铬诱导鸡胚成纤维细胞凋亡作用研究

为探讨铬对鸡胚成纤维细胞的凋亡作用,以鸡胚成纤维细胞为研究材料,应用酶标仪、倒置相差显微镜、激光共聚焦显微镜及流式细胞仪,分析细胞毒性、细胞形态、凋亡率、细胞周期、线粒体膜电位、胞内钙离子稳态及活性氧水平。结果显示:0.5、1.0和2.0μmol/L CrCl_3促使细胞活性下降、呈现凋亡形态学变化,细胞周期阻滞于G1期,细胞凋亡率呈时间-剂量依赖性,细胞线粒体膜电位下降,胞内游离钙离子浓度增大,胞内活性氧增多。铬可通过影响细胞周期进程,降低线粒体膜电位,干扰胞内钙离子稳态,氧化损伤来诱导鸡胚成纤维细胞凋亡。     

哺乳动物精子线粒体研究进展

哺乳动物精子线粒体是维持精子活力的关键细胞器，对精子超激活运动、获能、顶体反应及受精等过程起到重要的调节作用。哺乳动物精子线粒体特有的形态特征与特异性酶异构体使其具有独特动力学和调节特性。精子线粒体中发生的氧化磷酸化过程是维持精子运动的重要途径，该过程产生的活性氧对精子功能的维持具有重要作用，但过量可能导致精子损伤，加速精子凋亡。哺乳动物精子质膜磷脂酰丝氨酸外翻和相关半胱氨酸蛋白酶激活级联反应引起细胞凋亡。区别于体细胞线粒体，精子线粒体钙信号可能并未参与精子固有的凋亡途径，作为衡量线粒体功能的敏感指标，其对线粒体膜电位和耗氧量的检测研究至关重要。哺乳动物精子线粒体具有自身的遗传系统，线粒体基因拷贝数可能作为无创衡量精子质量和受精能力的标记。作者重点阐述了哺乳动物精子线粒体的结构、线粒体鞘的形成及其生物功能，包括发生在线粒体中的氧化磷酸化过程、活性氧对精子的利与弊、线粒体参与钙稳态与细胞凋亡过程；介绍了线粒体膜电位和耗氧量的检测，简述了线粒体基因组的研究进展，为进一步探讨线粒体所涉及的精子功能机制奠定基础。     

MCU在低氧诱导肉鸡心肌细胞线粒体损伤中的作用

旨在探讨线粒体钙单向转运体（mitochondrial calcium uniporter,MCU）介导线粒体Ca²⁺转运是否参与低氧诱导的肉鸡心肌细胞线粒体损伤。试验通过分离白羽肉鸡鸡胚原代心肌细胞,在低氧条件下（3% O₂,5% CO₂,92% N₂）培养24、48、72 h,同时使用RU360抑制MCU的表达并在低氧条件下处理72 h后,使用流式细胞术检测细胞内Ca²⁺浓度、线粒体Ca²⁺浓度、线粒体活性氧水平和线粒体膜电位,检测MCU及其调节因子的mRNA和蛋白表达。结果显示,通过差速贴壁方法培养的心肌细胞纯度可达90%以上;低氧培养24 h,诱导心肌细胞MCU、MICU1 mRNA表达增加（P<0.05）,胞浆和线粒体内Ca²⁺浓度显著上升（P<0.05）,线粒体膜电位增加（P<0.05）;低氧培养48 h,诱导MCUR1和MICU1 mRNA表达减少（P<0.05）,细胞Ca²⁺浓度上升（P<0.05）;低氧培养72 h,诱导MCU mRNA表达增加（P<0.01）,细胞和线粒体内Ca²⁺增加（P<0.01）,线粒体膜电位下降（P<0.01）,活性氧增加（P<0.01）。低氧处理72 h后,与低氧组相比,RU360预处理组细胞和线粒体内Ca²⁺减少,线粒体膜电位上升,活性氧生成减少（P<0.01）,MCU mRNA表达减少（P<0.01）。结果表明：低氧诱导MCU上调导致线粒体钙超载,促使线粒体功能降低并发生损伤,进而心肌细胞发生损伤;抑制MCU表达可以减轻低氧诱导的心肌细胞线粒体钙超载,保护线粒体。     

哺乳动物精子线粒体研究进展

哺乳动物精子线粒体是维持精子活力的关键细胞器,对精子超激活运动、获能、顶体反应及受精等过程起到重要的调节作用。哺乳动物精子线粒体特有的形态特征与特异性酶异构体使其具有独特动力学和调节特性。精子线粒体中发生的氧化磷酸化过程是维持精子运动的重要途径,该过程产生的活性氧对精子功能的维持具有重要作用,但过量可能导致精子损伤,加速精子凋亡。哺乳动物精子质膜磷脂酰丝氨酸外翻和相关半胱氨酸蛋白酶激活级联反应引起细胞凋亡。区别于体细胞线粒体,精子线粒体钙信号可能并未参与精子固有的凋亡途径,作为衡量线粒体功能的敏感指标,其对线粒体膜电位和耗氧量的检测研究至关重要。哺乳动物精子线粒体具有自身的遗传系统,线粒体基因拷贝数可能作为无创衡量精子质量和受精能力的标记。作者重点阐述了哺乳动物精子线粒体的结构、线粒体鞘的形成及其生物功能,包括发生在线粒体中的氧化磷酸化过程、活性氧对精子的利与弊、线粒体参与钙稳态与细胞凋亡过程;介绍了线粒体膜电位和耗氧量的检测,简述了线粒体基因组的研究进展,为进一步探讨线粒体所涉及的精子功能机制奠定基础。     

DSS诱导血管内皮细胞ECV304凋亡及机制研究

为了研究葡聚糖硫酸钠(DSS)诱导血管内皮细胞:ECV304增殖和凋亡的影响。体外培养血管内皮细胞ECV304,采用不同浓度DSS诱导细胞,流式细胞术分析细胞凋亡、活性氧(ROS)、线粒体膜电位(MMP)、细胞周期;WST-1法检测细胞增殖抑制率。结果显示,随着DSS浓度的增加细胞凋亡率增加,并导致细胞GOG1期阻滞,细胞内活性氧含量减少,同时线粒体膜电位下降,细胞增殖抑制率明显升高。结果表明,DSS可以抑制血管内皮细胞ECV304增殖并诱导细胞凋亡。     

两种活性羰基类物质对 ECV304细胞的毒性研究

为探讨乙二醛（GO）和甲基乙二醛（MGO）对上皮细胞的细胞毒性及其机制,分别以 MTT 法检测两种活性羰基化合物（RCS）对 ECV304细胞活力的影响,评价其细胞毒性;DCFH-DA 探针法检测氧化应激;用罗丹明123检测线粒体膜电位及线粒体形态变化。结果表明,GO 和 MGO 对 ECV304细胞的 IC50分别为3．6 mmol／L±0．1 mmol／L 和1．3 mmol／L±0．1 mmol／L;氧化应激水平小幅度增高,线粒体膜电位随发生改变;线粒体形态发生改变。GO 和 MGO 的细胞毒性与氧化应激水平增加相关性较低而与线粒体损伤相关。     

T-2毒素对TM3细胞线粒体功能的影响

探讨T-2毒素对TM3细胞线粒体功能的影响。将不同浓度T-2毒素(0 nM、1 nM、10 nM、100 nM)作用于TM3细胞24 h,通过MTT法检测T-2毒素对TM3细胞增殖的影响;生化方法测定细胞内Ca~(2+)、活性氧、线粒体超氧化物、ATP的含量、Ca~(2+)-Mg~(2+)-ATPase和Na~+-K~+-ATPase的活性以及线粒体膜电位的变化。结果显示,T-2毒素对TM3细胞具有毒性作用,可引起细胞内Ca~(2+)含量增加,使细胞内活性氧和线粒体超氧化物的含量增加,降低线粒体膜电位、ATP的含量以及ATP依赖的Ca~(2+)-Mg~(2+)-ATPase和Na~+-K~+-ATPase的活性,进而影响线粒体功能的发挥。     

线粒体自噬调节NLRP3炎症小体活性改善动物健康的作用机制

线粒体在氧化磷酸化过程中会产生少量的活性氧(ROS),而当线粒体应激时,线粒体去极化、ROS产生增加造成线粒体损伤,导致线粒体活性氧(mtROS)的表达水平增高;ROS作为激活NLRP3的重要介质,促进NLRP3炎症小体的组装及激活,引发动物机体如乳腺炎、子宫内膜炎和神经退行性疾病等多种疾病。作为一种选择性自噬,线粒体自噬发挥其生物学功能的机制是通过清除受损和多余线粒体,调节NLRP3炎症小体的活性,维持细胞正常生理功能。本文基于线粒体自噬调节NLRP3炎症小体对动物机体健康的影响进行综述,以期深入了解线粒体自噬在NLRP3炎症小体引发的相关疾病中的影响,为动物机体健康提供新的防治靶点。     

镉对体外培养大鼠肾小管上皮细胞的毒性损伤及乙酰半胱氨酸的保护效应

在建立肾小管上皮细胞体外培养模型的基础上,通过在培养液中添加不同浓度的醋酸镉和乙酰半胱氨酸(NAC),用CCK-8法测定细胞存活率,流式细胞仪检测细胞凋亡、细胞内活性氧与线粒体膜电位,探讨了镉对体外培养大鼠肾小管上皮细胞的毒性损伤及NAC的保护效应。结果显示,作用12 h,各染毒组与对照组相比,细胞存活率和线粒体膜电位显著下降(P0.05或P0.01),细胞凋亡率、坏死率、乳酸脱氢酶(LDH)漏出率和细胞内活性氧含量均显著升高(P0.05或P0.01),呈剂量-效应关系;而NAC保护组与染毒组相比,细胞存活率和细胞凋亡率均有显著差异(P0.05),细胞坏死率无明显差异(P0.05)。结果表明,本试验所选择的镉浓度引起的肾小管上皮细胞死亡是以凋亡为主,氧化应激直接参与镉对肾小管上皮细胞的毒性损伤,NAC对肾小管上皮细胞凋亡有一定的保护效应。     

谷胱甘肽对铜孵育的肉鸡肝线粒体膜电势及膜通透性的保护作用

线粒体是细胞内主要的能量形成场所,也是各种损伤最为敏感的细胞器之一,所以无论在生理上或病理上都具有十分重要的意义。大量的研究表明,细胞凋亡的最早期阶段在细胞核病理改变出现之前,线粒体膜电位就已下降,膜电位消失亦是线粒体损伤的一个重要标志。线粒体跨膜电位一旦崩溃,则细胞凋亡不可逆转;线粒体膜内外的各种物质进出线粒体的通道被称之为线粒体膜通透性转变孔(mitochondrialpermeability transition pore,MPTP),是线粒体内外信息交流的中心枢纽,其开放状态指示着线粒体正常功能的发挥与否;铜是细胞色素氧化酶和铜蓝蛋白等     

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

环境雌激素灭多威对牛卵母细胞体外培养质量的影响

试验旨在研究体外培养液中添加灭多威（methomyl，MET）对牛卵母细胞质量的影响。通过免疫荧光染色和实时荧光定量PCR等方法检测牛卵母细胞的卵丘细胞扩展程度、第一极体排出率、活性氧（reactive oxygen species，ROS）水平、谷胱甘肽（glutathione，GSH）水平、线粒体膜电位（ΔΨm）以及凋亡相关基因的表达进行评价。结果显示，不同浓度MET不影响卵丘细胞的扩展程度，但200 μmol/L MET处理组卵母细胞的第一极体排出率显著低于对照组（P<0.05）。与对照组相比，200 μmol/L MET处理组的卵母细胞ROS水平显著升高，GSH水平、线粒体膜电位（ΔΨm）均显著降低（P<0.05）。此外，本研究还发现，200 μmol/L MET处理组卵母细胞中促凋亡基因Caspase-3和Bax的mRNA转录水平显著升高，抗凋亡基因Bcl-xl的mRNA转录水平显著降低（P<0.05）。上述结果表明，MET可以通过增加氧化应激和细胞凋亡来降低体外培养牛卵母细胞的质量。     

玉米赤霉烯酮对小鼠睾丸间质细胞线粒体的损伤作用

将不同作用时间（时间梯度组）及不同质量浓度（质量浓度梯度组）的玉米赤霉烯酮（Zearalenone,ZEA）作用于体外培养的原代小鼠睾丸间质细胞,时间梯度组设0（对照组）、6、12、24h4个观察组,染毒质量浓度为2.5mg/L;质量浓度梯度组设0（对照组）、5、10、20mg/L ZEA 4个观察组,染毒时间为12h。采用流式细胞技术测定线粒体膜电位和透射电子显微镜观察细胞超微结构的方法观测ZEA对睾丸间质细胞线粒体的损伤作用。结果显示,睾丸间质细胞线粒体膜电位2.5mg/L ZEA暴露6h较对照组显著下降（P〈0.05）,暴露12h及24h较对照组都有极显著下降（P〈0.01）;ZEA质量浓度为5mg/L组较对照组显著下降（P〈0.05）,10mg/L组和20mg/L组较对照组都有极显著下降（P〈0.01）。细胞超微结构分析显示,线粒体的空泡化和内质网断裂的程度与ZEA的暴露存在时间-效应关系和质量浓度-效应关系。结果表明,ZEA可导致睾丸间质细胞线粒体损伤,这是ZEA抑制睾丸间质细胞分泌睾酮的一个重要原因。     

Direct embryotoxicity of chromium (III) exposure during preimplantation development

Chromium in its trivalent form (chromium (III)) is an essential component of a balanced diet, and its deficiency disturbs glucose and lipid metabolism in humans and animals. The prevailing view is that chromium (III) is notably less toxic than chromium (VI), which is genotoxic and carcinogenic. Thus, the biotransformation of environmental chromium (VI) to chromium (III) is a promising and environmentally friendly detoxification method. However, increasing evidence suggests that chromium (III) induces considerable cytotoxicity. However, the toxicity of chromium (III) to early embryos remains largely unknown. In the present study, we used in vitro fertilization (IVF) to produce mouse embryos and identified the direct embryotoxicity of chromium (III). On exposure to high concentrations of CrCl₃, blastocyst formation almost completely failed and a large proportion of embryos were arrested at the 2- to 4-cell stage. At low concentrations of CrCl₃, IVF embryos showed a significant decrease in blastocyst formation, reduced total cell numbers, aberrant lineage differentiation, increased oxidative stress, and apoptosis. We also found that chromium (III) exposure during the preimplantation stage, even at low concentrations, led to impaired post-implantation development. Thus, our study substantiates the direct embryotoxicity of chromium (III) during preimplantation development and prolonged impairment of development potential. The results further highlight the potential adverse effects of chromium (III) on public reproductive health with respect to increased environmental enrichment of and dietary supplementation with chromium (III) complexes.     

The suppressive effect of dietary coenzyme Q10 on mitochondrial reactive oxygen species production and oxidative stress in chickens exposed to heat stress

This study was conducted to determine if dietary supplementation with coenzyme Q₁₀ (CoQ₁₀), which can act as a potent antioxidant and is an obligatory cofactor of mitochondrial uncoupling protein, suppresses the heat stress (HS)‐induced overproduction of mitochondrial reactive oxygen species (ROS) and oxidative damage in the skeletal muscle of birds. The carbonyl protein content of skeletal muscle was significantly higher in birds exposed to HS treatment (34°C, 12 h) than in thermoneutral birds (25°C). This increase was suppressed by CoQ₁₀ supplementation (40 mg/kg diet). Succinate‐supported mitochondrial ROS production was increased by HS treatment, and this increase was also suppressed by CoQ₁₀ supplementation. In contrast, CoQ₁₀ supplementation did not affect the HS‐induced decrease in mitochondrial proton leak. The mitochondrial membrane potential (ΔΨ), to which HS‐induced ROS production was previously shown to be sensitive, tended to be increased by HS treatment, but this rise in ΔΨ was not affected by CoQ₁₀ supplementation. Taken together, these results suggest that dietary CoQ₁₀ supplementation attenuates HS‐induced oxidative damage to skeletal muscle, by preventing the overproduction of succinate‐supported mitochondrial ROS in a manner that is independent of ΔΨ. © 2015 Japanese Society of Animal Science     

Aflatoxin B1 impairs mitochondrial functions,activates ROS generation,induces apoptosis and involves Nrf2 signal pathway in primary broiler hepatocytes

Aflatoxin B1 (AFB1) is known as a mycotoxin that causes various health problems in animals, but the precise mechanism of AFB1 on mitochondrial functions and apoptosis in primary broiler hepatocytes (PBHs) is not clear. The objective of this study was to investigate the effects of AFB1 on the mitochondrial functions, reactive oxygen species (ROS) generation, apoptosis and nuclear factor erythroid 2‐like factor 2 (Nrf2)‐related signal pathway in PBHs. Here, the mitochondrial membrane potential (MMP), ROS generation, antioxidative genes and apoptosis in PBHs induced by AFB1 were investigated. The results showed that AFB1 evoked mitochondrial ROS generation, decreased MMP and induced apoptosis in PBHs. AFB1 increased the percentage of apoptotic cells, and expression of caspase‐9 and caspase‐3, upregulated messenger RNA (mRNA) expression of Nrf2 and downregulated mRNA expressions of NAD(P)H: quinine oxidoreductase 1, superoxide dismutase and Heme oxygenase 1 in PBHs. The expression of Bax was also observed in cytoplasm. These findings suggested AFB1 results in a significant impairment of mitochondrial functions, activates ROS generation, induces apoptosis, and is involved in Nrf2 signal pathway through mitochondria ROS‐dependent signal pathways in PBHs.     

Induction of lipid peroxidation in mice by hexavalent chromium and its relation to the toxicity

Comparative effects of hexavalent (K2Cr2O7:Cr(VI)) and trivalent chromium (Cr(NO3)3:Cr(III)) on the development of lipid peroxidation, and the relationship between the lipid peroxidation and damage to tissues were studied using male ddY strain mice. The animals were administered with either of two chemicals at a dose of 20 mg Cr/kg by a single intraperitoneal injection. The results obtained were as follows: (1) Lipid peroxidation in the liver, as measured by the synthesis of thiobarbituric acid reactive substances (TBARS), showed a significant increase at 24 and 48 hr after Cr(VI) injection, while in the kidney it was observed only at 48 hr. In the mice administered with Cr(III), TBARS formation in the liver went down below the control levels, while no change was observed in the kidney. (2) Chromium contents in the liver and kidney showed a maximum level at 6 hr after injection of Cr(VI) and then those declined to the half of the maximum level at 48 hr, respectively. Chromium contents in the liver and kidney of the mice injected with Cr(III) were lower than those injected with Cr(VI) during the experimental period. (3) Increases of TBARS formation in the liver, chromium content in the liver and kidney, and ornithine carbamyl transferase (OCT) activity indicative of the liver cell damage, and urea nitrogen content in the serum, indicative of the kidney damage, observed at 24 hr after injection of Cr(VI) were inhibited by simultaneous injection of 100 mg/kg of L-ascorbic acid, as antichrome agent, respectively. These observations might suggest a possible causative role of lipid peroxidation in Cr(VI) toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mdivi-1, mitochondrial fission inhibitor,impairs developmental competence and mitochondrial function of embryos and cells in pigs

Mitochondria are highly dynamic organelles that undergo constant fusion/fission as well as activities orchestrated by large dynamin-related GTPases. These dynamic mitochondrial processes influence mitochondrial morphology, size and function. Therefore, this study was conducted to evaluate the effects of mitochondrial fission inhibitor, mdivi-1, on developmental competence and mitochondrial function of porcine embryos and primary cells. Presumptive porcine embryos were cultured in PZM-3 medium supplemented with mdivi-1 (0, 10 and 50 μM) for 6 days. Porcine fibroblast cells were cultured in growth medium with mdivi-1 (0 and 50 μM) for 2 days. Our results showed that the rate of blastocyst production and cell growth in the mdivi-1 (50 μM) treated group was lower than that of the control group (P < 0.05). Moreover, loss of mitochondrial membrane potential in the mdivi-1 (50 μM) treated group was increased relative to the control group (P < 0.05). Subsequent evaluation revealed that the intracellular levels of reactive oxygen species (ROS) and the apoptotic index were increased by mdivi-1 (50 μM) treatment (P < 0.05). Finally, the expression of mitochondrial fission-related protein (Drp 1) was lower in the embryos and cells in the mdivi-1-treated group than the control group. Taken together, these results indicate that mdivi-1 treatment may inhibit developmental competence and mitochondrial function in porcine embryos and primary cells.