神经生长因子对大鼠脑皮质神经细胞凋亡的影响

目的研究神经生长因子（ＮＧＦ）对大鼠脑皮质神经细胞凋亡的影响。方法用原代培养的大鼠胎鼠脑皮质细胞,建立了Ｎ－甲基－Ｄ－门冬氨酸（ＮＭＤＡ）、缺氧／缺糖诱导的神经细胞凋亡模型,并观察了ＮＧＦ的保护作用。结果ＮＭ－ＤＡ３００μｍｏｌ·Ｌ－１与细胞作用２０ｍｉｎ及细胞缺氧／缺糖培养１０ｈ可诱导细胞凋亡。提取细胞ＤＮＡ进行琼脂糖凝胶电泳呈现典型的ＤＮＡ梯状条带,应用流式细胞仪检测,在Ｇ１峰前出现一个“Ｇ１亚峰”,凋亡细胞所占比例为５０．２％及４５．７％,同时出现细胞核染色体聚集、断裂及细胞空泡样变等形态变化。ＮＧＦ（５０,１００μｇ·Ｌ－１）能使ＤＮＡ梯状电泳条带不明显或消失,降低凋亡细胞数,并使细胞超微结构损伤减轻或基本恢复正常。结论ＮＧＦ能拮抗ＮＭＤＡ及缺氧／缺糖诱导的神经细胞凋亡。     

(-)-S·R-蝙蝠葛苏林碱对谷氨酸引起的大鼠大脑皮质神经元损伤的保护作用

用细胞培养方法，在原代培养的大鼠皮质神经细胞上，观察了（－）Ｓ·Ｒ蝙蝠葛苏林碱对谷氨酸引起的神经元损伤的保护作用。以Ｆｕｒａ２／ＡＭ为Ｃａ２＋的荧光指示剂，用ＡＲＣＭＭＩＣ阳离子测定系统观察（－）Ｓ·Ｒ蝙蝠葛苏林碱对谷氨酸诱发大鼠脑皮质神经细胞内Ｃａ２＋升高的影响。结果表明（－）Ｓ·Ｒ蝙蝠葛苏林碱能剂量依赖性地抑制谷氨酸的神经毒作用，对谷氨酸诱发的神经细胞内游离Ｃａ２＋升高有明显的抑制作用。提示蝙蝠葛苏林碱对缺血性脑损伤有保护作用。     

溴氰菊酯对大鼠中枢神经系统一氧化氮合成酶活性的影响

本研究在发现溴氰菊酯（ＤＭ）可使突触前膜谷氨酸释放增加的基础上，探讨了ＤＭ对大鼠中枢神经系统不同部位的一氧化氮合成酶（ＮＯＳ）活性的影响。利用不同性质阻断剂，对影响ＮＯＳ活性的可能机理进行了研究。结果发现，ＤＭ可引起大脑皮层、海马及小脑部位的ＮＯＳ活性显著增加。Ｌ－硝基精氨酸是ＮＯＳ的竞争性抑制剂，它可抑制由ＤＭ所致ＮＯＳ活性的升高。尼莫地平是一种电压依赖性钙通道阻断剂，它也可以有效的抑制ＤＭ所致ＮＯＳ活性的增加。认为ＤＭ导致神经毒性的机理可能是通过谷氨酸过量释放，诱导ＮＯＳ活性的升高，造成神经元的损伤。     

阿米替林对谷氨酸损伤中枢神经细胞的保护作用

目的：研究阿米替林（Ami)对谷氨酸损伤培养大鼠皮层神经细胞的保护作用。方法：分离培养15－18d胎龄的大鼠神经细胞，加入谷氨酸（Glu)，造成神经细胞的损伤，测定乳酸脱氢酶，一氧化氮，丙二醛及超氧化物歧化酶的含量，观察谷氨酸对神经细胞的损伤作用及阿米替林的保护作用。结果：加入谷氨酸后，神经细胞出现明显损伤性变化，死亡率升高，培养上清液中乳酸脱氢酶（LDH），一氧化氮（NO）含量增加，细胞匀浆中丙二醛（MDA）生成增加，超氧化物歧化酶（SOD）含量明显减少，Ami10^-8-10^-6mol/L^-1能不同程度地减轻上述损伤性变化。结论：Ami对谷氨酸所致的神经细胞损伤具有保护作用。其作用机制可能与抗脂质过氧化作用有关。     

丁基苯酞对氯化钾及 N-甲基-D-门冬氨酸诱导的大鼠皮质神经细胞损伤的保护作用

用原代培养大鼠皮质神经细胞的方法，观察ｌ丁基苯酞（ｌＮＢＰ）和ｄ丁基苯酞（ｄＮＢＰ）对ＫＣｌ及Ｎ甲基Ｄ门冬氨酸（ＮＭＤＡ）诱导的大鼠皮质神经细胞损伤的保护作用。结果表明：ｌＮＢＰ和ｄＮＢＰ能剂量依赖性地抑制ＮＭＤＡ诱导的大鼠皮质神经细胞内乳酸脱氢酶的释放，降低细胞死亡率，改善受损细胞的形态；此外ｌＮＢＰ和ｄＮＢＰ还能明显减轻ＫＣｌ诱导的神经细胞损伤。提示：ｌＮＢＰ和ｄＮＢＰ对ＫＣｌ及ＮＭＤＡ诱导的大鼠皮质神经细胞损伤有明显保护作用。     

脱氢表雄酮硫酸酯对体外大鼠脑皮层神经细胞拟老化反应的影响

用原代培养Ｗｉｓｔａｒ大鼠胎鼠脑皮层神经细胞，从形态学和生化学方面研究自由基、无血清培养对神经元的损伤以及脱氢表雄酮硫酸酯（ＤＨＥＡＳ）的保护作用。结果显示，神经细胞与自由基作用及在无血清培养条件下，细胞生存率明显下降，培养液的上清液中乳酸脱氢酶（ＬＤＨ）活性显著提高，丙二醛（ＭＤＡ）含量明显增加，超氧物岐化酶（ＳＯＤ）和谷胱甘肽过氧化物酶（ＧＳＨＰｘ）活力显著下降。相差显微镜下观察，神经细胞突起回缩，颗粒增加，折光性下降。ＤＨＥＡＳ剂量依赖地提高神经细胞损伤后细胞生存率，降低ＬＤＨ的释放及ＭＤＡ的生成，并显著提高抗氧化酶活性。结果提示，ＤＨＥＡＳ可能通过抑制脂质过氧化物生成及提高抗氧化酶活性来保护拟衰老反应中的大脑皮层神经细胞。     

脑缺血再灌注损伤后神经细胞凋亡与褪黑素的影响

目的：观察局灶性脑缺血再灌注损伤（CI-RI）后神经细胞凋亡与褪黑素（MT）的影响,探讨相关机制。方法：线栓法制作大鼠大脑中动脉闭塞2h/再灌注24h模型,再灌注0、1、2、6hi.p.MT。TTC染色观察脑梗死体积;TUNEL法检测神经细胞凋亡;免疫组化法检测脑组织Bcl-2和Bax蛋白表达;无机磷酸法测定脑组织钙调神经磷酸酶（CaN）活性;毛细管区带电泳（CZE）法测定脑组织ATP含量。结果：MT10、20mg/kg均可显著缩小脑梗死体积;MT20mg/kg可显著降低脑组织缺血半暗带区（IP）神经细胞凋亡、增高Bcl-2/Bax比值,显著抑制损伤侧脑组织CaN活性,升高脑组织ATP含量。结论：MT抑制CIRI后脑组织神经细胞凋亡,使脑梗死体积减小,其机制与上调Bcl-2/Bax比值、抑制CaN活性升高、增加ATP保有量均有关。     

Effect of nerve growth factor on glutamate-induced neurotoxicity in cerebral cortical neuronal cultures

在原代培养的大鼠胎鼠脑皮质神经元，观察神经生长因子（ＮＧＦ）对谷氨酸损伤的影响．谷氨酸（０．２－０．８ｍｍｏｌ·Ｌ－１）促进神经元死亡及乳酸脱氢酶（ＬＤＨ）释放，增加丙二醛（ＭＤＡ）含量．ＮＧＦ３－１００μｇ·Ｌ－１浓度依赖地抑制谷氨酸引起的神经元死亡及ＬＤＨ和ＭＤＡ的增加．ＮＧＦ３０μｇ·Ｌ－１提高超氧化物歧化酶和谷胱甘肽过氧化酶活力．ＮＧＦ浓度依赖地增加谷氨酸引起的抗氧化酶水平降低．结果提示ＮＧＦ通过抑制脂质过氧化物生成及提高抗氧化酶活性来保护大脑皮质细胞抗谷氨酸毒性．     

芸香甙对脑缺血再灌损伤的保护作用

目的研究芸香甙（Ｒｕ）对脑缺血再灌损伤的保护作用。方法小鼠脑缺血再灌模型通过结扎双侧颈总动脉并尾部放血０．３ｍｌ，再灌后，记录异常神经症状和断头后张口喘气时间，测定脑组织中乳酸脱氢酶（ＬＤＨ）含量；利用４血管模型，大鼠前脑缺血３０ｍｉｎ后再灌注４０ｍｉｎ。取脑组织测定ＬＤＨ、过氧化物歧化酶（ＳＯＤ）、丙二醛（ＭＤＡ）、一氧化氮（ＮＯ）及脑水肿。结果Ｒｕ（５０，１００ｍｇ·ｋｇ－１，ｉｖ），显著改善小鼠脑缺血再灌损伤后的异常神经症状和抑制断头后张口喘气时间的缩短及脑组织中ＬＤＨ的减少。Ｒｕ显著抑制大鼠脑缺血再灌损伤所致的脑水肿形成和脑组织中ＬＤＨ、ＳＯＤ、ＭＤＡ及ＮＯ的变化。结论Ｒｕ对脑缺血再灌损伤有显著的保护作用，其机制与抗自由基和ＮＯ有关。     

低剂量砷暴露小鼠脑组织核酸损伤的免疫组织化学观察

地方性砷中毒是世界性的公共卫生问题，其中砷暴露对人群神经系统有害影响已受到社会的极大关注动物实验表明，砷可以通过血脑屏障进入脑实质．慢性砷暴露的豚鼠和大鼠脑中砷浓度与暴露呈显著正相关。有报道，即使砷在饮用水卫生标准浓度范围内，也能影响体内氧化还原平衡，增高脑脂质过氧化水平，降低谷胱甘肽（GSH）浓度和抗氧化酶活力，使脑组织受到自由基损害，引起神经细胞异常凋亡。但是，砷诱发的神经系统损伤机制目前还不十分清楚。     

Damage effects of dust storm PM2.5 on DNA in alveolar macrophages and lung cells of rats.

The aim of the present study was to investigate in vitro toxicological effects of PM2.5 suspensions, their water-soluble fraction and solvent-extractable organics from dust storm on the viability and DNA of rat alveolar macrophages and in vivo toxicological effects of PM2.5 suspensions on DNA of lung cells of rats. PM2.5 samples from dust storm and normal weather were collected in Baotou city, Inner Mongolia Autonomous Region, and Wuwei city, Gansu Province, China, in March, 2004. DNA damage was detected with single cell gel electrophoresis technique and cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The results showed that: (1) In vitro, PM2.5 suspensions, their water-soluble fraction and solvent-extractable organics from both dust storm and normal weather caused a decrease of the cell viability and an increase of DNA damage of rat alveolar macrophages in a dose-response manner; for both Baotou city and Wuwei city, the samples of normal weather showed higher DNA damage than those of dust storm at the highest treated dosage; for both normal weather PM2.5 and dust storm PM2.5, their solvent-extractable organics showed higher DNA damage than the water-soluble fraction. (2) In vivo, PM2.5 from both dust storm and normal weather caused an increase of DNA damage of rat lung cells in a dose-response manner. (3) Baotou city is one of the heavy industrial cities, while Wuwei is one of agricultural cities in Northwest region of China. The effects induced by normal weather samples in Baotou city slightly higher than those in Wuwei city on DNA damage, though there was no significant difference was found between two cities. These results lead to conclusions that dust storm PM2.5 as well as normal weather PM2.5 could lead to DNA damage and the organic compounds and the insoluble particle-core might be the main contributors to DNA damage. Our results suggest that the risk of health effects may be greater during dust storms because dust storm PM2.5 whose airborne mass were much higher. Further studies are needed to determine the components of dust storm particles that may contribute to the particle toxicity.     

6-Mercaptopurine (6-MP) induces p53-mediated apoptosis of neural progenitor cells in the developing fetal rodent brain

6-Mercaptopurine (6-MP), a DNA-damaging agent, induces apoptosis of neural progenitor cells, and causes malformation in the fetal brain. The aim of the present study is to clarify the molecular pathway of 6-MP-induced apoptosis of neural progenitor cells in the fetal telencephalon of rats and mice. p53 protein is activated by DNA damage and induces apoptosis through either the intrinsic pathway involving the mitochondria or the extrinsic pathway triggered by death receptors. In this study, the expression of puma and cleaved caspase-9 proteins, which are specific intrinsic pathway factors, increased in the rat telencephalon after 6-MP treatment. 6-MP-induced apoptosis of neural progenitor cells was completely absent in p53-deficient mice. On the other hand, the expression of Fas protein, an extrinsic pathway factor, did not change throughout the experimental period in the rat telencephalon treated with 6-MP. The number of apoptotic neural progenitor cells was similar among Fas-mutated lpr/lpr and wild-type mice, suggesting that the Fas pathway does not play a significant role in 6-MP-induced apoptosis of neural progenitor cells. These results may suggest that the p53-mediated intrinsic pathway is essential for 6-MP-induced apoptosis of neural progenitor cells in the developing telencephalon of rats and mice.     

Activation of caspase-3 in HL-60 cells treated with pyrithione and zinc

The transition metal zinc (Zn) is an endogenous regulator of apoptosis. The ability of Zn to modulate apoptosis is believed to be mediated by the regulation of caspase activity. Previously, we reported that an acute influx of labile Zn induced apoptosis via activation of caspase in human leukemia HL-60 cells treated with a Zn ionophore (Py, pyrithione) and Zn at 1 and 25 microM, respectively. In the present study, we investigated the involvement of caspase-3 in Py (1 microM)/Zn (25 microM)-induced apoptosis in HL-60 cells. Pro-caspase-3 is an inactive form of caspase-3. The processing of pro-caspase-3, a sign of caspase-3 activation, occurred 6 h after treatment with Py/Zn. Proteolysis of poly (ADP-ribose) polymerase (PARP), a substrate of caspase-3, was also observed 6 h after treatment with Py/Zn. We also confirmed the elevation of caspase-3 activity as an index of the cleavage of amino acid sequences recognized by activated caspase-3. An inhibitor of caspase-3 attenuated the appearance of the DNA ladder. Taken together, these results indicate that the activation of caspase-3 is partly responsible for the induction of apoptosis in Py/Zn-treated HL-60 cells.     

Antileukemic action of (−)-epicatechin in the spleen of rats with acute myeloid leukemia

The aim of this study was to investigate whether (−)-epicatechin (EC) can induce DNA damage and apoptosis of cancer cells in the spleen of rat with acute myeloid leukemia. Healthy and leukemic rats were given EC by gavage at a dose of 40 mg/kg b.w. for 22 consecutive days. Spleen cells were subjected for analysis of DNA damage and apoptosis. The amount of DNA damage was estimated by the comet assay, while apoptosis was examined by flow cytometry using Annexin V staining. Leukemic cells were identified in the spleen cells by indirect immunofluorescence using RM-124 antibody followed by flow cytometry analysis. The results show that EC did not affect DNA damage in the splenocytes of healthy rats, but significantly increased the extent of DNA strand breaks in the spleen cells of leukemic animals. EC administration to leukemic rats induced a significant increase in the level of Annexin V-positive leukemic cells, but the level of non-leukemic Annexin V-positive cells remained unchanged in comparison to control. The percentage of leukemic cells decreased significantly under EC influence comparing to the untreated group. The results of the study reveal that EC could be used as an effective supplement of standard therapy against acute myeloid leukemia.     

银杏内酯B对背根神经节神经元谷氨酸损伤的保护作用

目的:观察银杏内酯B(Gin)对体外培养的胚胎大鼠背根神经节(DRG)神经元谷氨酸(Glu)损伤的保护作用.方法:Wistar胎鼠DRG神经元分散培养48 h后,用Glu(200 μmol/L)造成神经元损伤(Glu损伤组),并同时给予Gin 50 μmol/L(Gin保护组),观察添加Gin和末添加Gin孵育的Glu损伤的神经元活细胞生长状况,并且利用流式细胞仪检测各组神经元的凋亡率,共聚焦激光扫描显微镜(CLSM)检测各组细胞内钙离子荧光强度.结果:用Gin孵育的DRG神经元生长状况良好,接近于正常对照组细胞,较损伤组DRG神经元生长状态有明显改善;Glu损伤组DRG神经元细胞凋亡率(41.1%)高于对照组(2.5%)和Gin保护组(7.6%).Glu损伤组细胞内钙离子荧光强度较对照组明显增高(P＜0.01),Gin保护组细胞内钙离子荧光强度较Glu损伤组明显降低(P＜0.01),Gin保护组DRG神经元胞体内钙离子的荧光强度与正常对照组相比差异无统计学意义(P＞0.05).结论:Gin可通过减低钙离子内流,从而对Glu损伤的DRG神经元产生保护作用.     

IL-10 ameliorates PM2.5-induced lung injury by activating the AMPK/SIRT1/PGC-1α pathway

Exposure to fine particulate matter with a diameter ≤2.5 μm (PM2.5) can cause a number of respiratory diseases. However, there is currently no safe treatment for PM2.5-induced lung damage. This study investigated the protective effect of IL-10 against lung injury and the possible involvement of AMPK/SIRT1/PGC-1α signaling. The mean diameter, particle size distribution, and zeta potential of PM2.5 samples were assessed using a Zetasizer Nano ZS90 analyzer. Thereafter, Wistar rats were exposed to PM2.5 (1.8, 5.4, or 16.2 mg/kg) alone or high-dose PM2.5 with recombinant rat IL-10 (rrIL-10; 5 μg/rat). Treatment with rrIL-10 ameliorated PM2.5-induced acute lung injury, reduced mitochondrial damage, and inhibited inflammation, oxidative stress, and apoptosis in the PM2.5-treated rats. Moreover, the mRNA and protein expression of AMPK, SIRT1, and PGC-1α were upregulated by rrIL-10 treatment. In conclusion, rrIL-10 protected lung tissues against PM2.5-induced inflammation by reducing oxidative stress and apoptosis via activating AMPK/SIRT1/PGC-1α signaling.     

Aflatoxin G1-induced oxidative stress causes DNA damage and triggers apoptosis through MAPK signaling pathway in A549 cells

Our previous studies showed that Aflatoxin G₁ (AFG₁) could induce lung adenocarcinoma, and that the cancer cells originated from alveolar type II cells (AT-II cells). Recently, we found AFG₁ induced structural impairment in rat AT-II cells, which may account for an early event in lung tumorigenesis. However, the mechanism of AFG₁-induced AT-II cell damage remains unclear. DNA damage and apoptosis induced by oxidative stress are well accepted causes of cell damage. Thus, we explore whether AFG₁ activates the reactive oxygen species (ROS)/MAPK/apoptosis pathway to cause cell damage in human AT-II cells like the cell line (A549). We found AFG₁ induced oxidative stress by increasing ROS generation and caused DNA double-strand breaks (DSBs) by up-regulating γH2AX expression. AFG₁ also triggered apoptosis in A549 cells by regulating Fas/FasL, caspase-8, Bax, Bcl-2, and activating caspase-3. Pre-treatment with antioxidant n-acetyl-l-cysteine (NAC) reduced ROS generation and DNA DSBs, inhibited apoptosis, and increased cell viability in AFG₁-treated cells. Furthermore, we found AFG₁ activated ROS-mediated JNK and p38 pathways to induce cell apoptosis in A549 cells. In conclusion, our results indicate that AFG₁ induces oxidative DNA damage and triggers apoptosis through ROS-mediated JNK and p38 signaling pathways in A549 cells, which may contribute to AFG₁-induced AT-II cell damage.     

p53-Dependent apoptosis induced in human bronchial epithelial (16-HBE) cells by PM2.5 sampled from air in Guangzhou,China

Epidemiological studies have shown that air pollution particulate matter (PM) is associated with increased respiratory morbidity and mortality. However, the mechanisms are not fully understood. Oxidative stress-mediated apoptosis plays an important role in the occurrence of respiratory diseases. In this study, human bronchial epithelial (16-HBE) cells were exposed to different concentrations (16–128?µg/ml) of PM_2.5 for 24?h to investigate the apoptosis induced by PM_2.5. The results showed that PM_2.5 exposure significantly induced apoptosis, DNA strand breaks, and oxidative damage in a dose-dependent manner in 16-HBE cells. The expression of p53 and p73 increased significantly along with the dose of PM_2.5 in 16-HBE cells, whereas the expression of p21^Cip1/WAF1 decreased; the expression of mdm2 increased and then decreased, but not significantly. Taken together, these observations indicate that PM_2.5 may lead to oxidative damage and induce apoptosis through the p53-dependent pathway in 16-HBE cells. p53-dependent apoptosis mediated by DNA strand breaks may be an important mechanism of PM_2.5-induced apoptosis in 16-HBE cells.     

Compound K suppresses ultraviolet radiation-induced apoptosis by inducing DNA repair in human keratinocytes

Ultraviolet (UV)-induced DNA damage is a crucial molecular trigger for sunburn cell formation and skin cancer. Nucleotide excision repair (NER) is the main mechanism in repairing UVB-induced DNA damage of mammalian cells. The purpose of this study is to investigate the functional role of ginsenoside compound K on HaCaT cells (a keratinocyte-derived permanent cell line) irradiated by UV. Hoechst 33258 staining were performed in analyzing UV-induced apoptosis on keratinocytes which were treated with compound K. ImmunoDotBlot assay was used in detecting cyclobutane pyrimidine dimers, the main DNA damage. Western blot analysis was applied for analyzing XPC and ERCC1, two of the NER proteins. Compound K inhibited UV-induced apoptosis of keratinocytes and caused a notable reduction in UV-specific DNA lesions which was due to induction of DNA repair. In agreement with this, compound K induced the expression of particular components of the NER complex, such as XPC and ERCC1. Our results demonstrate that compound K can protect cells from apoptosis induced by UV radiation by inducing DNA repair.     

Hydrogen peroxide-induced oxidative damage and apoptosis in cerebellar granule cells: protection by Ginkgo biloba extract.

The ability of oxidative stress to induce apoptosis and the protective effects of Ginkgo biloba extract (EGb761) against this induction were studied in cultures of rat cerebellar granule cells. Cells were exposed to oxidative stress by treatment with 50 microm hydrogen peroxide+100 microm ferrous sulphate which generates hydroxyl radicals by Fenton reaction. Both morphological observation and biochemical analysis revealed that H(2)O(2)/FeSO(4)treatment induced apoptotic cell death in cerebellar granule cells, which was characterized by chromatin condensation and DNA fragmentation. During this process, the fluidity of the cell membrane decreased markedly, and the conformation of membrane proteins altered significantly. Pretreating cerebellar granule cells with the antioxidant EGb761 (Ginkgo biloba extract) effectively attenuated oxidative damage induced by H(2)O(2)/FeSO(4), and prevented cells from apoptotic cell death. The results suggested that EGb761 might be used as a potential drug for neuronal diseases associated with the excessive production of reactive oxygen species.