ROS在镉诱导HK-2细胞氧化损伤和凋亡中的作用研究

该文研究了活性氧(reactive oxygen species, ROS)在镉(Cadmium, Cd)诱导HK-2细胞氧化损伤和凋亡中的作用。不同浓度CdCl2处理HK-2细胞不同时间后,通过MTT法、DCFH-DA标记、JC-1染色、彗星实验和流式细胞术分别检测细胞活性、ROS、线粒体膜电位Δψm、DNA损伤及细胞凋亡情况。结果显示, CdCl2处理引起HK-2细胞形态皱缩、变圆,活性下降,且呈时间和剂量依赖性; CdCl2处理导致ROS水平升高、线粒体膜电位Δψm下降、DNA损伤和caspase-3活化,最终导致细胞凋亡,且60μmol/L处理组及高浓度组与对照组相比差异极显著(P0.01)。采用ROS清除剂NAC与CdCl2共处理细胞24 h,发现细胞形态明显恢复、ROS水平显著降低、线粒体膜电位Δψm显著升高、彗星尾部长度和DNA百分比显著下降、凋亡细胞减少(P0.01)。综上所述, ROS介导了Cd诱导的HK-2细胞氧化损伤和凋亡。     

柯里拉京对人肺癌细胞A549的抑制作用及其机制研究

该研究旨在探讨柯里拉京对人肺癌A549细胞凋亡的影响及其潜在作用机制。采用CCK-8细胞活性检测试剂盒检测柯里拉京对A549细胞活性的影响;通过流式细胞术检测细胞凋亡;JC-1线粒体膜电位检测试剂盒检测线粒体膜电位;免疫印迹法检测凋亡相关蛋白(bax、bcl-2、cleaved-caspase-3、cleaved-PARP)的表达量;通过DCFH-DA探针标记检测细胞内ROS水平。研究结果显示,柯里拉京处理能够剂量依赖性地抑制A549细胞的活性,并通过上调bax的表达、下调bcl-2的表达,破坏线粒体膜电位,促进有活性的cleaved-caspase-3以及cleaved-PARP的形成,诱导A549细胞凋亡。活性氧清除剂NAC能够明显逆转柯里拉京诱导的细胞凋亡。因此,柯里拉京可能通过调节胞内ROS水平诱导人肺癌细胞A549发生凋亡。     

西达本胺诱导胰腺癌细胞凋亡

目的：观察西达本胺对胰腺癌细胞BxPC-3和PANC-1生长抑制及诱导细胞凋亡作用,探讨西达本胺抗胰腺癌的机制。方法：西达本胺处理BxPC-3和PANC-1细胞后,用流式细胞术检测细胞的凋亡率,用罗丹明123和DCFH—DA染色方法测定细胞线粒体膜跨膜电位变化和活性氧（ROS）的产生,用Western印迹检测Bcl-2家族和γH2AX蛋白表达的变化。结果：西达本胺对胰腺癌细胞BxPC-3和PANC-1具有生长抑制和诱导细胞凋亡的作用,且呈时间和剂量依赖关系;处理72h后,胰腺癌细胞内ROS产生增强导致DNA损伤发生,且线粒体跨膜电位明显下降;促凋亡蛋白Bax的表达,抑制抑凋亡蛋白Bcl-2和Mcl—1的表达。结论：西达本胺具有抑制胰腺癌细胞增殖,诱导细胞凋亡的作用;西达本胺增强胰腺癌细胞内ROS的产生并导致DNA损伤,最终诱导细胞凋亡的发生。     

UVA对斜纹夜蛾SL细胞损伤研究

目的:研究了不同剂量长波紫外光(UVA)对斜纹夜蛾细胞(Spodoptera litura,SL)活力的影响及其机理。方法:四唑盐比色实验(MTT)法测定UVA对离体培养的SL细胞的细胞毒力;流式细胞术(FCM)检测UVA处理后SL细胞内活性氧水平(ROS)和线粒体膜电位变化;1%琼脂糖凝胶电泳检测UVA处理后SL细胞DNA损伤情况。结果:48 KJ/m2剂量UVA处理细胞后,细胞增殖抑制率明显上升,且增殖抑制率与UVA剂量呈正相关。12 KJ/m2UVA处理后2 h细胞内ROS水平显著升高,且与UVA剂量呈正相关。与2 h相比,相同剂量UVA处理后24 h和48 h的ROS水平有所降低。在144 KJ/m2剂量UVA处理下,细胞线粒体膜电位呈降低-恢复-再降低趋势。DNA琼脂糖凝胶电泳表明,不超过144 KJ/m2剂量UVA对细胞DNA不出现细胞凋亡典型症状的DNA ladder。结论:24 KJ/m2UVA是进行SL细胞光活化研究的最佳实验剂量。     

缺氧条件下GC-1细胞凋亡的作用机制研究

以小鼠精原细胞系GC-1细胞为研究对象,探讨缺氧条件下GC-1细胞凋亡潜在的分子机制。首先,采用CCK-8 (Cell Counting Kit-8)法检测不同缺氧时间处理下的细胞活力,以确定细胞缺氧损伤的时间;然后,通过化学荧光法检测GC-1细胞中活性氧(reactive oxygen species, ROS)的含量,采用JC-1法检测线粒体的膜电位,采用比色法检测ATP的含量,采用线粒体荧光探针法检测线粒体的数量与分布,并采用透射电镜观察细胞的超微结构;最后,利用实时荧光定量PCR检测线粒体信号通路相关基因胱天蛋白酶(caspase)-3、caspase-9、细胞色素c (cytochrome c, Cyt-c)、Bax和Bcl-2的m RNA表达水平。结果显示,缺氧36 h后,细胞活力为(60.36±5.40)%,符合后续实验需求;在缺氧条件下, GC-1细胞中的ROS含量显著增加, ATP含量显著下降,线粒体膜电位下降、数量减少,同时细胞中促凋亡相关基因的表达水平上调,抗凋亡因子Bcl-2的基因表达水平下调。实验结果初步表明,缺氧可导致GC-1细胞线粒体功能障碍, ROS/线...     

镉胁迫对猪肾PK-15细胞活性氧和抗氧化酶活性的影响

目的:探明氯化镉(Cd Cl_2)对猪肾PK-15细胞活性氧(ROS)生成和抗氧化酶活的影响。方法:用不同浓度Cd Cl_2(2、4、6、8μmol/L)处理PK-15细胞24 h,观察细胞的形态学变化,并用MTT法检测细胞活性;用不同浓度Cd Cl_2或不同浓度Cd Cl_2+NAC(500μmol/L)处理PK-15细胞24 h,观察对细胞形态和活性氧(ROS)生成的影响;收集不同浓度Cd Cl_2处理24 h的细胞,用试剂盒检测总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)的活性,以及还原型谷胱甘肽(GSH)的含量变化。结果:Cd Cl_2处理PK-15细胞24 h,与对照组相比,细胞活性显著降低且呈剂量-效应关系(P0.05,P0.01);随着Cd Cl_2浓度的升高,细胞逐渐皱缩、变圆,ROS荧光强度逐渐升高;Cd Cl_2+NAC处理组与Cd Cl_2处理组相比,细胞皱缩变圆程度明显降低且ROS荧光强度明显减弱;随着Cd Cl_2浓度增加,T-SOD活性及GSH含量显著提高(P0.05),CAT活性极显著下降(P0.01)。结论:镉胁迫PK-15细胞是通过氧化应激机制介导肾脏细胞损伤的。     

Sestrin2对热暴露肺上皮细胞凋亡的干预作用及其机制*

目的:探讨Sestrin2蛋白对热暴露肺上皮细胞凋亡的干预作用及其作用机制。方法:体外培养的Beas-2B细胞分为对照组(37℃)和热暴露组(39℃、40℃和41℃),在上述温度中暴露不同时间(0、3、6和12 h),胰酶消化后收集细胞,分别通过Western blot、荧光分光光度计、流式细胞仪等方法检测细胞中的Sestrin2、超氧化物歧化酶(SOD)、活性氧自由基(ROS)表达水平,细胞线粒体膜电位及细胞凋亡率。基因序列克隆入高表达质粒pcDNA 3.1⁺中,采用Lipfectamine 2000方法转染Beas-2B细胞,构建Sestrin2和SOD高表达细胞,观察细胞线粒体膜电位及细胞凋亡等指标的变化。结果:随着暴露温度的升高,与对照组相比,热暴露组细胞Sestrin2蛋白表达水平下降。在41℃热暴露Beas-2B细胞,不同时间点ROS水平显著上升,线粒体膜电位显著下降,细胞凋亡率增加。Sestrin2和SOD高表达细胞,在41℃暴露条件下,与对照组比较,ROS表达水平显著降低,线粒体膜电位下降幅度减小,热暴露导致细胞凋亡率降低。结论: Sestrin2能够通过线粒体膜电位和SOD缓解热暴露引起肺上皮细胞的凋亡,对Beas-2B细胞具有保护作用。     

姜黄素对H_2O_2诱导的HT29细胞氧化损伤的保护作用及机制研究

研究姜黄素对H_2O_2诱导HT29细胞氧化应激的保护作用及其可能的分子机制。分别采用低、中、高浓度姜黄素处理H_2O_2诱导的HT29细胞氧化损伤模型,并设置H_2O_2模型组和正常对照组;MTT法确定H_2O_2最佳损伤浓度和时间及姜黄素(2.5、5、10μmol/L)对H_2O_2诱导的HT29细胞活性的影响;Annexin V/PI双标记流式细胞术检测细胞凋亡情况;PI染色流式细胞术检测细胞周期变化;DCFH-DA荧光探针检测细胞内活性氧(ROS);JC-1染色检测细胞线粒体膜电位;比色法测定乳酸脱氢酶(LDH)释放量,以及丙二醛(MDA)、超氧化物歧化酶(SOD)、caspase-3和caspase-9的水平。结果显示姜黄素组(2.5、5、10μmol/L)明显提高H_2O_2诱导的HT29细胞的存活率(P0.05,P0.01);与H_2O_2模型组相比,姜黄素组细胞凋亡率降低(P0.01),增殖指数增高(P0.05,P0.01),LDH释放量和细胞内ROS降低(P0.05,P0.01),线粒体膜电位上升(P0.01),SOD活力增加(P0.01),MDA降低(P0.01),caspase-3和caspase-9活性增强(P0.01),并呈剂量-效应关系。结果表明姜黄素在一定剂量范围内对H_2O_2诱导的HT29细胞氧化损伤具有较好的保护作用,该作用可能与清除ROS,减轻DNA氧化损伤,抑制线粒体通路介导的细胞凋亡有关。     

镉诱导HEK293细胞凋亡及其线粒体凋亡途径

本课题研究了氯化镉(CdCl_2)诱导HEK293细胞(人胚胎肾细胞系)的凋亡,初步探讨了凋亡过程中Caspase-3、Bcl-2的变化和凋亡诱导因子(AIF)的转移以及它们的意义。MTT法检测CdCl_2对HEK293细胞增殖的抑制作用;通过倒置显微镜、电镜、琼脂糖凝胶电泳、流式细胞术、激光共聚焦观察细胞凋亡;应用Western blot法和荧光免疫法测定Caspase-3酶原、Bcl-2蛋白的变化以及检测AIF蛋白在细胞中的定位。结果显示:CdCl_2对HEK293细胞具有显著的生长抑制作用,并呈明显的剂量和时间依赖性。在琼脂糖凝胶电泳中,显示有凋亡细胞特有的DNA梯状条带,其中30μmol/L作用6-9h梯状条带最为清晰,时间过长或浓度过高则梯状条带逐渐模糊,表明镉浓度过高或处理时间过长,细胞有坏死。流式细胞仪检测也印证了这一结果。形态学观察可见明显的细胞凋亡特征。同时线粒体膜电位明显下降,发现Caspase-3酶原蛋白、Bcl-2蛋白含量减少,并具有时间依赖性;另外检测到线粒体AIF向细胞核转移。而Bcl-2转染后有一定的抑制凋亡作用。实验结果提示,CdCl_2能够诱导HEK293细胞凋亡,线粒体损伤导致AIF转移与细胞色素c释放,从而引发的非Caspases与Caspases凋亡途径可能在镉引发的细胞凋亡过程中起重要作用,而Caspase-3, Bcl-2起着重要的调控作用。     

大蒜素诱导人食管癌EC-109细胞凋亡

为寻找毒副作用小并且治疗效果好的抗癌药物,研究大蒜素对人食管癌EC-109细胞凋亡的影响,同时探讨了大蒜素引发细胞凋亡的可能机制。通过激光共聚焦显微镜观察细胞形态变化,琼脂糖凝胶电泳检测DNA片段化情况,流式细胞术检测细胞凋亡率和线粒体膜电位变化,qRT-PCR和Western blotting检测细胞凋亡相关基因Bax、Bcl-2的mRNA和蛋白表达水平。结果显示,大蒜素作用人食管癌EC-109细胞48 h后,线粒体膜电位显著降低,并且早期凋亡细胞和晚期凋亡细胞所占百分比均显著增加。同时,与对照组相比,Bax mRNA和蛋白水平均显著升高(p<0.05),Bcl-2 mRNA和蛋白水平均显著降低(p<0.05)。据此,本研究得出大蒜素可诱导人食管癌EC-109细胞凋亡,并呈剂量依赖性,有潜在的药用价值。     

Globular adiponectin-induced RAW 264 apoptosis is regulated by a reactive oxygen species-dependent pathway involving Bcl-2

Globular adiponectin (gAd), a truncated form of adipocyte-derived cytokine, stimulates RAW 264 cells to produce reactive oxygen species (ROS), which trigger an apoptotic cascade. In this study, we investigated the generation of intracellular and mitochondrial ROS in gAd-stimulated RAW 264 cells. Treatment with gAd efficiently induced the generation of intracellular and mitochondrial ROS, as detected by dichlorodihydrofluorescein diacetate and MitoSOX fluorescence, respectively. Furthermore, gAd treatment significantly increased 8-oxoguanine, a specific indicator of oxidative DNA damage. The transfection of RAW 264 cells with iNOS- and gp91^phox-specific small interfering RNA reduced markedly the generation of intracellular, but not mitochondrial, ROS. Quantitative PCR revealed that the expression ratio of Bcl-2 to Bax was reduced in a time-dependent manner in gAd-treated RAW 264 cells. The overexpression of Bcl-2 markedly inhibited gAd-induced apoptosis in RAW 264 cells and also reduced both the intracellular and the mitochondrial ROS generation induced by gAd treatment. Moreover, the overexpression of Bcl-2 significantly suppressed gAd-induced NO secretion and NOS activity. In addition, the inhibition of NOS activity partially reduced the oxidative DNA damage induced by gAd. Taken together, these results demonstrate that the gAd-induced apoptotic pathway acting via ROS/RNS generation involves Bcl-2.     

Cadmium-bound metallothionein induces apoptosis in rat kidneys, but not in cultured kidney LLC-PK1 cells

The ability of cadmium-bound metallothionein(Cd-MT) to induce apoptosis was investigated in vivo and in vitro. Administration of purified Cd-MT (0.15 mg MT bound Cd per kg body weight) to the rat induces DNA fragmentation, a biochemical characteristic of apoptosis in the kidney at 16 h, which was detectable by ethidium bromide staining on an agarose gel. It was still detected 24 h after administration. Induction of apoptosis by Cd-MT was specific to kidney; it was not observed in cerebrum, cerebellum, heart, lung, liver, testis, dorsolateral prostate, and ventral prostate. In contrast, addition of Cd-MT (0.01-100 microM) to the cultured porcine kidney LLC-PK1 cells failed to induce apoptosis under the condition where cadmium chloride (10 microM) did. There was no additivity of induction of apoptosis by CdCl2 (10 microM) in the presence of Cd-MT (0.01-100 microM). To examine the effect of intracellular MT on cadmium-induced apoptosis in cultured cells, new cell lines were established, which constitutively produce MT, being termed as Cd(r)-LLC-PK1 cells since Cd-MT exogenously added had much less permeability to the cultured cells. Followed by exposure of wild-type LLC-PK1 cells to 50 microM CdCl2 for 24 h, the surviving cells(Cd(r)-LLC-PK1 cells) induce MT at the level of 1.9 microg/2 x 10(6) cells. In Cd(r)-LLC-PK1 cells, 10 microM CdCl2 failed to induce apoptosis, but 60 microM CdCl2 could exert the apoptotic response, indicating that intracellular MT which was induced by CdCl2 did not facilitate CdCl2-elicited apoptosis. Furthermore, chromatin in rat kidneys was condensed by Cd-MT, but not that in LLC-PK1 cells. Thus, Cd-MT induces apoptosis in rat kidneys, but not in the cultured renal cells, suggesting that the ionic form of cadmium was required for programmed cell death.     

口蹄疫病毒诱导宿主细胞凋亡的研究

本文报道了口蹄疫病毒（Foot-and-Mouth Disease Virus,FMDV）在体外诱导PK-15细胞凋亡的研究结果。采用Hoechst33258荧光探针、DNA凝胶电泳、脱氧核糖核酸转移酶介导的缺口末端标记（TUNEL）技术均检测到了典型的细胞凋亡。结果显示使用感染性滴度为4.8lgTCID50/mL的口蹄疫病毒感染PK-15细胞,在培养32?h后荧光探针检测呈现典型的凋亡细胞核固缩和梅花状碎裂核,并伴随有凋亡小体出现,凋亡率约为20%;DNA凝胶电泳显示ladder梯带;末端标记检测到强绿色荧光标记物结合于凋亡细胞核上。研究结果提示口蹄疫病毒可以在体外诱导宿主细胞凋亡,细胞凋亡是其致细胞病变死亡的重要途径之一。     

The protective effect of EGF-activated ROS in human corneal epithelial cells by inducing mitochondrial autophagy via activation TRPM2

Oxidative stress is a major pathogenesis of some ocular surface diseases. Our previous study demonstrated that epidermal growth factor (EGF)-activated reactive oxygen species (ROS) could protect against human corneal epithelial cell (HCE) injury. In the present study, we aimed to explore the role and mechanisms of oxidative stress and mitochondrial autophagy in HCE cells subjected to scratch injury. CCK-8 assays, EdU assays, Western blot analysis, wound-healing assays, and flow cytometry were conducted to determine cell viability, proliferation, protein expression, cell apoptosis, and intracellular ROS levels, respectively. The results showed that EGF could promote damage repair and inhibit cell apoptosis in scratch injured HCE cells by upregulating ROS (**p < .01, ***p < .001). EGF also induced mitochondrial autophagy and alleviated mitochondrial damage. Interestingly, the combination of the mitochondrial autophagy inhibitor and mitochondrial division inhibitor 1 (MDIVI-1) with EGF could reduce cell proliferation, viability, and the ROS level (*p < .05, **p < .01, ***p < .001). Treatment using the ROS inhibitor N-acetyl- l -cysteine abrogated the increase in mitochondrial membrane potential after EGF treatment. (*p < .05). Taken together, these findings indicated that EGF plays an important role in HCE damage repair and could activate ROS to protect against HCE injury by inducing mitochondrial autophagy via activation of TRPM2.     

Oxidative Stress and Ca2+ Signals Involved on Cadmium-Induced Apoptosis in Rat Hepatocyte

Cadmium (Cd) is an important industrial and environmental pollutant. In animals, the liver is the major target organ of Cd toxicity. In this study, rat hepatocytes were treated with 2.5～10 μM Cd for various durations. Studies on nuclear morphology, chromatin condensation, and apoptotic cells demonstrate that Cd concentrations ranging within 2.5～10 μM induced apoptosis. The early-stage marker of apoptosis, i.e., decreased mitochondrial membrane potential, was observed as early as 1.5 h at 5 μM Cd. Significant (P?P?2+ concentration ([Ca²⁺] _i ) of Cd-exposed cells significantly increased (P?2+] _i may play an important role in apoptosis. Overall, these results showed that oxidative stress and Ca²⁺ signaling were critical mediators of the Cd-induced apoptosis of rat hepatocytes.     

Mitochondrial oxidative burst involved in apoptotic response in oats

Apoptotic cell response in oats is induced by victorin, a host-selective toxin secreted by Cochliobolus victoriae and thought to exert toxicity by inhibiting mitochondrial glycine decarboxylase (GDC) in Pc-2/Vb oats. We examined the role of mitochondria, especially the organelle-derived production of reactive oxygen species (ROS), in the induction of apoptotic cell death. Cytofluorimetric analysis showed that victorin caused mitochondrial deltaPsim breakdown and mitochondrial oxidative burst. Ultrastructural analysis using a cytochemical assay based on the reaction of H2O2 with CeCl3 detected H2O2 eruption at permeability transition pore-like sites on the mitochondrial membrane in oat cells treated with victorin. ROS generation preceded the apoptotic cell responses seen in chromatin condensation and DNA laddering. Both aminoacetonitrile (a specific GDC inhibitor) and antimycin A (a mitochondrial complex III inhibitor) also induced mitochondrial H2O2 eruption, and led to the apoptotic response in oat cells. ROS scavengers such as N-acetyl-l-cysteine and catalase suppressed the mitochondrial oxidative burst and delayed chromatin condensation and DNA laddering in the victorin- or antimycin A-treated leaves. These findings indicate possible involvement of mitochondria, especially mitochondrial-derived ROS generation, as an important regulator in controlling apoptotic cell death in oats.