微囊藻毒素缓解过氧化氢胁迫下铜绿微囊藻损伤的初步研究

过氧化氢可抑制藻类生长, 同时会导致微囊藻毒素(Microcystins, MCs)的释放, 实验设置4个处理组探讨了外源微囊藻毒素MC-LR对H2O2胁迫下铜绿微囊藻生理生化变化的影响。结果表明: 在H2O2胁迫下, 微囊藻的生长和光合活性受到显著抑制, 藻细胞存活率降低, ROS含量明显增加, SOD活性上升。与单独H2O2胁迫相比, 加入MC-LR能增加微囊藻细胞的存活率。250 mol/L H2O2处理24h和48h后, 在培养基中加入200 ng/mL MC-LR可以缓解H2O2对铜绿微囊藻光合系统PSII活性的抑制作用。当微囊藻暴露于250 mol/L H2O2环境中时, 添加了MC-LR处理组藻细胞中的ROS含量明显减少(P0.05)。在相同浓度H2O2且加入了外源MC-LR后藻细胞SOD活性下降(P0.05)。因此, 微囊藻毒素MC-LR可缓解250 mol/L H2O2引起的氧化损伤并增强微囊藻自身的生存能力。研究结果有利于阐明H2O2胁迫影响产毒蓝藻生长代谢的途径及MCs生物学意义。       

微囊藻毒素-LR对小鼠原代肝细胞线粒体功能的影响

目的 蓝藻水华引起的微囊藻毒素污染是世界性关注话题之一,微囊藻毒素LR(MC-LR)具有强特异性肝毒性,但其引起肝损伤的确切机制尚未完全阐明.为解决这一问题,本研究从细胞分子层面探讨MC-LR造成肝细胞线粒体功能改变的分子机制.方法 提取小鼠原代肝细胞,加入梯度剂量的MC-LR(2.5～10 nmol/L)作用48 h...     

微囊藻毒素的毒性效应与蛋白磷酸酶2A

蛋白磷酸酶2A(protein phosphatase 2A,PP2A)是蛋白磷酸酶家族的主要成员,在蛋白质可逆磷酸化过程中与蛋白激酶一样起着举足轻重的作用。自然界存在很多天然毒素可特异性地作用于PP2A从而影响体内蛋白质的可逆磷酸化,其中微囊藻毒素由于急性肝毒性和强促癌活性日益引起关注。尽管确切的机制仍未探明,但从目前的研究来看,微囊藻毒素产生毒性的机制可能与其引起细胞氧化应激、DNA损伤、细胞骨架的破坏以及诱导细胞凋亡相关。而PP2A在氧化应激、DNA损伤修复及维持细胞骨架稳态中起着重要作用,并能调控凋亡相关激酶CaMKII和Bcl-2家族蛋白,这对更好地理解微囊藻毒素LR如何通过影响PP2A而产生毒作用提供了新思路。     

牛磺酸对HepG2细胞甘油三酯合成的影响研究

本文探讨牛磺酸对HepG2细胞甘油三酯合成的影响,为牛磺酸预防/改善机体高脂状态的深入研究提供参考。在DMEM培养基中添加0.05 mmol/L油酸建立高甘油三酯细胞模型,分别以终浓度为1、5、10、20 mmol/L的牛磺酸处理细胞24、48、72 h,测定细胞内甘油三酯水平;并检测5 mmol/L牛磺酸作用24 h后细胞内固醇调节元件结合蛋白1c(SREBP-1c)及脂肪合成相关酶乙酰辅酶A合成酶(AceCS)、乙酰辅酶A羧化酶(ACC)、脂肪酸合成酶(FAS)、长链酰基辅酶A合成酶1(ACSL1)的蛋白表达水平。1 mmol/L牛磺酸作用72 h,5和10 mmol/L牛磺酸作用24、48、72 h,20 mmol/L牛磺酸作用24和48 h均可使高脂HepG2细胞内甘油三酯水平显著下降(P<0.05);5 mmol/L牛磺酸作用24 h,高脂HepG2细胞的SREBP-1c、FAS、ACC、AceCS1、ACSL1表达明显减少(P<0.05),磷酸化ACC表达显著增加(P<0.05)。结论:牛磺酸通过调控SREBP-1c及其下游靶基因而抑制高脂HepG2细胞脂...     

和厚朴酚对人肺癌A2细胞增殖和凋亡的影响

本研究的目的在于研究不同浓度和厚朴酚对人肺癌A2细胞增殖和凋亡的影响,分析凋亡的可能机制。通过台盼蓝拒染法检测和厚朴酚对细胞增殖的影响,荧光显微镜下观察细胞形态的变化,流式细胞术检测细胞凋亡率和细胞周期的变化,qRT-PCR和Western blotting分别检测Bax、Bcl-2基因mRNA和蛋白表达情况。结果表明,在一定范围内,和厚朴酚对人肺癌A2细胞增殖有抑制作用且呈时间和剂量依赖性,其作用人肺癌A2细胞24 h、48 h和72 h的IC50值分别为44.03 nmol/L、26.51 nmol/L和19.54 nmol/L。不同浓度的和厚朴酚作用人肺癌A2细胞48 h后,细胞出现明显的凋亡特征,早期凋亡细胞增多,且G1期和G2期细胞减少,S期细胞增多。与对照组相比,Bax mRNA和蛋白表达均显著升高(p0.05),Bcl-2mRNA和蛋白表达均显著降低(p0.05)。本研究表明一定浓度范围的和厚朴酚能抑制人肺癌A2细胞增殖,诱导细胞凋亡且呈时间和剂量依赖性,并可上调Bax基因表达,下调Bc L-2基因表达。     

蛋白磷酸酶2A的结构、功能和活性调节

蛋白磷酸酶 2A(proteinphosphatase 2A ,PP2A)是主要的丝 /苏氨酸蛋白磷酸酶 ,拥有众多不同基因编码的亚基 ,分别组成多种不同的PP2A全酶 ,参与细胞周期、DNA复制、信号转导、细胞分化和细胞恶性转化等多种细胞生物学事件 ,并和神经退行性疾病、肿瘤等多种疾病的发生、发展有关。PP2A调节亚基的组织特异性表达和细胞内定位 ,催化亚基羧基末端的磷酸化和甲基化 ,第二信使神经酰胺 (ceramide)、天然小分子抑制剂等都能够调节PP2A的活性。     

微囊藻毒素对典型微生物生长及生理生化特性的影响

研究了不同浓度微囊藻毒素对典型微生物大肠杆菌和枯草芽孢杆菌生长及生理生化特性的影响。微囊藻毒素对大肠杆菌和枯草芽孢杆菌的生长和细胞活性具有一定的剂量效应,较高浓度微囊藻毒素对其生长和活性有短时间的抑制作用,随着处理时间的延长,细胞的生长和活性逐渐恢复。细胞内可溶性糖和可溶性蛋白的含量,处理组和对照组相比均有先上升后下降的趋势。结果表明,微囊藻毒素的处理对大肠杆菌和枯草芽孢杆菌具有一定的胁迫作用,细胞通过调节细胞内可溶性蛋白和可溶性糖的含量来抵抗外界胁迫,但随着处理时间的延长,细菌逐渐适应了这种胁迫,恢复正常的生长。     

大田软海绵酸对FL细胞DNA的损伤及凋亡相关蛋白表达的影响

本文旨在探讨大田软海绵酸对人羊膜细胞DNA的损伤及凋亡相关蛋白表达的影响。实验用0、20、40、608、0、100 nmol/L OA诱导FL细胞4h后,检测DNA损伤程度的彗星实验表明,OA对FL细胞DNA的损伤随染毒浓度的升高而增加。蛋白免疫印迹法显示凋亡相关蛋白Bcl-2、Bax和p53的表达与染毒浓度呈负相关;用100 nmol/L OA分别诱导2h、4h、8h后发现,三种蛋白的表达与染毒时间也呈负相关。由此可知在OA诱导的FL细胞凋亡中,损伤DNA,降低Bcl-2蛋白的表达可能参与了凋亡的部分作用,而Bax和p53蛋白则可能与OA诱导的细胞增殖有关。     

高铁酸钾/PAC 氧化-混凝去除水体中铜绿微囊藻

铜绿微囊藻（Microcystis aeruginosa）是常见的水华种类。水华爆发时生态系统的结构和功能遭到破坏,同时产生毒素,因此研究微囊藻及其毒素的去除具有重要的意义。研究不同浓度的高铁酸钾与PAC 高铁酸钾与PAC 单独和协调去除水体中铜绿微囊藻和微囊藻毒素（Microcystin-LR,MC-LR）的去除效果。结果表明,采用高铁酸盐预氧化铜绿微囊藻,再通过PAC混凝协同作用,明显优于单独使用PAC混凝效果。20 mg×L-1高铁酸钾处理后水体中MC-LR含量降低,当高铁酸钾浓度升高至40 mg×L-1时候MC-LR 含量增加,但高铁酸钾浓度升高（100 mg×L-1）水体中MC-LR 减少;当8 mg×L-1高铁酸钾和8-16 mg×L-1PAC 联合作用时,不仅絮凝沉降藻细胞,而且高铁酸钾氧化水体中MC-LR,通过镜检发现不会造成藻细胞破裂。     

Ser/Thr蛋白磷酸酶PPP家族结构研究新进展

PP1, PP2A以及PP2B同属于丝/苏氨酸蛋白磷酸酶的PPP家族, 在体内参与调节多种重要的生理功能, 包括细胞周期、细胞生长及分化等过程. 它们之间的同源性相对较高, 空间结构更为保守. PP1和PP2B的结构解析研究进展较快, 但是由于PP2A结构的复杂性以及调节亚基的多样性而相对进展较慢. 最近发现PP2A是由结构亚基、催化亚基先组成二聚体的核心酶结构然后再与调节亚基组成三聚体全酶. 主要对丝/苏氨酸蛋白磷酸酶的亚基组成和结构异同等方面进行综述.     

Microcystin-LR Induced Reactive Oxygen Species Mediate Cytoskeletal Disruption and Apoptosis of Hepatocytes in Cyprinus carpio L.

Microcystins (MCs) are a group of cyclic hepatotoxic peptides produced by cyanobacteria. Microcystin-LR (MC-LR) contains Leucine (L) and Arginine (R) in the variable positions, and is one of the most common and potently toxic peptides. MC-LR can inhibit protein phosphatase type 1 and type 2A (PP1 and PP2A) activities and induce excessive production of reactive oxygen species (ROS). The underlying mechanism of the inhibition of PP1 and PP2A has been extensively studied. The over-production of ROS is considered to be another main mechanism behind MC-LR toxicity; however, the detailed toxicological mechanism involved in over-production of ROS in carp (Cyprinus carpio L.) remains largely unclear. In our present study, the hydroxyl radical (•OH) was significantly induced in the liver of carp after a relatively short-term exposure to MC-LR. The elevated reactive oxygen species (ROS) production may play an important role in the disruption of microtubule structure. Pre-injection of the antioxidant N-acetyl-cysteine (NAC) provided significant protection to the cytoskeleton, however buthionine sulfoximine (BSO) exacerbated cytoskeletal destruction. In addition, the elevated ROS formation induced the expression of apoptosis-related genes, including p38, JNKa, and bcl-2. A significant increase in apoptotic cells was observed at 12 - 48 hours. Our study further supports evidence that ROS are involved in MC-LR induced damage to liver cells in carp, and indicates the need for further study of the molecular mechanisms behind MC-LR toxicity.     

Subchronic microcystin-LR exposure increased hepatic apoptosis and induced compensatory mechanisms in mice

Acute lethal cytotoxicity of microcystin-LR (MC-LR), a toxin produced by fresh-water cyanobacteria, has been attributed to protein phosphatases type 1 and type 2A (PP1/PP2A) inhibition and reactive oxygen species (ROS) generation. However, the effects and molecular mechanisms of prolonged, sublethal MC-LR exposure are less known. We studied mice intraperitonealy injected with saline or 25 μg MC-LR/kg for 28 days (every 2 days). MC-LR induced apoptosis in liver and not in kidneys or heart of treated animals. Liver also showed decreased α-tubulin levels (45.56% ± 7.65% of controls) and activation of p38-MAPK and CaMKII pathways (137.93% ± 11.64% and 419.35% ± 67.83% of the control group, respectively). PP1/PP2A activity decreased from 1.82 ± 0.23 (controls) to 0.91 ± 0.98 mU/mg (MC-LR-treated mice); however, no difference in total Ser/Thr phosphatase activity was found between both the groups. The results demonstrated that apoptosis and cytoskeleton disruption contributed to the hepatic cytotoxic effects of subchronic MC-LR administration. These effects occurred in association with sustained activation of signaling cascades and development of compensatory mechanisms to maintain total Ser/Thr phosphatase activity.     

Bioaccumulation, oxidative stress and HSP70 expression in Cyprinus carpio L. exposed to microcystin-LR under laboratory conditions

Microcystin-LR (MC-LR) produced by cyanobacteria are potent specific hepatotoxins. So far the pathogenesis of environmental MC-LR toxicity to aquatic organisms has not been fully elucidated. In the present study the accumulation of MC-LR was investigated in various organs/tissues of Cyprinus carpio L. (C. carpio) following exposure to MC-LR for 14 d at environmentally relevant concentrations (0.1 to 10 μg L(-1)). Results showed that the presence of MC-LR enhanced toxin accumulation in all investigated organs and the highest accumulation was found in the liver of fish exposed to 5.0 μg L(-1) of MC-LR. An EPR analysis indicated ·OH intensity in liver was significantly induced at 0.1 μg L(-1) of MC-LR and then restored when the MC-LR concentration was greater than 0.1 μg L(-1). After 14-day exposure, MC-LR (1.0-10.0 μg L(-1) of MC-LR) caused a pronounced promotion of glutathione S-transferase (GST) activity and a depletion of reduced glutathione (GSH) content in fish liver, which indicated that GSH was involved in detoxification of MC-LR and the conjugation reaction of MC-LR and GSH occurred. A mild oxidative damage was evidenced by the accumulation of malondialdehyde (MDA) level at 5.0 μg L(-1) of MC-LR exposure, but which was restored when the MC-LR concentration was increased to 10.0 μg L(-1). The responses of antioxidant enzymes and the induction of HSP70 expression might contribute to MC-LR tolerance of C. carpio. However, the protein phosphatase (PP) activities were strikingly inhibited in all treated groups. Thus, the overall toxicity of environmental MC-LR on C. carpio seems to be initiated in the liver via both the ROS pathway and the PP inhibition pathway, and the latter might be more important when ambient MC-LR concentration is greater than 0.1 μg L(-1). More importantly, these results can help to support the evaluation on the potential effects of MC-LR under common environmental concentrations.     

Identification of ATP-synthase as a novel intracellular target for microcystin-LR

Microcystins (MCs) are a group of closely related cyclic heptapeptides produced by a variety of common cyanobacteria. These are potent and highly specific hepatotoxins, the toxicity of which is based upon their inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases. Apart from protein phosphatases, it is not known whether these phosphatase-inhibiting peptides could bind any other cellular proteins. We wanted to determine whether any possible unknown MC-adducts could explain the apoptotic effects observed at high concentrations of MCs. The question of other possible cellular proteins binding to MCs is also relevant when these compounds are employed for affinity purification of protein phosphatases. In MC-treated cell lysates, antibodies to MC recognized three protein adducts of 35-37 and 55 kD. By immunochemical and proteomics approaches, these proteins were identified as the catalytic subunits of type-1 and type-2A protein phosphatases and the ATP-synthase beta-subunit. The latter target could be associated with the suggested apoptosis-inducing potential of MCs.     

流感病毒感染巨噬细胞对缺氧诱导因子-1α诱导炎症反应通路的机制研究

为探索缺氧诱导因子(hypoxia inducible factor,HIF)-1α诱导甲型流感病毒毒株感染小鼠巨噬细胞引起炎症反应的具体机制,本研究以甲型H1N1流感病毒(简称H1N1)株A/PR/8感染小鼠巨噬细胞RAW264.7后,在显微镜下观察其在感染后的表型变化,分别在不同时间段收集样本,通过聚合酶链反应(polymerase chain reaction,PCR)检测HIF-1α、干扰素(interferon,IFN)-γ、白细胞介素(interleukin,IL)-6、肿瘤坏死因子(tumor necrosis factor,TNF)-α和M蛋白(M protein,MBP)mRNA的变化,通过蛋白质印迹法(Western blot, WB)检测HIF-1α、核转录因子-κB(nuclear factor-κB,NF-κB)、促分裂原活化的蛋白激酶(mitogenactivated protein kinase,MAPK)、蛋白激酶B(protein kinase B,Akt)以及M蛋白的变化。随后,加入抑制剂2-MeOE-2(10 nmol/L)进行抑制试验,采用PCR和WB检测HIF-1α表达被抑制后上述炎症因子mRNA表达水平及炎症蛋白通路的变化。结果显示,H1N1PR8感染小鼠巨噬细胞RAW264.7后,H1N1PR8复制率在24 h达到峰值,HIF-1α mRNA在感染6 h后开始升高,12 h迅速上升,24 h达到峰值。IFN-γ、IL-6、TNF-α mRNA变化趋势基本与HIF-1α一致,但在感染12 h并未进入快速上升期。HIF-1α蛋白在感染后6 h表达明显增多,24 h达到峰值,与mRNA变化水平基本一致。NF-κB通路蛋白在感染12 h后明显增多,48 h开始减少。加入抑制剂2-MeOE-2后,培养感染细胞24 h,抑制剂组IL-6、TNF-α mRNA水平较对照组显著下降(P<0.05),抑制剂组NF-κB通路蛋白较对照组表达下降。本研究结果表明,小鼠巨噬细胞被H1N1感染后,HIF-1α可能通过激活NF-κB通路促进IL-6、TNF-α等炎症因子的分泌参与炎症反应。     

AngⅡ和PKC对心肌细胞AngⅡ 1型受体的转录调节

利用体外培养的心肌细胞,观察血管紧张素Ⅱ（ＡｎｇⅡ）和蛋白激酶Ｃ（ＰＫＣ）在诱导ＡｎｇⅡ１型受体（ＡＴ１）基因表达及蛋白质代谢中的作用．研究结果表明：ＡｎｇⅡ可诱导ＡＴ１ｍＲＮＡ水平一过性下调,呈时间及剂量依赖性,１０ｎｍｏｌ／ＬＡｎｇⅡ刺激细胞６ｈ,引起ＡＴ１ｍＲＮＡ水平降低幅度最大,降至对照的５１．６％±９．５％,然后逐渐回升,２４ｈ恢复至对照水平．３０μｍｏｌ／ＬＨ－７（ＰＫＣ抑制剂）能阻断ＡｎｇⅡ诱导的ＡＴ１ｍＲＮＡ水平的下调．０．３μｍｏｌ／Ｌ的ＰＭＡ（ＰＫＣ激活剂）单独应用可诱导ＡＴ１ｍＲＮＡ水平下调达对照的４３％±８％,加入ＡＴ１拮抗剂ＤＭＰ８１１及Ｄｕｐ７５３均可阻断ＡｎｇⅡ诱导的ＡＴ１ｍＲＮＡ水平的下调．１０ｎｍｏｌ／Ｌ的ＡｎｇⅡ刺激心肌细胞９６ｈ可使蛋白含量降低至对照的７３．４％±５．６％,而加药持续刺激１４４ｈ可使蛋白含量较对照增加３３．８％±６．３％,Ｈ－７不能阻断ＡｎｇⅡ诱导的蛋白含量降低,但可有效地抑制蛋白含量的增加．以上结果提示：ＡｎｇⅡ对心肌细胞ＡＴ１基因的转录和细胞的蛋白代谢有调节作用,而ＰＫＣ则参与了ＡｎｇⅡ的这种调节作用