过氧化氢致H9c2大鼠心肌细胞DNA损伤作用

目的 研究不同浓度过氧化氢(H₂O₂)对H9 c2大鼠心肌细胞DNA损伤及诱导细胞凋亡的作用。方法 H9 c2心肌细胞处理组分别暴露于50,100,200,400μmol/L H₂O₂中1,3,6,12,24 h后,采用四甲基偶氮噻唑蓝(MTT)法检测细胞存活率;采用丫啶橙/溴乙锭(AO/EB)双染技术观察细胞凋亡;利用单细胞凝胶电泳技术(SCGE)观察细胞DNA损伤。结果 H₂O₂可以引起H9 c2心肌细胞损伤,随时间呈剂量效应关系(P<0.05),其中24 h,400μmol/L剂量组细胞存活率最低达40.6%。H₂O₂可诱导H9 c2心肌细胞出现凋亡并观察到明显的凋亡形态学的改变,与阴性对照组比较,差异有统计学意义(P<0.05),其中6 h时,100μmol/L剂量组细胞凋亡最明显,凋亡率达22.2%。H₂O₂可引起H9 c2心肌细胞DNA损伤,且损伤随剂量增加而增大(P<0.01),以400μmol/L剂量组最为明显,细胞DNA损伤率可达64.0%。结论 H₂O₂造成H9 c2心肌细胞氧化损伤和DNA损伤,随着其暴露剂量和时间的不同表现为凋亡和坏死。     

视黄酸诱导H9c2细胞p38 MAPK转位的研究

目的了解H9c2心肌细胞中p38MAPK的分布及其在全反式视黄酸(atRA)诱导下的转位。方法对H9c2细胞进行饥饿加药,应用激光扫描共聚焦显微镜对细胞内的p38进行定位扫描。通过对p38荧光强度的核浆比进行分析,得出atRA与SB202190对H9c2细胞内p38核转位的影响。结果在未受刺激细胞内,p38较多分布在胞浆;经atRA分别刺激细胞5min,30min后,胞浆中p38的荧光强度均变弱,核浆比显示实验组与对照组间有显著性差异。SB202190能明显抑制atRA诱导的p38核转位(P<0.01),并且,由SB202190抑制组与对照组的p38核浆比能看出,SB202190降低了26.1%的p38核转位。结论atRA能诱导心肌细胞中p38的核转位,p38信号转导通路可能参与心脏的发育调控。     

全反视黄酸调节心脏特异转录因子蛋白表达及其机制

目的探讨全反视黄酸(atRA)对心脏特异转录因子(dHAND)蛋白表达的调节作用和分子机制。方法用atRA处理H9c2细胞,然后用Western blot分析和免疫组化方法来检测dHAND表达。结果在H9c2细胞中,atRA可抑制dHAND蛋白表达,这一抑制作用具有时间和剂量依赖性。Western blot分析和免疫组化检测均证实,5μnol／L 的atRA处理H9c2细胞2h可以显著降低dHAND和ET-1蛋白的表达。用BQ-123阻断ET -1／ETAR信号通路后,dHAND表达上调。结论在H9c2细胞中,atRA通过ET-1／ ETAR信号传导通路来抑制dHAND表达。     

镉致大鼠心肌细胞H9c2凋亡作用

目的 研究不同浓度氯化镉(CdCl2)引起大鼠心肌细胞H9c2凋亡作用。方法 采用姬姆萨染色法观察氯化镉对细胞毒作用的形态学改变;采用Annexin V-FITC/propidium iodide(PI)流式细胞技术(flowcytometric,FCM)检测氯化镉对H9c2细胞凋亡的影响;采用流式细胞术法检测线粒体膜电位(MMP)的变化;采用免疫细胞化学法检测细胞色素C表达。结果 5,10,30,50,80μmol/L的氯化镉分别作用H9c2细胞6,12,24 h可以诱导细胞凋亡;随着镉剂量的加大及作用时间的延长,线粒体膜电位明显下降,与阴性对照比较,差异有统计学意义(P<0.05);随着镉剂量增加,CytC表达增强。结论 镉可以通过线粒体途径诱导H9c2细胞凋亡。     

血红素加氧酶-1对H9c2心肌细胞氧化应激损伤的保护作用研究

目的:探讨血红素加氧酶-1(HO-1)对H9c2心肌细胞氧化应激损伤的保护作用及其作用机制。方法:建立过氧化氢(H2O2)诱导的H9c2心肌细胞氧化应激损伤模型,给予HO-1诱导剂氯化血红素(Hemin)预处理,或给予HO-1抑制剂锌原卟啉(Znpp-IX)共孵育。双波长法检测HO-1活性;四甲基偶氮唑盐(MTT)法检测细胞存活率;Hoechst33342染色和Caspase-3活性检测评估细胞凋亡;硫代巴比妥酸显色法检测丙二醛(MDA)含量,氮蓝四唑显色法检测总超氧化物歧化酶(SOD)活性;Western Blot法检测NF-κB蛋白表达水平。结果:与H2O2组相比,Hemin显著增加细胞HO-1活性,且该作用能被Znpp-IX阻断。与H2O2组相比,HO-1活性增加能够显著下调细胞凋亡率和Caspase-3活性,降低细胞MDA含量,增加总SOD活性,且上述作用能被Znpp-IX逆转。此外,HO-1活性增加能够显著抑制H2O2诱导的NF-κB激活,且该作用能被Znpp-IX逆转。结论:Hemin诱导的HO-1活性增加可能通过抑制NF-κB激活,维持细胞氧化还原平衡状态,抑制H2O2诱导的H9c2心肌细胞氧化应激损伤。     

镉暴露对HepG2细胞NRF2信号通路影响

目的 探讨镉暴露对HepG2细胞转录因子NF-E2相关因子2（NRF2）信号通路的影响。方法 采用甲臢比色法测定CdCl₂（0、1、2.5、5、10、25、50、100、200 μmol/L）处理24 h后,HepG2细胞活力变化;应用蛋白免疫印迹法检测CdCl₂（1、2、5、10、20 μmol/L）处理细胞6 h后,NRF2蛋白水平;采用RT-qPCR方法检测10 μmol/L CdCl₂处理细胞2、4、6、12、24 h后,GCLC、GCLM、HO1 和 AKR1C1 mRNA水平变化,检测CdCl₂（1、2、5、10、20 μmol/L）处理细胞6 h后,GCLC、GCLM、HO1和AKR1C1 mRNA水平变化。结果 HepG2细胞活力随镉处理剂量升高而降低（P<0.05）;与对照组（0.60±0.01）比较, 1、2、5、10、20 μmol/L镉处理组HepG2细胞NRF2蛋白表达水平[分别为（0.65±0.01）、（1.37±0.04）、（1.94±0.05）、（2.24±0.07）、（2.22±0.05）]均明显升高（P<0.05）;与对照组比较,镉处理6 h时,HepG2细胞内GCLC、GCLM、HO1和AKR1C1 mRNA水平[分别为（45.76±7.04）、（114.21±5.23）、（59.52±1.50）、（674.13±27.12）]明显升高（P<0.05）;与对照组比较,5 μmol/L镉处理组HepG2细胞内GCLC和GCLM mRNA水平[分别为（24.77±2.16）、（29.93±0.67）]升高,2 μmol/L镉处理组HepG2细胞内HO1和AKR1C1 mRNA水平[分别（28.55±2.02）、（186.32±12.63）]升高（P<0.05）。结论 镉暴露能激活HepG2细胞系中NRF2信号通路。     

大蒜素抑制H2O2诱导的心肌细胞凋亡的实验研究

目的 探究大蒜素对过氧化氢（Hydrogen Peroxide, H2O2）诱导的H9c2细胞凋亡的影响。方法 H2O2和不同浓度大蒜素分别作用于H9c2细胞,Western blot检测对Caspase-3表达的影响,流式细胞术检测对细胞凋亡的影响,RT-PCR和Western blot检测对血红素氧合酶1(Heme Oxygenase-1,HO-1)表达的影响。分别通过HO-1抑制剂ZnPP9(Zinc protoporphyrin IX)和PI3K/AKT抑制剂LY294002,MTT检测对细胞增殖活性的影响,Western blot检测对p-Akt、 核转录因子Nrf2(nuclear factor-erythroid 2 related factor 2)和HO-1等表达的影响。结果 对照组和100 μM H2O2+大蒜素(0、5、10、20、40 mg/L)组细胞凋亡率分别为(4.83±0.66)%、(68.38±6.97)%、(61.19±6.01)%、(47.32±5.21)%、(22.07±3.73)%和(20.05±2.93)%、与Caspase-3蛋白表达趋势一致,并具有浓度依赖性(P＜0.05)。大蒜素作用下,HO-1 mRNA和蛋白表达亦呈浓度依赖性增加(P＜0.05)。H2O2组细胞相对增殖活性降低至(61.38±1.76)%(P＜0.05),大蒜素处理使其上调至(81.64±3.79)%(P＜0.05),而ZnPP9可阻断该上调,为(69.82±2.22)%(P＜0.05)。大蒜素明显增加p-Akt、Nrf2和HO-1等表达,可被LY294002阻断(P＜0.05)。结论 大蒜素可通过Akt/Nrf2途径上调HO-1表达并抑制H2O2诱导的H9c2细胞凋亡。     

低水平铅暴露儿童的GH/IGF-1轴变化及其与ALAD基因多态性的关系

目的:探讨低水平铅暴露儿童的生长激素/胰岛素样生长因子-1(GH/IGF-1)轴的变化及其与δ-氨基-γ-酮戊酸脱氢酶(ALAD)基因多态性的关系。方法:用钨舟原子吸收光谱法测定来自深圳市区242例学龄前儿童静脉血铅水平(BLL)。按血铅水平将患儿分为两组,A组BLL<50μg/L,B组BLL≥50μg/L。对两组对象的身高(H)、血红蛋白水平(Hb)、ALAD基因、生长激素(GH)、胰岛素样生长因子-1(IGF-1)和胰岛素样生长因子结合蛋白-3(IGFBP-3)水平进行比较。身高测量和血红蛋白检测用常规方法进行;ALAD基因多态性检测采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)法;生长激素测定采用化学发光法;IGF-1和IGFBP-3测定采用ELISA法。结果:242例儿童的血铅水平范围为8～146μg/L,几何均数47μg/L。BLL≥50μg/L者占43%,≥100μg/L者占0.8%。两组儿童的IGFBP-3水平差异有统计学意义,B组明显低于A组。身高和IGF-1略有差异,但无统计学意义。基因多态性分析显示,223例为ALAD1-1型,19例为ALAD1-2型,未发现ALAD2-2型。ALAD2基因出现的频率为7.85%。两组儿童突变基因的频率分布差异无统计学意义。结论:本研究发现即使低水平铅暴露也与儿童血IGFBP-3水平明显降低有关,表明铅中毒损害儿童GH/IGF-1轴功能。在低水平铅暴露状态下,ALAD基因的多态性对儿童血铅水平并无明显影响。可能只有在高水平铅暴露时,ALAD基因变异才会对儿童铅中毒易感性发挥作用。     

Lactobacillus casei alters hPEPT1-mediated glycylsarcosine uptake in Caco-2 cells

Augmentation of the normal flora of the gastrointestinal tract with probiotic bacteria is currently under investigation as a therapeutic tool for several diseases. However, it is unknown whether probiotic bacteria such as Lactobacillus casei alter the expression and function of intestinal transport proteins such as hPEPT1. The effects of 24 and 48 h incubation of Caco-2 cells with 10(8)/L L. casei on the hPEPT1-mediated uptake rate of 20 micro mol/L [(3)H]glycylsarcosine were examined. Dipeptide uptake did not differ from the control at 24 h (15.9 +/- 2.4 vs. 11.5 +/- 1.4 cm.s(-1).mg protein(-1)); however, a significant increase in uptake occurred after 48 h of L. casei treatment (23.7 +/- 1.5 vs. 12.0 +/- 1.9 cm.s(-1).mg protein(-1); P = 0.005). hPEPT1 involvement was confirmed in experiments using excess substrate. Increased uptake of [(3)H]glycylsarcosine appeared to be the result of the direct interaction of the bacteria with Caco-2 cells because conditioned medium had no effect on dipeptide uptake. hPEPT1 mRNA levels did not differ at any time point. These results show that prolonged incubation of Caco-2 cells leads to increased hPEPT1 activity and that this occurs by a mechanism distinct from increased gene expression.     

环磷酰胺处理对小鼠受精卵和二细胞期胚胎组蛋白H3K9乙酰化修饰的影响

目的用环磷酰胺处理雄性小鼠,是否会对受精后的受精卵及早期胚胎的表观遗传特征组蛋白H3K9乙酰化修饰产生明显影响。方法 5只性成熟ICR雄性小鼠用环磷酰胺注射处理4 d时间作为环磷酰胺处理组,另外5只雄鼠注射相应体积的生理盐水作为正常对照组。雄鼠与雌鼠交配后收集原核期受精卵和二细胞期胚胎,然后用间接免疫荧光染色技术结合激光共聚焦扫描显微镜技术检测胚胎细胞中组蛋白H3K9乙酰化修饰的分布和水平。结果正常对照组中,受精卵的雌雄原核均呈现较明显的H3K9乙酰化染色,至二细胞期时,单个核内的染色水平有所下降,H3K9乙酰化在细胞核内除核仁外的区域相对均匀分布。在环磷酰胺处理组中,大部分原核期受精卵和二细胞期胚胎组蛋白H3K9乙酰化水平和分布模式与正常对照组胚胎没有明显差异,只有少部分(25%)受精卵呈现比较大的雄原核以及相对较低的乙酰化染色水平;37%二细胞期胚胎发生碎化,形成2～3个相对大一些的乙酰化染色水平很低的细胞以及若干小的球状体。结论高剂量环磷酰胺处理雄鼠可以在一部分受精卵和早期卵裂胚中导致表观遗传特征和胚胎发育的异常。     

禽流感H9N2型病毒致BALB/c小鼠感染模型的建立与研究

目的建立H9N2型禽流感病毒BALB/c小鼠模型。方法从江苏某农场新分离得到禽流感病毒A/Chicken/Jiangsu/07/2002（H9N2），以逆转录聚合酶链反应(RT PCR)法对该病毒的HA、NA基因进行鉴定和测序以确证。病毒经狗肾传代细胞(MDCK)3次单克隆纯化,之后肺对肺传代感染BALB/c小鼠，通过体重丢失、死亡率、半数致死量(LD50)评价病毒的感染性和模型的建立。结果A/Chicken/Jiangsu/07/2002（H9N2）型禽流感病毒通过肺对肺传代能感染小鼠，并且毒性逐渐增强，4次传代后能使小鼠致死，10次传代后病毒的LD50为10-2.17。结论H9N2型禽流感病毒能在实验条件下感染哺乳类，并建立了研究禽流感病毒的BALB/c小鼠模型。     

Xenoestrogen-induced ERK-1 and ERK-2 activation via multiple membrane-initiated signaling pathways

Xenoestrogens can mimic or antagonize the activity of physiological estrogens, and the suggested mechanism of xenoestrogen action involves binding to estrogen receptors (ERs). However, the failure of various in vitro or in vivo assays to show strong genomic activity of xenoestrogens compared with estradiol (E2) makes it difficult to explain their ability to cause abnormalities in animal (and perhaps human) reproductive functions via this pathway of steroid action. E2 has also been shown to initiate rapid intracellular signaling, such as changes in levels of intracellular calcium, cAMP, and nitric oxide, and activations of a variety of kinases, via action at the membrane. In this study, we demonstrate that several xenoestrogens can rapidly activate extracellular-regulated kinases (ERKs) in the pituitary tumor cell line GH3/B6/F10, which expresses high levels of the membrane receptor for ER-alpha (mER). We tested a phytoestrogen (coumestrol), organochlorine pesticides or their metabolites (endosulfan, dieldrin, and DDE), and detergent by-products of plastics manufacturing (p-nonylphenol and bisphenol A). These xenoestrogens (except bisphenolA) produced rapid (3-30 min after application), concentration (10(-14)-10(-8) M)-dependent ERK-1/2 phosphorylation but with distinctly different activation patterns. To identify signaling pathways involved in ERK activation, we used specific inhibitors of ERs, epidermal growth factor receptors, Ca2+ signaling, Src and phosphoinositide-3 kinases, and a membrane structure disruption agent. Multiple inhibitors blocked ERK activation, suggesting simultaneous use of multiple pathways and complex signaling web interactions. However, inhibitors differentially affected each xenoestrogen response examined. These actions may help to explain the distinct abilities of xenoestrogens to disrupt reproductive functions at low concentrations.     

Icariin Protects H9c2 Rat Cardiomyoblasts from Doxorubicin-Induced Cardiotoxicity: Role of Caveolin-1 Upregulation and Enhanced Autophagic Response

Doxorubicin (Doxo) is a widely used antineoplastic drug which often induces cardiomyopathy, leading to congestive heart failure through the intramyocardial production of reactive oxygen species (ROS). Icariin (Ica) is a flavonoid isolated from Epimedii Herba (Berberidaceae). Some reports on the pharmacological activity of Ica explained its antioxidant and cardioprotective effects. The aim of our study was to assess the protective activities of Ica against Doxo-detrimental effects on rat heart-tissue derived embryonic cardiac myoblasts (H9c2 cells) and to identify, at least in part, the molecular mechanisms involved. Our results showed that pretreatment of H9c2 cells with 1 μM and 5 μM of Ica, prior to Doxo exposure, resulted in an improvement in cell viability, a reduction in ROS generation, the prevention of mitochondrial dysfunction and mPTP opening. Furthermore, for the first time, we identified one feasible molecular mechanism through which Ica could exerts its cardioprotective effects. Indeed, our data showed a significant reduction in Caveolin-1(Cav-1) expression levels and a specific inhibitory effect on phosphodiesterase 5 (PDE5a) activity, improving mitochondrial function compared to Doxo-treated cells. Besides, Ica significantly prevented apoptotic cell death and downregulated the main pro-autophagic marker Beclin-1 and LC3 lipidation rate, restoring physiological levels of activation of the protective autophagic process. These results suggest that Ica might have beneficial cardioprotective effects in attenuating cardiotoxicity in patients requiring anthracycline chemotherapy through the inhibition of oxidative stress and, in particular, through the modulation of Cav-1 expression levels and the involvement of PDE5a activity, thereby leading to cardiac cell survival.     

miR-19b下调大鼠H9c2心肌细胞Kir2.1蛋白表达

目的:检测miR-19b对大鼠H9c2心肌细胞Kir2.1表达的影响,探讨miRNA参与房颤发生发展的机制。方法:培养大鼠H9c2心肌细胞,构建miR-19b过表达质粒、miR-19b干扰质粒、阴性序列质粒,通过慢病毒载体感染大鼠H9c2心肌细胞;倒置荧光显微镜观察感染效率,qRT-PCR检测H9c2细胞中miR-19b mRNA表达水平。qRT-PCR检测H9c2细胞中Kir2.1mRNA水平。Western blot检测H9c2细胞中Kir2.1蛋白表达水平。结果:miR-19b慢病毒载体成功感染H9c2细胞;qRT-PCR检测,与阴性序列组相比,miR-19b过表达组Kir2.1 mRNA表达水平降低,miR-19b干扰组Kir2.1 mRNA表达水平升高;Western blot检测,与阴性序列质粒组相比,miR-19b过表达组Kir2.1蛋白表达水平降低,miR-19b干扰组Kir2.1蛋白表达水平升高。结论:miR-19b抑制KCNJ2的转录,下调Kir2.1蛋白表达,提示mi-19b可能参与房颤的发生发展。     

Age-related changes in memory and effector T cells responding to influenza A/H3N2 and pandemic A/H1N1 strains in humans

Age-dependent changes in the cellular immune response have been mainly described in CD8+ T cells, with relative sparing of CD4+ T cells. We show that in older compared to young adults, effector memory and effector CD8+ T-cell subsets responding to influenza A/H3N2 challenge have diminished cytolytic activity. In contrast, effector CD4+ T-cell subsets in older adults share similar phenotypic and functional characteristics with those from young adults. Further, we observed a diminished cytolytic T-cell response to both seasonal influenza A/H3N2 and pandemic H1N1 (pH1N1) strains in older compared to young adults who had received seasonal influenza vaccine. These results are consistent with the observed rates of serious complications from seasonal and pandemic influenza infections in different age groups, and suggest that CD4+ T cells may provide a compensatory response to influenza infection when CD8+ T cells become compromised during the aging process.     

Chronic prenatal ethanol exposure alters expression of central and peripheral insulin signaling molecules in adult guinea pig offspring

Maternal ethanol consumption during pregnancy can produce a range of teratogenic outcomes in offspring. The mechanism of ethanol teratogenicity is multi-faceted, but may involve alterations in insulin and insulin-like growth factor (IGF) signaling pathways. These pathways are not only important for metabolism, but are also critically involved in neuronal survival and plasticity, and they can be altered by chronic prenatal ethanol exposure (CPEE). The objective of this study was to test the hypothesis that CPEE alters expression of insulin and IGF signaling molecules in the prefrontal cortex and liver of adult guinea pig offspring. Pregnant Dunkin-Hartley-strain guinea pigs received ethanol (4 g/kg maternal body weight/day) or isocaloric-sucrose/pair-feeding (nutritional control) throughout gestation. Fasting blood glucose concentration was measured in male and female offspring at postnatal day 150–200, followed by euthanasia, collection of prefrontal cortex and liver, and RNA extraction. IGF-1, IGF-1 receptor (IGF-1R), IGF-2, IGF-2 receptor (IGF-2R), insulin receptor substrate (IRS)-1, IRS-2, and insulin receptor (INSR) mRNA expression levels were measured in tissues using quantitative real-time PCR. The mean maternal blood ethanol concentration was 281 ± 15 mg/dL at 1 h after the second divided dose of ethanol on GD 57. CPEE resulted in increased liver weight in adult offspring, but produced no difference in fasting blood glucose concentration compared with nutritional control. In the liver, CPEE decreased mRNA expression of IGF-1, IGF-1R, and IGF-2, and increased IRS-2 mRNA expression in male offspring only compared with nutritional control. Female CPEE offspring had decreased INSR hepatic mRNA expression compared with male CPEE offspring. In the prefrontal cortex, IRS-2 mRNA expression was increased in CPEE offspring compared with nutritional control. The data demonstrate that CPEE alters both central and peripheral expression of insulin and IGF signaling molecules at the mRNA level, which may be related to metabolic dysregulation in adult offspring. Furthermore, altered insulin and IGF signaling may be a mechanism of ethanol neurobehavioral teratogenicity.     

Prenatal chlorpyrifos exposure alters motor behavior and ultrasonic vocalization in cd-1 mouse pups

Background  

Chlorpyrifos (CPF) is a non-persistent organophosphate (OP) largely used as pesticide. Studies from animal models indicate that CPF is a developmental neurotoxicant able to target immature central nervous system at dose levels well below the threshold of systemic toxicity. So far, few data are available on the potential short- and long-term adverse effects in children deriving from low-level exposures during prenatal life and infancy.     

Ethanol exposure alters K+- but not bradykinin-induced dopamine release in PC12 cells

The effects of ethanol on [3H]dopamine release were investigated in cultured PC12 cells using two methods to stimulate dopamine release: exposure to depolarizing concentrations of extracellular K+ and incubation with the highly active secretagogue, bradykinin. Both K+ and bradykinin dose-dependently increased [3H]dopamine release. The mean +/- S.E.M. EC50 for K+ was 35.8 +/- 1.2 mM; for bradykinin it was 1.07 +/- 0.23 x 10(-7) M. The characteristics of the bradykinin-stimulated dopamine release showed it to be dependent on extracellular Ca2+ and was attenuated by 1 mM Co2+ or 1 mM Ni2+. However, release was unaffected by either the voltage-dependent Ca2+ channel antagonist, verapamil, or the dihydropyridine (DHP) Ca2+ channel agonist, BAY K 8644. In contrast, 1 mM Co2+ completely blocked, verapamil inhibited and BAY K 8644 augmented K+-stimulated [3H]dopamine release. PC12 cells acutely exposed to ethanol (100 and 200 mM) showed diminished K+-stimulated [3H]dopamine release but an unaltered bradykinin-stimulated response. Cells exposed to 200 mM ethanol for 6 days showed significantly enhanced [3H]dopamine release in response to high concentrations of K+ but no changes were observed in their response to bradykinin. These data provide evidence that ethanol, within the same cell, can differentially affect neurotransmitter release, dependent upon the secretagogue used.