挽救激酶(reperfusion injury salvage kinase,RISK)信号通路在1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)后适应减轻H9c2心肌细胞缺氧/复氧损伤中的作用。方法将H9c2细胞随机分为7组,即(1)正常对照组(C);(2)缺氧/复氧组(H/R);(3)S1P组;(4)S1P+LY294002组(S1P+LY);(5)LY组;(6)S1P+PD98059组(S1P+PD);(7)PD组。采用MTT法测定各组细胞存活率;比色法测定丙二醛(MDA)含量、总超氧化物歧化酶(TSOD)和锰超氧化物歧化酶(Mn-SOD)活力;采用激光共聚焦显微镜技术检测细胞内游离钙离子浓度变化;流式细胞仪检测心肌细胞凋亡率;Western blot法测定Akt和ERK1/2蛋白的磷酸化水平。结果与H/R组比较,S1P组可明显增加H9c2细胞缺氧/复氧损伤后细胞存活率及凋亡率,增加TSOD及Mn-SOD活力,降低MDA含量,降低钙离子浓度,增加Akt和ERK1/2磷酸化水平,而PI3K/Akt信号通路阻断剂LY294002或ERK1/2阻断剂PD98059可阻断S1P对H9c2的上述作用。结论 S1P能够减轻H9c2细胞缺氧/复氧损伤,加入PI3K/Akt抑制剂LY294002和ERK1/2抑制剂PD98059均使S1P的保护作用被取消,表明S1P通过RISK信号通路发挥抗H9c2细胞缺氧/复氧损伤作用。     

基于网络药理学探究血尿胶囊治疗急性肾盂肾炎的作用机制

目的:采用网络药理学和实验验证的方法探究血尿胶囊治疗急性肾盂肾炎(APN)的作用机制.方法:通过APN大鼠模型研究血尿胶囊对APN的作用效果.使用中药系统药理学数据库与分析平台(TCMSP),化学专业数据库,中医证候关联(SymMap)数据库检索菝葜、薏苡仁、棕榈子的化学成分,PharmMapper与SwissTarg...     

Brassinin Induces Apoptosis in PC‐3 Human Prostate Cancer Cells through the Suppression of PI3K/Akt/mTOR/S6K1 Signaling Cascades

The oncogenic PI3K/Akt/mammalian target of rapamycin (mTOR) signaling axis and its downstream effector, the ribosomal protein S6 kinase 1 (S6K1) play a key role in mediating cell survival in various tumor cells. Here, we investigated the effects of brassinin (BSN), a phytoalexin first identified as a constituent of cabbage, on the PI3K/Akt/mTOR/S6K1 activation, cellular proliferation, and apoptosis in PC‐3 human prostate cancer. BSN exerted a significant dose‐dependent cytotoxicity and reduced constitutive phosphorylation of Akt against androgen‐independent PC‐3 cells as compared to androgen‐dependent LNCaP cells. Moreover, knockdown of androgen receptor (AR) by small interfering RNA enhanced the potential effect of BSN on induction of apoptosis in LNCaP cells. BSN clearly suppressed the constitutive activation of PI3K/Akt/mTOR/S6K1 signaling cascade, which correlated with the induction of apoptosis as characterized by accumulation of cells in subG1 phase, positive Annexin V binding, TUNEL staining, loss of mitochondrial membrane potential, down‐regulation of antiapoptotic and proliferative proteins, activation of caspase‐3, and cleavage of PARP. Additionally, BSN could block broad‐spectrum inhibition of PI3K/Akt/mTOR/S6K1 axes, and aberrant Akt activation by pcDNA3‐myr‐HA‐Akt1 plasmid could not prevent the observed suppressive effect of BSN on constitutive mTOR activation. Finally, overexpression of Bcl‐2 also attenuated BSN‐mediated apoptosis in PC‐3 cells. Taken together, our findings suggest that BSN can interfere with multiple signaling cascades involved in tumorigenesis and might be provided as a potential therapeutic candidate for both the prevention and treatment of prostate cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

LncRNA MEG3靶向miR-181a-5p调控宫颈癌细胞放射敏感性

目的 研究LncRNA MEG3对宫颈癌细胞放射敏感性的影响,并探讨其作用机制。方法 运用qRT-PCR法检测放射抗性和放射敏感性宫颈癌细胞中LncRNA MEG3的表达;将过表达对照组(转染pcDNA 3.1)、过表达LncRNA MEG3组(转染pcDNA 3.1-LncRNA MEG3)、抑制miR-NC组(转染anti-miR-NC)、抑制miR-181a-5p组(转染anti-miR-181a-5p)、过表达LncRNA MEG3+过表达miR-NC组(共转染pcDNA 3.1-LncRNA MEG3和anti-miR-NC)、过表达LncRNA MEG3+过表达miR-181a-5p组(共转染pcDNA 3.1-LncRNA MEG3和anti-miR-181a-5p),均用脂质体法转染至SiHa细胞;克隆形成实验检测细胞的存活分数;流式细胞术检测细胞的凋亡率;双荧光素酶报告基因检测实验检测细胞的荧光活性;Western blot检测细胞中PTEN、p-Akt、Akt的蛋白表达。结果 与放射敏感组相比,放射抗性宫颈癌组织中LncRNA MEG3的表达明显降低(P<0.05),其表达量与宫颈癌细胞的放射敏感性呈正相关;过表达LncRNA MEG3、抑制miR-181a-5p均可显著增强宫颈癌细胞SiHa放射敏感性,促进凋亡(P<0.05);野生型LncRNA MEG3细胞的荧光活性受miR-181a-5p的抑制。过表达miR-181a-5p逆转了LncRNA MEG3对宫颈癌细胞放射增敏和促凋亡作用及对PTEN/Akt信号通路的调控。结论 长链非编码RNA LncRNA MEG3可增强宫颈癌细胞放射敏感性,其机制可能与靶向miR-181a-5p调控PTEN/Akt 信号通路有关,可为提高宫颈癌的预后提供新方向。     

miR-145调控肝癌腹水模型Th9细胞升高的机制研究

目的:探讨miR-145调控肝癌腹水模型Th9细胞升高的作用机制。方法:构建小鼠H22肝癌腹水模型。造模两周后处死小鼠并分离出脾脏组织,流式细胞术分析肝癌腹水组（MA组）与正常对照组（Control 组）小鼠脾脏Th9细胞表达水平。ELISA法检测脾脏IL-9表达水平。RT-PCR法检测脾脏miR-145表达水平。Western Blot法检测脾脏中PI3K/Akt/mTOR/P70S6K/HIF-1α相关蛋白表达水平。分选小鼠脾脏CD4+T细胞,随机分为miR-145 mimics组和NC组,分别应用miR-145-5P mimics、阴性对照寡核苷酸（negative control,NC）进行转染,RT-PCR检测各组miR-145、HIF-1α mRNA及IL-9 mRNA表达水平。结果:与Control 组相比,MA组Th9细胞及其细胞因子IL-9表达均升高（P<0.05）,miR-145表达降低（P<0.05）,p-PI3K、p-Akt、mTOR、p-mTOR、p-P70S6K、HIF-1α蛋白均升高（P<0.05）。与NC组相比,miR-145 mimics组miR-145显著升高,HIF-1α mRNA及IL-9 mRNA表达下降。结论:肝癌腹水中Th9细胞升高可能与miR-145下降导致的PI3K/Akt/mTOR/P70S6K/HIF-1α通路激活有关。     

基于SNP芯片研究PI3K/Akt/mTOR通路上自噬相关基因SNPs与胃癌的关联

目的： 探讨与胃癌发生相关的PI3K/Akt/mTOR通路上新的与自噬相关基因单核苷酸多态性位点（SNPs）,为寻找有价值的胃癌发生相关的分子标志物提供新的依据。方法： 采用1：1配对病例-对照研究的方法。通过KEGG pathway网站和Gene Ontology、Ensemble数据库及HaploView、STRING、Cytoscape软件联合SNP芯片筛选目标SNPs,采用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）对筛检出来的SNPs位点在来自福建省仙游县的622例胃癌患者和622例健康人群基因组中进行验证。结果： SNP芯片及生物信息学分析筛选出IRS1 rs10205233、PIK3CD rs3934934、PIK3R1 rs706711、PIK3R1 rs706714和AKT1 rs35285446为候选位点。扩大样本验证发现IRS1 rs10205233的多态性变异（C > T）显著降低了胃癌的发病风险[共显性模型、显性模型的OR（95% CI）分别为0.761（0.595,0.975）、0.764（0.601,0.973）]。进一步分层分析,该位点显性模型、隐形模型、共显性模型以及等位基因在贲门癌和非贲门癌人群中均未见统计学差异（P > 0.05）。并未见其他位点与患胃癌风险的关联有统计学意义。对PIK3R1基因2个位点（rs706711、rs706714）的单体型分析也未见统计学差异。结论： IRS1 rs10205233位点与福建省胃癌高发区仙游县的胃癌发生存在关联,T等位基因可能是胃癌发生的一个遗传保护因素。     

A novel function of bamboo extract in relieving lipotoxicity

Lipotoxicity is closely related to the etiology and complications of type 2 diabetes mellitus. This study investigated the protective effect of an extract from bamboo Phyllostachys edulis against palmitic acid (PA)-induced lipoapoptosis. The lipo-detoxification function of the bamboo extract (BEX) was evaluated using cell culture models. Cell viability was measured by MTT assay and cell apoptosis was monitored by Annexin V staining. Cellular uptake of fluorescent free fatty acid (FFA) analog was measured by flow cytometry. Protein levels of total protein kinase B (Akt) and phosphorylated Akt (p-Akt) were measured by western blotting. The results show that co-incubating BEX with mouse myoblast C2C12 cells had no effect on the cellular uptake of FFA, but dramatically decreased PA-induced cell apoptosis and protected cell viability. A similar antilipotoxicity effect of BEX was observed in other mammalian cells. BEX significantly decreased the protein levels of both Akt and p-Akt in C2C12 cells under normal cell culture conditions but not under lipotoxic conditions, indicating the regulatory effect of BEX on cell signaling pathways and its response to a high FFA environment. This study demonstrated a novel function of bamboo extract in preventing lipotoxicity in mammalian cells, implicating a promising phytotherapeutic approach for lipo-detoxification.     

哮喘大鼠肺组织Akt蛋白表达上调对IL-4,IL-12和IL-13的影响

目的：研究Akt蛋白对支气管哮喘大鼠IL-4,IL-12和IL-13表达的影响。方法：雄性SD大鼠24只,随机分为3组：对照组（C组）、哮喘组（A组）、渥曼宁青霉素组（Wortmannin,W组）,每组8只。以卵白蛋白（OVA）致敏和激发建立大鼠哮喘模型。各组大鼠于末次激发后24h,放血处死。酶联免疫吸附试验（ELISA）测定BALF中IL-4,IL-13和IL-12的浓度;免疫组化法测定肺组织中p-Akt蛋白的表达;Western blot测定肺组织匀浆中p-Akt蛋白的含量。结果：A组p-Akt蛋白的表达明显高于C组,W组p-Akt蛋白的表达低于A组,但仍高于C组;BALF中IL-4,IL-13的含量A组明显高于C组,W组IL-4,IL-13的含量低于A组,但仍高于C组,BALF中IL-12的含量A组明显低于C组,W组IL-12的含量低于C组,但仍高于A组。结论：哮喘大鼠模型中出现了Akt蛋白的激活,细胞因子失衡可能与Akt蛋白的激活有关。     

三七总皂苷抑制心肌细胞凋亡的作用机制研究

目的 探讨三七总皂苷(Panax notoginseng saponin,PNS)对无糖无血清/缺氧诱导的心肌细胞凋亡的抑制作用.方法 将心肌H9e2细胞株分为正常对照组(control)、模型组(model)和PNS不同浓度(0.05、0.25、2.25 g/L)组.对照组采用含10%胎牛血清(FBS)的DMEM/F12培养基于普通二氧化碳培养箱培养,模型组采用无糖无血清培养基培养,同时进行缺氧处理诱导细胞凋亡,给药组按照模型组方法处理的同时给予不同浓度的PNS.细胞处理结束后收集细胞,Annexin V/PI染色后流式细胞仪检测不同浓度PNS干预后各组细胞的凋亡率.DCFH-DA荧光探针检测细胞内活性氧的水平.Western blot检测Akt和磷酸化Akt(P-Akt)的表达水平.结果 PNS能够抑制缺血环境诱导的心肌细胞凋亡,且呈剂量依赖性.对照组、模型组和PNS处理组细胞凋亡率分别为(8.01±1.44)%、(65.71±3.36)%(vs control P<0.001)、(65.00±1.24)%、(52.51±1.76)%(vs model P<0.01)、(23.99±0.76)%(vs model P<0.001).DCFH-DA染色结果显示,模型组细胞能够观察到明显的绿色荧光信号,而PNS组荧光信号与模型组比较明显减弱,说明PNS具有清除ROS的作用.Western blot结果显示与对照组比较,模型组p-Akt蛋白表达水平明显降低;与模型组比较,PNS组p-Akt蛋白表达水平显著升高,而PI3K特异性抑制剂LY294002明显抑制PNS促进p-Akt表达的作用.结论 PNS通过促进p-Akt的活性从而抑制缺血诱导的心肌细胞凋亡.     

Akt和8－OHdG在铁过载肝细胞中的表达

目的：铁过载可以促进肝细胞增生甚至肝的纤维化,但其中的确切机制尚不清楚。本文通过喂食大鼠羰基铁致铁过载,后行大部分肝切除建立动物模型,检测不同时间点的目标蛋白来研究铁过载对肝细胞增殖影响的机制。方法：通过免疫组织化学方法分别检测了Akt和8－OHdG水平。结果：正常组0小时肝实质细胞的核中8－OHdG的表达。24小时8－OHdG的表达显著增加并且达到顶峰。实验组同样在细胞核中有表达,12小时8－OHdG的表达显著增加并且达到顶峰,在这之后这种表达逐渐减弱（图1F）。正常组肝实质细胞细胞质中Akt有表达,在36小时Akt表达最强。在实验组0小时同样检测出 Akt表达,到24小时这种表达最为显著。结论：铁过载的肝细胞通过 PI3K／AKt信号通路引起了肝细胞的再生。