自噬下降参与了β1肾上腺素受体自身抗体诱导的H9c2心肌细胞凋亡

目的 探讨自噬下降对β₁肾上腺素受体自身抗体(β₁adrenergic receptor autoantibodies, β₁-AA)诱导的H9c2心肌细胞凋亡的作用。方法 使用1 μM的β₁-AA对H9c2心肌细胞进行处理,Real time-PCR检测自噬相关基因LC3 mRNA表达的改变,Western blot检测LC3、Beclin 1以及P62蛋白表达水平的变化;使用流式细胞术、Caspase-3活性检测以及Hoechst 33258染色反映细胞凋亡情况;使用经典的自噬抑制剂3-甲基腺嘌呤(3-Methyladenine, 3-MA)预处理30 min以及mTOR通路抑制剂雷帕霉素(rapamyein,RAPA)预处理1 h后,再给予细胞β₁-AA处理,检测此时细胞凋亡的变化情况。结果 1 μM的β₁-AA干预H9c2心肌细胞6 h可以明显降低细胞存活率;β₁-AA干预细胞6 h、12 h、24 h,LC3 mRNA水平依次降低,LC3II和Beclin 1蛋白水平依次降低,P62蛋白水平逐渐增加,表明β₁-AA可以引起心肌细胞自噬水平逐渐下降;同时发现,β₁-AA干预细胞6 h、12 h,细胞凋亡水平明显升高;使用经典自噬抑制剂3-MA预处理心肌细胞30 min抑制自噬后,H9c2心肌细胞凋亡水平较β₁-AA单独处理组显著增加,相反使用经典mTOR通路抑制剂RAPA预处理心肌细胞1 h上调自噬后,心肌细胞凋亡水平较β₁-AA单独处理组明显下降,说明自噬水平改变可以影响心肌细胞凋亡水平。 结论 自噬下调能够增加β₁-AA诱导的H9c2心肌细胞凋亡。     

哈巴苷对H2O2诱导的大鼠心肌细胞H9c2氧化应激损伤的保护作用

目的探究哈巴苷(HG)对大鼠心肌细胞氧化应激损伤的作用。方法将H9c2细胞随机分为对照组(H9c2组)、哈巴苷组、H2O2组和H2O2+哈巴苷组,用H2O2处理细胞,复制氧化损伤模型。用哈巴苷(4μmoL)处理细胞,CCK8检测细胞增殖,Hoechst染色检测细胞凋亡,二氯荧光乙酰乙酸盐(DCF)法检测细胞内活性氧(ROS),根据试剂盒说明书检测上清液中超氧化物歧化酶(SOD)、丙二醛(MDA)和谷胱甘肽(GSH)浓度,Westernblot检测细胞凋亡相关蛋白Bcl-2、Bax、Caspase-3和Caspase-9的表达。结果与H9c2组比较,H2O2组心肌细胞增殖倍数明显降低,哈巴苷作用细胞4d后,H2O2+哈巴苷组心肌细胞增殖倍数明显高于H2O2组;同时,与H9c2组比较,H2O2组细胞凋亡率明显升高;与H2O2组比较,H2O2+哈巴苷组细胞凋亡率显著降低;H2O2还能显著诱导H9c2细胞Bax、Caspase-3和Caspase-9的表达,抑制Bcl-2表达;HG能显著减弱H2O2诱导Caspase-3和Caspase-9表达和抑制Bcl-2表达的作用;此外,H2O2组细胞内ROS活性及上清液中SOD和GSH浓度明显低于H9c2组,MDA浓度明显高于H9c2组,H2O2+哈巴苷组细胞内ROS活性及SOD和GSH浓度明显高于H2O2组,MDA浓度明显低于H2O2组。结论哈巴苷能通过抑制氧化应激抑制心肌细胞H9c2凋亡。     

紫花牡荆素抑制氧化应激诱导的HUVEC细胞凋亡

目的　研究紫花牡荆素（CAS）对H₂O₂　氧化应激诱导的HUVEC细胞凋亡的影响及其可能机制。方法　体外培养HUVEC细胞,在添加H₂O₂　氧化应激诱导之前先加入不同浓度的CAS　（5、10和20　μmoL/L）　预孵30　min,用MTT法观察各组细胞生长活性;用AO/EB染色和FCM法检测HUVEC细胞凋亡情况及其凋亡率;用Western　blot检测细胞凋亡相关蛋白表达活性。结果　MTT提示与H₂O₂组相比较,CAS呈浓度和时间依赖性增加H₂O₂氧化应激诱导的HUVEC细胞的生长活性（P<0.05）,降低HUVEC细胞的凋亡数量及凋亡率（P<0.05）;同时下调Cytochrome　C、Caspase-9和Caspase-3蛋白表达降低（P<0.05）,激活Bcl-2蛋白表达（P<0.05）,而Bax蛋白表达不变（P>0.05）,Bcl-2/Bax上升（P<0.05）。结论　CAS拮抗H₂O₂氧化应激诱导的HUVEC细胞凋亡可能与其调节内源性线粒体凋亡途径有关。     

柚皮素对高糖诱导的H9c2心肌细胞凋亡及Nrf2/ARE信号通路的影响

目的]探讨柚皮素(NAR)对高糖(HG)诱导的大鼠心肌细胞(H9c2)凋亡及核因子E2相关因子2(Nrf2)/抗氧化反应元件(ARE)信号通路的影响。 [方法]将培养的H9c2细胞分为对照组(正常糖量)、HG组(35.5 mmol/L葡萄糖)、HG＋NAR低、中、高浓度组(35.5 mmol/L葡萄糖＋6.25、12.5、25.0 μmol/L NAR)。用噻唑蓝法检测H9c2细胞增殖活性;流式细胞术检测细胞凋亡;二氯二氢荧光素二乙酸酯荧光探针染色法检测细胞内活性氧(ROS)水平;Western blot法检测Nrf2、血红素加氧酶1(HO-1)、Bax、Caspase-3、Bcl-2蛋白表达水平。 [结果]经HG处理的H9c2细胞增殖活性、Nrf2、HO-1、Bcl-2蛋白表达水平显著降低,细胞凋亡率、ROS水平、Bax、Caspase-3蛋白表达水平显著升高(P＜0.05)。经HG与NAR共同处理H9c2细胞后,NAR低、中、高浓度组H9c2细胞增殖活性、Nrf2、HO-1、Bcl-2蛋白表达水平显著升高,细胞凋亡率、ROS水平、Bax、Caspase-3蛋白表达水平显著降低(P＜0.05)。 [结论]NAR可抑制HG诱导的H9c2细胞凋亡,其作用机制可能与激活Nrf2/ARE信号通路密切相关。     

miR-152-3p调控硫氧还蛋白结合蛋白表达对过氧化氢诱导的内皮祖细胞凋亡的影响

目的]探讨miR-152-3p靶向硫氧还蛋白结合蛋白(TXNIP)对过氧化氢(H₂O₂)诱导的内皮祖细胞(EPC)凋亡的影响。 [方法]从健康人外周血分离与鉴定EPC,建立H₂O₂诱导的EPC损伤模型(500 μmol/L H₂O₂处理8 h),RT-qPCR检测EPC中miR-152-3p表达;EPC中过表达miR-152-3p或抑制TXNIP表达或同时过表达miR-152-3p和TXNIP,再使用500 μmol/L H₂O₂处理8 h,分别检测miR-152-3p表达水平、细胞凋亡率及TXNIP、Bax、Bcl-2、Caspase-3蛋白表达水平;双荧光素酶报告基因实验验证miR-152-3p与TXNIP的关系。 [结果]从健康人外周血成功分离EPC,miR-152-3p在H₂O₂诱导的EPC中表达减少65.0%(P＜0.001);与对照组相比,模型组EPC凋亡率及Bax、Caspase-3蛋白表达水平增加895.1%、352.0%、290.3%,Bcl-2蛋白表达水平降低79.4%(均P＜0.001);与miR-NC组相比,miR-152-3p mimic组EPC凋亡率及Bax、Caspase-3蛋白表达水平降低55.7%、60.9%、56.8%(P＜0.001),Bcl-2蛋白表达水平增加389.5%(均P＜0.001);与si-NC组相比,si-TXNIP组EPC凋亡率及TXNIP、Bax、Caspase-3蛋白表达水平降低40.2%、57.5%、59.8%、55.4%(均P＜0.001),Bcl-2蛋白表达水平增加313.0%(P＜0.001);与miR-152-3p mimic＋pcDNA组相比,miR-152-3p mimic＋TXNIP组EPC凋亡率及TXNIP、Bax、Caspase-3蛋白表达水平增加86.8%、184.8%、137.7%、109.2%(P＜0.001),Bcl-2蛋白表达水平降低69.1%(P＜0.001);双荧光素酶报告基因实验结果显示,miR-152-3p可靶向负调控TXNIP表达。 [结论]miR-152-3p在H₂O₂诱导的EPC中低表达,过表达miR-152-3p可通过靶向抑制TXNIP表达抑制H₂O₂诱导的EPC凋亡。     

血管紧张素(1-7)抑制异丙肾上腺素诱导的H9c2心肌细胞凋亡

目的观察血管紧张素(1-7)[Ang(1-7)]对心肌细胞凋亡的影响,探讨其可能的作用机制。方法应用异丙肾上腺素(ISO)处理H9c2心肌细胞12 h建立心肌细胞凋亡模型,Ang(1-7)或PI3K抑制剂LY294002与H9c2心肌细胞共处理12 h观察对ISO诱导的心肌细胞凋亡的影响。显微镜下观察H9c2心肌细胞生长情况,采用MTS法检测各组细胞的相对细胞活性,TUNEL法检测各组细胞凋亡率。JC-1荧光探针检测线粒体膜电位,Western blot检测cleaved Caspase-3、p-Akt及Akt蛋白的表达量。结果 ISO呈浓度依赖性抑制H9c2心肌细胞的相对细胞活性,Ang(1-7)呈浓度依赖性逆转ISO诱导的H9c2心肌细胞相对活性的降低;与对照组比较,ISO组细胞凋亡率及cleaved Caspase-3蛋白表达量显著增加,线粒体膜电位和p-Akt蛋白表达量显著降低;Ang(1-7)可抑制ISO诱导的H9c2心肌细胞凋亡率的增加,减少cleaved Caspase-3蛋白的表达,增加线粒体膜电位和p-Akt蛋白表达量。LY294002预处理后Ang(1-7)对ISO诱导的H9c2心肌细胞的保护作用明显减弱,表现为心肌细胞凋亡率及cleaved Caspase-3蛋白的表达量明显增加,p-Akt蛋白表达量减少。结论 ISO能诱导H9c2心肌细胞线粒体途径的细胞凋亡,而Ang(1-7)能抑制ISO诱导的线粒体途径的细胞凋亡。PI3K/Akt信号通路可能在Ang(1-7)抑制ISO诱导的H9c2心肌细胞凋亡中起到关键作用。     

人参皂苷Rb1对H9c2细胞缺氧复氧的作用

目的观察人参皂苷Rb1对H9c2细胞缺氧复氧的作用。方法采用人参皂苷Rb1对H9c2细胞缺氧复氧进行预处理,通过检测氧化应激、凋亡指标观察人参皂苷Rb1的作用。将细胞随机分为对照组、缺氧复氧组、缺氧复氧加人参皂苷Rb1(50、100、200μmol/L),按实验设计因素处理后,通过蛋白电泳检测凋亡相关蛋白caspase-3,检测氧化应激指标丙二醛(MDA)、超氧化物歧化酶(SOD)、活性氧(ROS),TUNEL法检测细胞凋亡。结果缺氧复氧处理可使H9c2心肌细胞的ROS和MDA水平显著增加,SOD活性显著下降,上调caspase-3的表达,增加H9c2心肌细胞的凋亡。人参皂苷Rb1预处理可减少缺氧复氧H9c2细胞MDA表达量,增加SOD活性,降低ROS水平,且人参皂苷Rb1预处理可减少缺氧复氧H9c2细胞caspase-3的表达量及凋亡细胞数量。结论人参皂苷Rb1可降低缺氧复氧对H9c2心肌细胞的氧化应激损伤及凋亡,从而对缺氧复氧H9c2心肌细胞起保护作用。     

重组人生长停滞特异性蛋白6对缺氧/复氧原代心肌细胞及H9c2细胞损伤的影响

目的研究外源重组人生长停滞特异性蛋白6(rhGas6)对缺氧/复氧(H/R)诱导的原代乳鼠心肌细胞及H9c2细胞损伤的影响。方法体外培养新生Wistar大鼠心肌细胞及H9c2细胞,随机各分为3组:正常组、H/R组及rhGas6预处理组(rhGas6组)。观察各组细胞形态及心肌细胞搏动频率,检测细胞存活率、乳酸脱氢酶(LDH)浓度及半胱氨酸蛋白酶(Caspase-3)活性变化,流式细胞仪检测细胞凋亡率。结果原代心肌细胞及H9c2细胞H/R组均较正常组细胞活力下降,凋亡率、LDH及Caspase-3水平均较正常组增高。而Gas6组与H/R组比较,细胞活力增加,凋亡率、LDH及Caspase-3水平均减低(P<0.05)。结论 rhGas6对H/R诱导的原代心肌细胞及H9c2细胞损伤均发挥着潜在的保护作用,并可能是通过抑制Caspase-3活性减少细胞凋亡发挥的。     

调控自噬对H9c2心肌细胞缺氧／复氧损伤的影响

目的探讨调控自噬水平对H9c2心肌细胞缺氧／复氧（H／R）损伤的影响及意义。方法将H9c2心肌细胞缺氧2h／复氧4h,建立H／R损伤模型。以3-甲基腺嘌呤（3-MA）为自噬特异抑制剂和雷帕霉素为自噬增强剂,试验随机分为四组：正常对照组（C组）、H／R组、H／R＋100mol／L3-MA组（M＋H／R组）、H／R＋100nmol／L雷帕霉素（R＋H／R组）,应用MTT法检测细胞活力,透射电镜检测心肌细胞自噬小体,流式细胞技术检测细胞凋亡比例,Westernblot法检测自噬相关蛋白LC3、Beclin1,凋亡相关蛋白Bcl-2、Bax及下游活性片段Caspase-9、Caspase-3蛋白表达。结果H／R组明显诱导H9c2心肌细胞自噬发生、细胞活力下降、凋亡增加（P〈O．01）．Westernblot检测发现,Bax、Caspase-9、Caspase-3活性片段蛋白表达明显增加,Bcl-2表达明显抑制,Bax／Bcl-2比值、活化蛋白Caspase-3、Caspase-9表达增加（P〈O．01）;M＋H／R组H／R损伤作用明显减弱,线粒体凋亡通路及下游蛋白表达抑制（P〈O．01）;而R＋H／R组线粒体凋亡通路进一步激活,促进细胞凋亡发生（P〈O．01）。结论自噬在H9c2心肌细胞H／R损伤中起到致命性作用,抑制自噬可保护心肌细胞H,R氧化应激损伤,其机制与抑制线粒体凋亡通路有关。     

H2S拮抗HUVEC衰老与促进SIRT1巯基化和减少FOXO1乙酰化有关

目的]探讨沉默信息调节因子1(SIRT1)/叉头转录因子O1(FOXO1)在H₂S拮抗H₂O₂诱导内皮细胞衰老过程中的作用。 [方法]建立内皮细胞衰老模型,通过衰老相关β-半乳糖苷酶(SA-β-gal)染色在光学显微镜下观察到的蓝染细胞数(即衰老细胞)计算阳性细胞率。采用Western blot检测细胞P21、P53、纤溶酶原激活物抑制剂1(PAI-1)、叉头转录因子O1(FOXO1)、乙酰化FOXO1(ac-FOXO1)、锰超氧化物歧化酶(MnSOD)及过氧化氢酶的蛋白表达水平,采用生物素转换法测定S-巯基化SIRT1的表达,采用活性氧(ROS)检测定量评估细胞内ROS水平。 [结果]经100 μmol/L H₂O₂处理可显著提高SA-β-gal染色阳性细胞率和P21、P53、PAI-1的蛋白表达,提示衰老细胞模型成功建立,而100 μmol/L NaHS可明显拮抗这一作用,SA-β-gal染色阳性细胞数明显下降(P＜0.01),P21、P53、PAI-1的蛋白表达显著降低(P＜0.01)。与对照组相比,H₂O₂组SIRT1、FOXO1、ac-FOXO1、MnSOD及过氧化氢酶的蛋白表达显著降低(P＜0.05或P＜0.01),ac-FOXO1/FOXO1比值显著增加(P＜0.01),ROS水平明显升高(P＜0.01)。与H₂O₂组相比,NaHS＋H₂O₂组SIRT1、S-巯基化SIRT1、FOXO1、ac-FOXO1、MnSOD及过氧化氢酶的蛋白表达显著升高((P＜0.05或P＜0.01),ac-FOXO1/FOXO1比值显著下降(P＜0.01),ROS水平明显降低(P＜0.05)。 [结论]H₂S可拮抗H₂O₂诱导的HUVEC衰老,其机制与促进SIRT1巯基化和减少FOXO1乙酰化有关。     

H2-Antagonists and carmustine

Summary The highly metabolized nitrosourea carmustine (BCNU) is an anticancer agent which alkylates DNA and is metabolized to both active and inactive species by cytochrome P-450 enzymes. Other highly metabolized anticancer drugs have altered toxicities when some histamine H₂ antagonists are coadministered. To test this hypothesis with BCNU, DBA/2J male mice were given a single injection of cimetidine (CMT 100 mg/kg) or ranitidine (RNT 25 mg) at various times up to 30 min before, or up to 60 min after a BCNU injection. Spleen colony assays for normal bone marrow stem cell viability showed enhanced toxicity for BCNU when CMT was administered concomitantly. In P-388 leukemia-bearing DBA/2J mice, both CMT and RNT significantly enhanced the antitumor effects of BCNU doses of 30 mg/kg. Pharmacokinetic analyses of BCNU elimination in Cd-1 mice showed marked prolongation of BCNU elimination and increased (BCNU concentration) x time products when CMT was concomitantly administered. These results demonstrate that enhanced BCNU bone marrow toxicity and antitumor activity is produced by CMT. The effect appears to be related to impaired drug clearance when the two agents are administered concurrently. RNT slightly enhanced BCNU antileukemic effects, but it did not significantly alter BCNU myelotoxicity nor drug elimination patterns.     

Histamine H2-receptor antagonists

The first histamine H2-receptor antagonists were developed in the early 1970s, and they have a dominant role in today's management of peptic ulceration. The original regimens using either cimetidine or ranitidine attempted to control acidity across the 24 hours, but more 'modern' regimens use a large single dose of the H2-blocker in the evening, which produces a pulse of decreased intragastric acidity during the night with a normal acidity in the daytime. High-dose regimens using a new generation of extremely potent histamine H2-receptor antagonists may improve ulcer healing rates at 4 weeks, and may be particularly useful for the management of either severe oesophagitis or intractable duodenal ulceration.     

H2-Kb and H2-Db regulate cerebellar long-term depression and limit motor learning

There are more than 50 class I MHC (MHCI) molecules in the mouse genome, some of which are now known to be expressed in neurons; however, the role of classical MHCI molecules in synaptic plasticity is unknown. We report that the classical MHCI molecules, H2-K^b and H2-D^b, are co-expressed by Purkinje cells (PCs). In the cerebellum of mice deficient for both H2-K^b and H2-D^b (K^bD^b−/−), there is a lower threshold for induction of long-term depression (LTD) at parallel fiber to PC synapses. This change may be a result of additional glutamate release observed at K^bD^b−/− CF to PC synapses, which are thought to “train” the cerebellar circuit. A behavioral correlate of cerebellar LTD is motor learning; acquisition and retention of a Rotarod behavioral task is significantly better in K^bD^b−/− mice than in WT cohorts. These physiological and behavioral phenotypes in K^bD^b−/− mice reveal a surprising role for classical MHCI molecules in synaptic plasticity and motor learning.     

The effect of histamine, H1 and H2 agonists, and H1 and H2 antagonists on postprandial pancreatic polypeptide release in dogs

The influence of histamine, H1 and H2 agonists (2-AETD and dimaprit), and H1 and H2 antagonists (mepyramine and cimetidine) on the postprandial release of pancreatic polypeptide (PP) was assessed in five Labrador retrievers. Infusions of histamine, 0.25 mumol kg-1 h-1; 2AETD, 4 mumol kg-1 h-1; and dimaprit, 1 mumol kg-1 h-1, enhanced postprandial release (131-189% of control values). The response to 2-AETD and dimaprit was significantly different from control values (p less than 0.05). An increase was observed even when 50 mg mepyramine and 400 mg cimetidine were injected as intravenous boluses (131-139% of control values (p less than 0.05]. The action of the antagonists may be explained by partial agonism or histamine release induced by these agents. It is concluded that postprandial PP release is stimulated by H1 and H2 receptors.     

The tail domain of lamin Dm0 binds histones H2A and H2B

In multicellular organisms, the higher order organization of chromatin during interphase and the reassembly of the nuclear envelope during mitosis are thought to involve an interaction between the nuclear lamina and chromatin. The nuclear distribution of lamins and of peripheral chromatin is highly correlated in vivo, and lamins bind specifically to chromatin in vitro. Deletion mutants of Drosophila lamin Dm0 were expressed to map regions of the protein that are required for its binding to chromosomes. The binding activity requires two regions in the lamin Dm0 tail domain. The apparent Kd of binding of the lamin Dm0 tail domain was found to be approximately 1 microM. Chromatin subfractions were examined to search for possible target molecules for the binding of lamin Dm0. Isolated polynucleosomes, nucleosomes, histone octamer, histone H2A/H2B dimer, and histones H2A or H2B displaced the binding of lamin Dm0 tail to chromosomes. This displacement was specific, because polyamines or proteins such as histones H1, H3, or H4 did not displace the binding of the lamin Dm0 tail to chromosomes. In addition, DNA sequences, including M/SARs, did not interfere with the binding of lamin Dm0 tail domain to chromosomes. Taken together, these results suggest that the interaction between the tail domain of lamin Dm0 and histones H2A and H2B may mediate the attachment of the nuclear lamina to chromosomes in vivo.     

Histone H2A subtypes associate interchangeably in vivo with histone H2B subtypes.

The yeast Saccharomyces cerevisiae contains two primary sequence subtypes of histone H2B (H2B1 and H2B2) and of H2A (H2A1 and H2A2). Mutants in each of the H2B subtypes have been used to show previously that yeast cells lacking one or the other, but not both, of the H2B proteins are viable. Because H2A protein interacts in the nucleosome with H2B, we wished to determine whether specific H2A subtypes must interact with specific H2B subtypes. We describe experiments in which frameshift mutations were introduced into both of the H2A genes in vitro and the mutant genes integrated into the yeast genome, replacing the wild-type H2A genes by a subsequent recombination. Using these mutant (hta1- and hta2-) strains we find that neither H2A gene has a unique essential function during any phase of the yeast life cycle, although strains homozygous for hta1- grow more slowly. However, one functional H2A gene is required for viability because cells mutant in both H2A genes arrest at spore germination prior to bud separation. By combining these H2A mutations with the H2B mutations obtained previously, we show that all combinations of H2A and H2B subtypes produce viable cells. From these genetic experiments and electrophoretic analysis of the histone proteins of these mutants we conclude that the H2A subtypes can associate interchangeably with the H2B subtypes.