外源DMSO和ASA对微囊藻毒素胁迫下烟草细胞ROS生成和抗氧化系统的影响

采用2μg/mL微囊藻毒素-RR(MC-RR)、2μg/mL MC-RR 0.5%二甲基亚砜(DMSO)和2μg/mL MC-RR 2 mmol/L抗坏血酸(ASA)分别处理烟草悬浮细胞,研究上述各处理对烟草悬浮细胞活性氧(ROS)产生和抗氧化系统的影响。结果表明,与对照相比,MC-RR单独处理后烟草悬浮细胞中ROS、膜脂过氧化产物丙二醛(MDA)和细胞内源ASA的含量及超氧化物歧化酶(SOD)和过氧化物酶(POD)的活性明显升高,还原型谷胱甘肽(GSH)的含量有一个先降后升的变化过程。在分别加入外源抗氧化剂DMSO或ASA后,细胞内ROS和MDA含量下降,ASA、GSH含量和SOD、POD酶活性基本可恢复到对照水平。以上结果说明,微囊藻毒素单独处理细胞可造成氧化胁迫,其所诱导的ROS的大量积累很有可能是其产生细胞毒害的关键因子,外源抗氧化剂ASA和DMSO可缓解MC-RR对细胞的毒害作用,对细胞起一定保护作用。     

微囊藻毒素-LR对小鼠肝细胞线粒体功能的影响

目的：探究微囊藻毒素-LR对小鼠肝细胞线粒体功能的影响。方法：采用BALB／c小鼠作为模型动物,随机分为3组：A组,空白对照组,正常饮用水;B组,添加5g门L微囊藻毒素．LR的饮用水;C组,添加30gm微囊藻毒素-LR的饮用水。分组喂养3个月,分离小鼠肝脏、提取线粒体,采用线粒体荧光探针JC-1测定线粒体膜电位（MMP）,qRT-PCR检测自噬相关基因Beclinl和Lc3α的转录水平,WesternBlot检测细胞色素c的释放,电镜观察线粒体的形态和内部结构。结果：微囊藻毒素-LR处理组的小鼠肝细胞线粒体膜电位明显下降,自噬相关基因Lc3α的转录水平上升,细胞色素C由线粒体释放到胞浆,电镜观察线粒体形态异常、内部结构被破坏。结论：微囊藻毒素-LR对小鼠肝细胞线粒体有较强的毒性作用,并引发线粒体自噬。     

鲤肝细胞抗氧化系统对微囊藻毒素毒性的反应

用10μg／L的微囊藻毒素LR(Microcystin—LR,MC—LR)处理鲤肝细胞培养物,检测鲤肝细胞抗氧化系统的6项指标。结果表明,MC—LR处理后活性氧(ROS)含量明显升高,还原型谷胱甘肽(GSH)含量迅速下降,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性明显升高,谷胱甘肽过氧化物酶(GSH—Px)活性在MC—LR处理15min后也有明显上升,但谷胱甘肽S-转移酶(GST)活性在MC—LR处理后没有明显变化。另外,还从氧自由基理论解释了微囊藻毒素造成鲤肝细胞损伤的可能机理。     

一种检测微囊藻毒素LR诱导大鼠肾细胞凋亡的方法

微囊藻毒素(Microcystins)是一种具有生物活性的单环七肽毒素,主要由微囊藻产生。其中5个氨基酸为固定组成成分,另外2种氨基酸是可变的,由此衍生出众多的同类物,微囊藻毒素LR(Microcystin LR,MCLR)是其中具有代表性的一种。同位素示踪显示,静脉注射、腹腔注射和口服3种不同途径进入小鼠体内的125I-MCLR 70%以上分布在肝脏和肾脏,揭示这两个脏器是它的靶器官。本实验中采用正常大鼠的肾细胞来研究MCLR对细胞的影响,利用流式细胞仪PI/Annexin V双染色法检测MCLR能否引起细胞凋亡的改变,为进一步探讨MCLR对细胞的损伤及作用机制提供依据。       

微囊藻毒素-LR 对小鼠肝细胞线粒体功能的影响*

摘要目的：探究微囊藻毒素-LR 对小鼠肝细胞线粒体功能的影响。方法：采用BALB/c 小鼠作为模型动物,随机分为3 组：A 组, 空白对照组,正常饮用水;B 组,添加5 g/L微囊藻毒素-LR 的饮用水;C 组,添加30 g/L 微囊藻毒素-LR 的饮用水。分组喂养3 个月,分离小鼠肝脏、提取线粒体,采用线粒体荧光探针JC-1 测定线粒体膜电位（MMP）,qRT-PCR检测自噬相关基因Beclin1 和 Lc3琢的转录水平,Western Blot检测细胞色素C的释放,电镜观察线粒体的形态和内部结构。结果：微囊藻毒素-LR 处理组的小 鼠肝细胞线粒体膜电位明显下降,自噬相关基因Lc3琢的转录水平上升,细胞色素C由线粒体释放到胞浆,电镜观察线粒体形态 异常、内部结构被破坏。结论：微囊藻毒素-LR 对小鼠肝细胞线粒体有较强的毒性作用,并引发线粒体自噬。     

滇池水华蓝藻铜锈微囊藻和绿色微囊藻的生长生理特性及毒素分析

从滇池分离纯化了两种常见水华微囊藻即铜锈微囊藻和绿色微囊藻。在常规培养条件下,两种藻类在对数生长期的生长速率μ值分别为0.61和0.63;早期生长的抑制光强不大于100μmol m~(-2)s~(-1)。铜锈微囊藻主要产生3种微囊藻毒素:MYCST-RR,MYCST-YR和MYCST-LR,绿色微囊藻产生的主要微囊藻毒素为[Dha~7]-MYCST-RR,和[Dha~7]-MYCST-LR,另含有少量的[Dha~7]-MYCST-YR。在低光强15μE m~(-2)s~(-1)时,毒素含量每毫克干重细胞达到3.127μg微囊藻毒素,当光强达到100μE m~(-2)s~(-1)时,毒素含量降低到每毫克干重细胞1.971μg;光强对毒素形成的影响受到温度的调节,而温度对毒素形成的影响不大。探讨了两种微囊藻细胞在不同光照强度下叶绿素荧光比值Fv/Fm的变化,此比值的变化可以间接反映细胞受外界光照强度抑制程度。     

表没食子儿茶素没食子酸酯对人结肠癌HT-29细胞G1期的阻滞作用

目的:研究表没食子儿茶素没食子酸酯(Epigallaocatechin-3-gallate,EGCG)时人结肠癌HT-29细胞增殖的影响.方法:实验分为EGCG不同浓度处理组和阴性对照组.采用MTT比色法检测EGCG(30μg/mL、40μg/mL、50μg/mL、60μg/mL、70μg/mL)对HT-29细胞的生长影响;应用流式细胞术分析EGCG对HT-29细胞周期分布的影响;免疫印迹观测EGCG对HT-29细胞p38MAPK、cyclinD1蛋白表达的影响.结果:MTT比色结果显示.不同浓度EGCG(30μg/ml、40μg/ml、50μg/ml、60μg/ml)对HT-29细胞具有明显的生长抑制作用,并呈剂量-效应依赖关系(P<0.05);流式细胞术分析显示,EGCG诱导人结肠癌细胞G1期阻滞,且随着处理时间的延长,其诱导周期阻滞的效应越明显(P<0.05);蛋白免疫印迹显示.总的p38MAPK不随处理时间和浓度的改变而改变,但是磷酸化的p38MAPK蛋白的表达随处理时间和处理浓度的增加而明显增加,而CyclinD1蛋白的表达随处理浓度的增加而明显减少.结论:EGCG诱导HT-29细胞G1期阻滞,抑制细胞增殖,可能与活化p38MAPK,下调CyclinD1蛋白表达有关.     

微囊藻细胞抽提液对小鼠血液的亚慢性毒性

本文研究了注射含微囊藻毒素的微囊藻细胞抽提掖对小鼠血液以及免疫系统的亚慢性毒性作用。实验分为3个处理组和1个对照组（每组10只昆明小鼠,雌雄各半）,采用腹腔注射的染毒方法对3个处理组进行暴露,剂量分别为2.4、4.8 和 9.6 μg microcystin-LR/kg body weigh,对照组注射等量的生理盐水,连续注射14d。实验结果表明,14d 染毒后,小鼠的肝体比和脾体比都明显增大（p < 0.05）, 同时在9.6 μg/kg处理组,血清丙氨酸转移酶、天冬氨酸转移酶、乳酸脱氢酶和碱性磷酸酶活性与对照相比明显升高,但血清总蛋白、白蛋白和白蛋白/球蛋白比率下降。这些指标的变化说明,含微囊藻毒素的微囊藻细胞提取液对处理组小鼠肝脏造成了损伤,肝组织学观察也印证了这个结果,在处理组小鼠肝组织有明显的水样变性。另外,9.6 μg/kg处理组小鼠血液白细胞数量比对照组明显减少。组织细胞学观察发现,处理组小鼠脾脏也有明显的损伤。该实验结果说明,含微囊藻毒素的微囊藻细胞抽提液对小鼠的血液和免役系统都产生了一定程度的损伤。     

铜锈微囊藻两种表型的生长生理特性及毒素组成比较分析

从滇池蓝藻水华中分离得到的铜锈微囊藻群体在实验室无机营养中解聚成单细胞,结果表明,群体微囊藻的生长速度明显低于单细胞微囊藻;前者具明显可见的胞外酸性多糖胶鞘,而单细胞则几乎没有;按常规方法分析比较两种细胞形态的毒性大小和毒素组成,发现群体微囊藻主要含有三种微囊藻毒素的异构体,而单细胞以MCLR为主;且单细胞微囊藻的毒性约为群体的10倍.二者的LDH和PGM同工酶酶谱也有差异.本研究为解释毒素的合成和调控机理提供了新的证据.       

锌离子存在下EGCG对前列腺癌细胞生长的影响

本文研究了锌离子存在下EGCG对前列腺癌细胞PC-3生长的影响．研究发现Zn^2＋可以增强EGCG抗癌活性,Zn^2＋存在下。EGCG处理后前列腺癌细胞PC-3克隆形成率显著下降。以RT—PCR、免疫组化方法研究Zn^2＋、EGCG对67kD层粘连蛋白受体（67kD Laminin Receptor,67LR）表达调控,结果表明Znn可通过上调67LR的表达,为EGCG提供更多作用的靶位点,增强EGCG对前列腺癌细胞PC-3的毒性作用。MMP-9是肿瘤侵袭转移过程中关键的基质金属蛋白酶。MMP-9活性与癌细胞的转移潜能密切相关。本文研究发现Zn^2＋、EGCG处理可通过抑制MMP-9活性,降低前列腺癌细胞PC-3的迁移率．其中80umol／LEGCG＋80umol／L Zn^2＋处理24h后显著抑制了PC-3细胞的迁移率。     

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.     

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.     

Feselol enhances the cytotoxicity and DNA damage induced by cisplatin in 5637 cells

Transitional cell carcinoma (TCC), which is the most common type of bladder cancer, shows resistance to chemotherapeutic agents due to the overexpression of drug efflux pumps. In this study, the effects of feselol, a sesquiterpene coumarin extracted from Ferula badrakema, on cisplatin cytotoxicity were investigated in 5637 cells, a TCC subline. Cell viability and DNA lesion were evaluated by thiazolyl blue tetrazolium bromide and comet assays, respectively. Feselol had no significant cytotoxic effect in 5637 cells but at 32 microg/mL it increased the cytotoxicity of 1 microg/mL cisplatin by 37% after 24 h. Furthermore, the comet assay revealed that DNA damage induced by cisplatin in 5637 cells is enhanced by 31% when used in combination with feselol. Therefore, feselol might be considered as an effective reversal agent for future in vivo and clinical studies.     

肝细胞生长因子对氧糖剥夺/再灌注神经元的保护作用

本文旨在探讨肝细胞生长因子（hepatocyte growth factor,HGF）对神经元氧糖剥夺/再灌注损伤的影响。取原代培养12d的Sprague-Dawley大鼠大脑皮层神经元,无糖、无氧（95%N2＋5%CO2）孵育2h后,换含25mmol/L葡萄糖的培养液、常氧培养0-24h,以MTT比色法检测细胞活力、乳酸脱氢酶（lactate dehydrogenase,LDH）漏出率作为细胞损伤指标,建立体外氧糖剥夺/再灌注损伤细胞模型;用流式细胞仪和Hoechst33258染色分析细胞凋亡率;用RT-PCR和Western blot分别检测大鼠脑皮层神经元HGF受体c-Met mRNA和蛋白的表达。于氧糖剥夺2h/再灌注24h处理前2h,加入不同终浓度（5-120ng/mL）的HGF,观察HGF对皮层神经元的影响。结果显示,c-Met表达于皮层神经元,氧糖剥夺2h/再灌注24h后,c-Met mRNA和蛋白表达均显著上调,神经元细胞活力明显降低,LDH漏出率和细胞凋亡率显著增高。HGF预处理明显促进氧糖剥夺/再灌注损伤神经元的存活,降低LDH漏出率,最大效应剂量为80ng/mL。流式细胞术和Hoechst33258染色结果均显示,HGF（80ng/mL）显著降低氧糖剥夺/再灌注神经元的细胞凋亡率。此外,c-Met抑制剂SU11274（5μmol/L）完全阻断HGF的神经保护作用。结果表明,HGF对皮层神经元氧糖剥夺/再灌注损伤具有直接的保护作用,呈一定的剂量依赖关系,并能有效对抗神经元凋亡。     

Influence of Microcystin-LR on the activity of membrane enzymes in rat intestinal mucosa

The objective of the present study was to evaluate the effects of microcystin-LR (MCLR) on the activity of membrane enzymes from intestinal mucosa. In addition, serum chemistry and peroxidative status of both serum and intestinal homogenate were evaluated after treatment with MCLR. Wistar rats were treated with intraperitoneal injection of either 100 microg pure MCLR/Kg body weight or saline solution. A significant increase in liver weight and altered serum enzyme activities were found in MCLR-treated rats, indicating damage to the liver in these rats, as previously suggested. A higher specific activity of sucrase (1.5-fold) was observed after the administration of MCLR, whereas other intestinal apical membrane enzymes, such as lactase, maltase and alkaline phosphatase were not modified by the treatment. The specific activities of acid phosphatase and succinate dehydrogenase, markers for lysosomal and mitochondrial membranes, respectively, were also increased (32% and 60%, respectively) in treated rats. The analysis of lipid peroxidation showed that the peroxidative status was increased in both serum and intestinal mucosa from MCLR-treated rats, reflecting an excess production of oxygen free radicals induced by this cyanobacterial toxin. In conclusion, this study shows that acute exposure to MCLR affects the intestinal physiology by modifying the intestinal peroxidation status as well as the activity of membrane enzymes.     

AMPK-mTOR-ULK1 axis activation-dependent autophagy promotes hydroxycamptothecin-induced apoptosis in human bladder cancer cells

10-hydroxycamptothecin (HCPT), a natural plant extract, exerts anticancer capacity. HCPT has been reported to induce apoptosis and autophagy in human cancer cells. The interaction between autophagy and apoptosis induced by HCPT and the molecular mechanism in bladder cancer cells were investigated in this study. Our results confirmed that HCPT suppressed cell viability and migration and caused cell-cycle arrest in T24 and 5637. Then, we used Z-VAD(OMe)-FMK to clarify that apoptosis induced by HCPT was mediated by caspase. Moreover, HCPT boosted autophagy through activating the AMPK/mTOR/ULK1 pathway. Blocking autophagy by 3-methyladenine, the adenosine monophosphate-activated protein kinase (AMPK) inhibitor dorsomorphin and siATG7 reversed HCPT-induced cytotoxicity. Conversely, rapamycin and the AMPK activator AICAR enhanced growth inhibition and cell apoptosis, suggesting that autophagy played a proapoptosis role. Taken together, our findings showed that HCPT-induced autophagy mediated by the AMPK pathway in T24 and 5637 cell lines, which reinforced the apoptosis, indicating that HCPT together with autophagy activator would be a novel strategy for clinical treatment in bladder cancer.     

A rapid competitive binding nonseparation electrochemical enzyme immunoassay (NEEIA) test strip for microcystin-LR (MCLR) determination

A competitive binding nonseparation electrochemical enzyme immunoassay (NEEIA) is described for the determination of microcystin-LR (MCLR) using a double-sided microporous gold electrode in cartridge-type cells. A gold film sputtered on one side of porous nylon membrane constitutes a working electrode, while another gold film formed on the opposite side serves as a pseudo reference electrode. After immobilizing MCLR antibody on working electrode by physical adsorption, the double-sided electrode was placed simply in a diffusion U-type or within a dry strip-type cell with a conjugate pad pre-loaded with a glucose oxidase labeled MCLR (GOx-MCLR) on working electrode side. Assays were performed in two steps: an MCLR-containing sample mixed with a known amount of GOx-MCLR conjugate either in buffer solution or in pre-loaded dry pad was incubated for an appropriate period (about 10 min) to induce competitive reaction with an immobilized anti-MCLR antibody on working electrode, and a fixed concentration of glucose solution (substrate) was then added to the backside of the working electrode. Due to the competitive nature of the assay, enzymatically generated product, hydrogen peroxide (H2O2), was detected at the working gold electrode (at +800 mV versus Au) by oxidation, and the magnitude of amperometric current was inversely proportional to the concentration of MCLR in the sample. The response time after substrate addition was about 30s. Mean recovery of MCLR added to tap water was 93.5%, with a coefficient of variation (CV) of 6.6%. The proposed competitive NEEIA system is in general comparable to existing heterogeneous enzyme immunoassays with a similar detection limit (100 pg/mL MCLR), and suitable for developing a disposable type biosensor for on-site monitoring of environment.     

Effect of Salvia miltiorrhiza on aflatoxin B1-induced oxidative stress in cultured rat hepatocytes

Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B₁ (AFB₁). Salvia miltiorrhiza (Sm), a herbal plant that has been used extensively in traditional Chinese medicine for treating cardiovascular and liver diseases, is believed to have some antioxidative capabilities. In this study, the protective effect of Sm against AFB₁-induced cytotoxicity was investigated in cultured primary rat hepatocytes. AFB₁-induced cytotoxicity and lipid peroxidation (LPO) were estimated by determination of lactate dehydrogenase (LDH) leakage and thiobarbituric acid reactive substances (TBARS) formation, respectively. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). In addition, changes of intracellular glutathione (GSH) content were also studied. Results showed that Sm was able to suppress the LDH leakage induced by AFB₁ in a dose-dependent manner. A dose-dependent inhibitory effect of Sm on AFB₁-induced LPO was also found in hepatocytes treated with Sm. It was further observed that Sm produced an inhibitory effect on ROS formation caused by AFB₁. Concomitantly, the GSH content in Sm-treated groups increased substantially compared to those without Sm treatment. These findings suggest that Sm can inhibit the cytotoxicity of AFB₁ through decreasing ROS formation, inhibiting LPO and preventing GSH depletion. The major component of the aqueous extract of Sm was identified by using high performance liquid chromatography (HPLC), proton magnetic resonance (¹H-NMR) and mass spectrum (MS). Analytical results suggested that D(+)β3,4-dihydroxyphenol lactic acid (DA) is the main compound of the aqueous extract of Sm.