内质网应激激活剂通过上调GRP78增加肺癌A549细胞对顺铂的敏感性

目的探讨内质网应激激活剂(TM)增加肺癌A549细胞对顺铂(DDP)敏感性的机制。方法 DDP和TM处理肺癌A549细胞,MTT法检测肺癌A549细胞存活率,流式细胞术检测细胞凋亡水平,Western印迹检测凋亡相关蛋白Cleaved Caspase-7表达。结果不同浓度的DDP(0,10,20,50μmol/L)作用于肺癌A549细胞24 h,细胞存活率以剂量依赖的方式下降;TM诱导内质网应激后,提高了DDP(20μmol/L)诱导的细胞凋亡率(55.23%±2.31%,P0.05);同时检测到凋亡相关蛋白Caspase-7表达增加。结论 DDP能够诱导肺癌A549细胞凋亡,但是TM通过上调GRP78增加了肺癌A549细胞对DDP敏感性,联合应用TM和DDP有望成为治疗肺癌的新策略。     

消癌解毒方通过内质网应激诱导肝癌HepG2细胞凋亡的研究

[目的]探讨内质网应激在消癌解毒方在人肝癌HepG2细胞凋亡中的作用。[方法]CCK-8法检测不同浓度的消癌解毒方处理HepG2细胞12、24、48 h后细胞的增殖情况;采用流式细胞术检测细胞消癌解毒方处理HepG2细胞24 h凋亡情况;应用qRT-PCR检测内质网应激上游分子标记GRP78、PERK、ATF-6、IRE-1的mRNA水平;采用Western blot方法分析消癌解毒方对PERK、ATF4、CHOP和TRB3蛋白的表达情况;并用CCK-8法检测内质网抑制剂4-苯基丁酸(4-PBA)对HepG2细胞凋亡率的影响。[结果]消癌解毒方抑制HepG2细胞的增殖,并且这种效应呈现时间和浓度依赖;ATF-6和IRE-1 mRNA水平无明显变化,GRP78和PERK mRNA水平较阴性对照组均显著增加;消癌解毒方可上调内质网应激通路的标志蛋白质PERK、ATF4、CHOP水平,增加下游TRB3蛋白的表达。4-PBA与消癌解毒方联合作用组的细胞凋亡率显著减少。[结论]消癌解毒方通过内质网应激诱导人肝癌HepG2细胞的凋亡,其可能是通过PERK通路上调内质网应激相关凋亡蛋白CHOP的表达。     

灰树花多糖对人肺腺癌细胞株A549线粒体的影响

目的检测人肺癌细胞株A549的细胞增殖、线粒体呼吸水平及线粒体膜电位,观察灰树花多糖对A549线粒体的影响。方法CCK-8法检测A549细胞增殖,线粒体呼吸系统检测细胞线粒体呼吸水平,Mito Tracker Red CMXRosb线粒体荧光探针、罗丹明123(Rh123)检测线粒体膜电位。结果 CCK-8检测细胞增殖显示0.4%的灰树花多糖即可抑制A549细胞的增殖,0.6%灰树花多糖作用4 h后,绝大多数A549细胞死亡,其结果与对照组相比较有显著差异(P<0.05);线粒体呼吸系统结果显示,0.4%灰树花多糖作用于A549细胞4 h后细胞呼吸微弱;荧光显微镜观察细胞状态、线粒体荧光探针染色显示,0.4%、0.6%灰树花多糖组部分细胞凋亡,细胞收缩呈圆形,强荧光表达;流式细胞仪检测线粒体膜电位显示,0.4%、0.6%灰树花多糖组荧光信号增强。结论灰树花多糖可以抑制A549的增殖,其作用机制与线粒体膜电位的耗散相关。     

姜黄素诱导A549细胞凋亡过程中活性氧水平和caspase-3活性的变化

目的 探讨姜黄素诱导人肺腺癌A549细胞凋亡的发生机制.方法 采用MTT法检测姜黄素对A549细胞增殖的影响;Hoechst33258荧光染色法观察姜黄素对A549细胞凋亡的影响; DCFH-DA为细胞内活性氧(ROS)探针,激光共聚焦扫描显微镜检测姜黄素对A549细胞ROS水平的影响,分光光度法测定姜黄素对A549细胞内caspase-3活性的影响.结果 不同浓度的姜黄素作用12、24、36、48 h后均可抑制人肺腺癌A549细胞的增殖并诱导其发生凋亡作用;激光共聚焦扫描显微镜下观察姜黄素作用后细胞ROS明显上升,细胞内caspase-3活性增强.结论 姜黄素对人肺腺癌A549细胞有明显的抑制生长和诱导凋亡作用,对细胞ROS和caspase-3活性的影响在其诱导A549细胞凋亡中发挥作用.     

白藜芦醇通过MCU抑制mPTP开放减轻H9c2细胞氧化应激损伤

目的 观察白藜芦醇(resveratrol)保护心肌细胞减轻氧化应激损伤的过程是否与线粒体通透性转换孔(mPTP)和线粒体钙离子单向转运蛋白(MCU)相关,并探讨可能的机制。方法 H9c2心肌细胞常规培养,随机分为对照组、过氧化氢组、白藜芦醇+过氧化氢组、MCU抑制剂(钌红)+白藜芦醇+过氧化氢组。微板法检测细胞外液中乳酸脱氢酶(LDH)的含量;Western印迹法检测内质网应激分子伴侣葡萄糖调节蛋白(GRP)78、GRP94和MCU蛋白表达;激光扫描共聚焦显微镜观察细胞内钙离子(Ca²⁺)、活性氧(ROS)和mPTP变化。结果 与对照组相比,过氧化氢显著增加细胞外液中LDH的含量、GRP78、GRP94和MCU蛋白表达、细胞内Ca²⁺和ROS含量,显著减少mPTP特异性探针TMRE的荧光强度(均P<0.05)。白藜芦醇明显抑制过氧化氢引起的变化,而钌红显著增强白藜芦醇的作用(均P<0.05)。结论 白藜芦醇通过MCU减少细胞内Ca²⁺超载和ROS生成进而抑制mPTP开放,减轻氧化应激引起的内质网应激损伤,保...     

参麦注射液对大鼠神经元内质网应激诱导凋亡的影响

目的 探讨参麦注射液对大鼠皮层神经元内质网应激诱导凋亡的保护作用.方法 体外培养SD乳鼠皮层神经元细胞,免疫组织化学、免疫荧光染色鉴定神经元纯度,流式细胞术AnnexinV、PI双标检测凋亡率及活性caspase-3、-9表达,Western印迹免疫分析GRP78、细胞色素C蛋白、Bcl-2蛋白表达,Fura-2/AM法荧光分光光度计检测细胞内钙浓度([Ca~(2+)]i).结果 SD乳鼠皮层神经元可纯化体外培养,2 μmol/L毒胡萝卜素作用神经元24、48 h细胞凋亡率分别是17.88%、21.38%,参麦治疗组分别是7.42%、8.16%,两组差异显著(P<0.05).胡萝卜素诱导神经元糖调节蛋白GRP78表达上调,活化caspase-3、-9,使细胞色素C蛋白表达增加,Bcl-2表达减少.参麦注射液促进细胞Bcl-2表达,抑制细胞色素C释放,减少活性caspase-3、-9含量,降低[Ca~(2+)]i浓度.结论 参麦注射液能抑制体外培养神经元内质网应激所致的凋亡可能与其降低[Ca~(2+)]i浓度有关.     

棕榈酸致大鼠胰腺腺泡细胞损伤中内质网应激通路的作用

目的 研究棕榈酸对大鼠胰腺腺泡细胞的损伤作用及内质网应激通路的作用.方法 采用胶原酶法体外分离大鼠胰腺腺泡细胞.将胰腺腺泡细胞与0.05和0.1 mmol/L棕榈酸分别共育1、2、3 h后,以未加棕榈酸的细胞为对照组,分别给予胆囊收缩素八肽(CCK-8)刺激,测定细胞淀粉酶释放比例;共聚焦显微镜动态观察胰腺腺泡细胞内钙离子浓度变化;透射电镜观察胰腺腺泡细胞凋亡;RT-PCR检测胰腺腺泡细胞内质网应激过程重要分子GRP78/Bip、XBP-1、GADD153/CHOP和caspase-12的基因表达.结果 体外分离的胰腺腺泡细胞与棕榈酸共同培育,给予100 pmol/L CCK-8刺激30 min后细胞淀粉酶释放比例随培育时间和棕榈酸浓度的增加而增加,且明显高于对照组(P＜0.05);细胞与0.1 mmol/L棕榈酸共同培育3 h,给予100 pmol/L CCK-8刺激后,可检测到GRP78/Bip、GADD153/CHOP、分裂的XBP-1和caspase-12的mRNA表达.透射电镜下观察到相应的胰腺腺泡细胞凋亡.细胞与0.1 mmol/L棕榈酸共同培育2、3 h后,分别给予100 pmol/L CCK-8刺激,发现细胞内钙荧光强度维持的高水平,较对照组明显增加(P＜0.05).结论 棕榈酸可增高大鼠胰腺腺泡细胞对CCK-8的敏感性,通过升高细胞内钙离子的浓度诱发内质网应激,从而引起细胞凋亡,加重胰腺腺泡细胞损伤.     

对比剂通过内质网应激诱导人肾小管上皮细胞凋亡

目的探讨内质网应激在对比剂诱导人肾小管上皮细胞(HKC)凋亡中的作用。方法在缺血、缺氧(0.2%胎牛血清,5%CO2-95%N2饱合混合气体)环境下,以HKC为研究对象,将HKC细胞株随机分为3组:空白组、对比剂组(加入120g I/L碘普罗胺预处理2h)和N-乙酰半胱氨酸(NAC)组(加入10mmol/L NAC预处理2h,再加120g I/L碘普罗胺处理2h)。细胞增殖试剂盒(CCK-8)法检测细胞增殖情况,流式细胞术检测细胞凋亡,实时荧光定量聚合酶链反应(real-time PCR)检测细胞中半胱氨酸蛋白酶-12(caspase-12)、葡萄糖调节蛋白-78(GRP78)和CAAT/增强子结合蛋白同源蛋白(CHOP)的mRNA表达水平,蛋白质印迹法(Western blot)检测细胞中caspase-12、GRP78和CHOP的蛋白表达水平。结果对比剂可呈时间依赖性抑制HKC活性,与0h相比,1hHKC活性显著降低(0.351±0.066比0.447±0.087,P0.05);流式细胞术结果示对比剂可促进缺血缺氧HKC凋亡(P0.05);real-time PCR结果示,对比剂组和NAC组GRP78和CHOP的mRNA水平明显高于空白组(P0.05),对比剂组caspase-12的mRNA水平明显高于空白组和NAC组(P0.05);Western blot结果示,对比剂明显增加了caspase-12、GRP78和CHOP的蛋白表达,而NAC明显降低了caspase-12的表达,但不能明显降低对比剂诱导的GRP78和CHOP高表达(P0.05)。结论在缺血缺氧条件下,对比剂诱导HKC活性下降、凋亡,此作用可能与细胞内氧化应激及内质网应激相关。     

PC12细胞氧糖剥夺不同时间段再灌注后内质网应激及细胞凋亡

目的观察大鼠肾上腺髓质嗜铬细胞瘤(PC12细胞)细胞氧糖剥夺(OGD)不同时间段再灌注(OGD/R)后内质网应激及凋亡的变化,探讨神经元OGD再灌注所致内质网应激与凋亡的关系。方法利用无糖DMEM及三气培养箱对PC12细胞进行OGD,不同时间段OGD后恢复正常培养24 h,根据不同OGD时间段,将细胞分为五组:control、3 h/24 h、6 h/24 h、9 h/24 h、12 h/24 h,OGD/R相应时间后,采用Western印迹检测内质网相关蛋白分子伴侣葡萄糖调节蛋白(GRP)78、蛋白激酶R样内质网激酶(PERK)及凋亡蛋白半胱氨酸蛋白酶(caspase)-3的表达,免疫荧光方法检测GRP78蛋白的表达情况,并利用流式细胞仪检测凋亡率。结果 OGD/R后,与control组相比,6 h/24 h、9 h/24 h、12 h/24 h组细胞中GRP78蛋白及caspase-3蛋白的表达水平明显增加(P<0.01,P<0.05),而PERK蛋白在各组中无明显变化。用Spearman相关性分析结果显示,capase-3蛋白与GRP78蛋白表达水平成呈正相关(R=0.561,P<0.05);流式细胞仪检测结果显示,与control组相比,各组的凋亡率明显增高(P<0.01)。结论 PC12细胞OGD/R后内质网应激及凋亡呈动态变化,且内质网应激蛋白GRP78与凋亡蛋白caspase-3的表达水平有关,这将对缺血再灌注后神经元损伤的研究具有一定的意义。     

GRP78的表达非小细胞性肺癌细胞A549对顺铂的敏感性

目的探讨下调GRP78的表达后是否增强非小细胞性肺癌细胞对顺铂的敏感性。方法通过蛋白印记的方法检测内质网应激后GRP78的表达变化;用流式细胞仪检测用siRNAGRP 78抑制GRP 78的表达上调后凋亡细胞的数目;用A549肿瘤细胞对裸鼠进行了皮下荷瘤,在体内检验GRP78的表达水平对顺铂耐药的影响;运用免疫组织化学的方法检测肺癌患者的肿瘤组织中GRP78的表达水平。结果内质网应激能上调GRP78的表达;抑制内质网应激诱导的GRP78的表达上调后,细胞对顺铂的敏感性增加;将GRP78基因表达干扰后,荷瘤组织的生长明显受到顺铂的抑制;20例肺癌患者的肿瘤组织与癌旁组织相比,存在GRP78的高表达。结论 GRP78的表达水平上调降低非小细胞性肺癌细胞对顺铂的敏感性,具体的参与调控的信号通路需要进一步的机制研究。     

Three layer functional model and energy exchange concept of aging process

Relying on a certain degree of abstraction, we can propose that no particular distinction exists between animate or living matter and inanimate matter. While focusing attention on some specifics, the dividing line between the two can be drawn. The most apparent distinction is in the level of structural and functional organization with the dissimilar streams of ‘energy flow’ between the observed entity and the surrounding environment. In essence, living matter is created from inanimate matter which is organized to contain internal intense energy processes and maintain lower intensity energy exchange processes with the environment. Taking internal and external energy processes into account, we contend in this paper that living matter can be referred to as matter of dissipative structure, with this structure assumed to be a common quality of all living creatures and living matter in general. Interruption of internal energy conversion processes and terminating the controlled energy exchange with the environment leads to degeneration of dissipative structure and reduction of the same to inanimate matter, (gas, liquid and/or solid inanimate substances), and ultimately what can be called ‘death.’ This concept of what we call dissipative nature can be extended from living organisms to social groups of animals, to mankind. An analogy based on the organization of matter provides a basis for a functional model of living entities. The models relies on the parallels among the three central structures of any cell (nucleus, cytoplasm and outer membrane) and the human body (central organs, body fluids along with the connective tissues, and external skin integument). This three-part structural organization may be observed almost universally in nature. It can be observed from the atomic structure to the planetary and intergalactic organizations. This similarity is corroborated by the membrane theory applied to living organisms. According to the energy nature of living matter and the proposed functional model, the decreased integrity of a human body's external envelope membrane is a first cause of the structural degradation and aging of the entire organism. The aging process than progresses externally to internally, as in single cell organisms, suggesting that much of the efforts towards the restoration and maintenance of the mechanisms responsible for structural development should be focused accordingly, on the membrane, i.e., the skin. Numerous reports indicate that all parts of the human body, like: bones, blood with blood vessels, muscles, skin, and so on, have some ability for restoration. Therefore, actual revival of not only aging tissue of the human body's membrane, but the entire human body enclosed within, with all internal organs, might be expected. We assess several aging theories within the context of our model and provide suggestions on how to activate the body's own anti-aging mechanisms and increase longevity. This paper presents some analogies and some distinctions that exist between the living dissipative structure matter and inanimate matter, discusses the aging process and proposes certain aging reversal solutions.     

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: Attempts to define mechanisms

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm²) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.     

Melatonin and photoperiodic time measurement: Seasonal breeding in the sheep

Abstract: Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status—the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.     

Serological survey of HIV and syphilis in pregnant women in Madagascar

Objectives Peripartal transmission of human immunodeficiency virus (HIV) and Treponema pallidum, the causative agent of syphilis, leads to severe consequences for newborns. Preventive measures require awareness of the maternal infection. Although HIV and syphilis testing in Madagascar could be theoretically carried out within the framework of the national pregnancy follow‐up scheme, the required test kits are rarely available at peripheral health centres. In this study, we screened blood samples of pregnant Madagascan women for HIV and syphilis seroprevalence to estimate the demand for systemic screening in pregnancy. Methods Retrospective anonymous serological analysis for HIV and syphilis was performed in plasma samples from 1232 pregnant women that were taken between May and July 2010 in Ambositra, Ifanadiana, Manakara, Mananjary, Moramanga and Tsiroanomandidy (Madagascar) during pregnancy follow‐up. Screening was based on Treponema pallidum haemagglutination tests for syphilis and rapid tests for HIV, with confirmation of positive screening results on line assays. Results Out of 1232 pregnant women, none were seropositive for HIV and 37 (3%) were seropositive for Treponema pallidum. Conclusions Our findings are in line with previous studies that describe considerable syphilis prevalence in the rural Madagascan population. The results suggest a need for screening to prevent peripartal Treponema pallidum transmission, while HIV is still rare. If they are known, Treponema pallidum infections can be easily, safely and inexpensively treated even in pregnancy to reduce the risk of transmission.     

Variabilité des concentrations plasmatiques de l’angiotensinogène et risque d’hypertension artérielle chez le rat transgénique

Aim

Genetic polymorphisms of the human angiotensinogen gene are frequent and may induce up to 30% increase of plasma angiotensinogen concentrations with a blood pressure increase of up to 5 mmHg. Their role for the pathogenesis of human arterial hypertension remains unclear. High plasma angiotensinogen levels could increase the sensitivity to other blood pressure stressors.

Methods

Male transgenic rats with a 9-fold increase of plasma angiotensinogen concentrations and male non-transgenic rats aged 10 weeks were treated or not with NG-Nitro-L-arginine-methyl ester for 3 weeks in their drinking water (n = 3/group). Systolic blood pressure and body weight were measured at baseline and at the end of the study when left ventricular weight and ventricular expression of angiotensin I-converting enzyme and procollagen Iα1 were determined (polymerase chain reaction).

Results

At baseline, transgenic rats had +18 mmHg higher bood pressure and –8% lower body weight compared to non-transgenic rats (P < 0.05) without significant changes for the vehicle groups throughout the study (P > 0.05). NG-Nitro-L-arginine-methyl ester increased blood pressure, left ventricular weight and left ventricular weight indexed for body weight by +41%, +17.6% and +18.6% (P < 0.05) in transgenic and +25%, +5.3% and +6.7% (P > 0.05) in non-transgenic rats compared to untreated animals, respectively. Cardiac gene expression showed no differences between groups (P > 0.05).

Conclusion

Increased plasma angiotensinogen levels may sensitize to additional blood pressure stressors. Our preliminary results point towards an independent role of angiotensinogen in the pathogenesis of human hypertension and associated end-organ damage.     

Morphological and immunocytochemical heterogeneity of cultured pinealocytes from one-week-and two-month-old rats: Planimetric and densitometric investigations

Abstract: In vitro preparations of rat pinealocytes are widely used for biochemical analyses of signal transduction processes. This paper deals with morphological and immunocytochemical features of such preparations. Special attention was paid to the problems of whether pinealocytes represent a heterogeneous cell population and how such heterogeneity may develop during ontogeny. The investigations were performed with cells which were obtained from the pineal organ of one-week-and two-month-old rats, attached to synthetic peptide-coated coverslips or tissue culture chamber slides, and maintained under in vitro conditions overnight. The attached cells were then fixed with paraformaldehyde. These preparations yielded monolayers of spherical cells of different sizes; most cells were isolated, but some of them were aggregated and formed small clusters. On the average, the cells from the one-week-old animals were smaller than the cells from the two-month-old animals. Immunocytochemical demonstration of S-antigen, a pinealocyte-specific marker, showed that the majority of the cells from two-month-old animals were intensely or moderately labelled. Pinealocytes from one-week-old animals were less S-antigen immunoreactive. Only very few cells (less than 1% displayed glial fibrillary acidic protein (GFAP)-immunoreactivity. Planimetric investigations of the cell size and semiquantitative densitometric investigations of the intensity of the S-antigen immunoreaction revealed that (i) pinealocytes kept in vitro form a heterogeneous cell population, and that (ii) this heterogeneity increases during postnatal development from one-week-old to two-month-old animals. Two groups of pinealocytes can be distinguished based on their developmental fate: pinealocytes of one group grow dramatically, but show only a moderate increase in S-antigen immunoreactivity, and pinealocytes of the other group retain their size, but display a distinct increment in S-antigen immunoreacti vitv.     

MUTATION FREQUENCY IN NURSES AND PHARMACISTS WORKING WITH CYTOTOXIC DRUGS

Individuals occupationally exposed to cytotoxic drugs may be at risk owing to the effects of these agents on DNA. As an index of DNA damage, in vivo mutations were measured in lymphocytes from 24 oncology nurses or pharmacists and 24 matched controls. Mutation frequency was significantly increased in exposed individuals and appeared to be related to duration of exposure. However, the overall magnitude of the increase was small and its biological significance remains to be determined.