左卡尼汀对过氧化氢诱导的心肌细胞凋亡的抑制作用(英文)

目的探讨左旋尼汀对心肌细胞凋亡的抑制作用及其可能的作用机制。方法采用培养的新生乳大鼠心肌细胞制备H2O2200μmol.L-1氧化损伤模型。实验分为正常对照组、H2O2200μmol.L-1模型组、左卡尼汀(0.3,0.6及1.2 mmol.L-1)组、左卡尼汀1.2 mmol.L-1+H2O2200μmol.L-1组。左卡尼汀0.3,0.6和1.2 mmol.L-1作用1 h后加入H2O2200μmol.L-1培养12 h,MTT法测定心肌细胞存活率,流式细胞仪测定心肌细胞的凋亡率,Western印迹法测定胱天蛋白酶3表达;用试剂盒方法检测过氧化物歧化酶(SOD)活性和丙二醛(MDA)水平。左卡尼汀1.2 mmol.L-1作用5 min后加入H2O2200μmol.L-1,采用Till阳离子测定系统监测5 min的心肌细胞[Ca2+]i瞬间变化。结果 H2O2200μmol.L-1作用于心肌细胞12 h能引起心肌细胞凋亡。左卡尼汀0.3,0.6和1.2 mmol.L-1能够不同程度的逆转由H2O2所致的损伤,使SOD活性明显增加,MDA水平明显降低(P<0.01)。左卡尼汀1.2 mmol.L-1作用最强,能够明显降低心肌细胞胱天蛋白酶3表达(P<0.01),明显降低H2O2引起的[Ca2+]i瞬间变化升高(P<0.01),但对于H2O2引起无钙血清培养的心肌细胞静息钙升高无影响。结论左卡尼汀能够抑制由H2O2引起的心肌细胞凋亡,该抑制作用可能与其保护细胞膜和减轻钙通道的损害、纠正[Ca2+]i瞬变失调及其抗氧化作用有关。     

ERK1/2信号通路介导丙泊酚对H_2O_2诱发的心肌细胞损伤保护作用

目的研究丙泊酚对H2O2诱发的心肌细胞损伤的保护作用及其作用机制。方法采用胰酶消化法获取大鼠胎鼠心肌细胞,以H2O2损伤心肌细胞获得心肌缺血再灌注损伤实验模型;加入不同浓度丙泊酚(12.5、25、50μmol·L-1),MTT法检测细胞存活率;用硫代巴比妥酸法、黄嘌呤氧化酶法分别测定各组细胞培养液中丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性;流式细胞术检测细胞凋亡水平;Western blotting检测细胞中ERK1/2、Bcl-2及Bax的表达。结果丙泊酚(12.5、25、50μmol·L-1)能抑制由H2O2导致的细胞死亡(P<0.01);减少MDA的产生(P<0.01),提高SOD活性(P<0.01);25、50μmol·L-1丙泊酚能抑制由H2O2导致的细胞凋亡(P<0.05);25μmol·L-1丙泊酚作用于细胞后能激活ERK1/2磷酸化,增加Bcl-2且减少Bax的表达。结论丙泊酚通过激活ERK1/2磷酸化对H2O2诱发的心肌细胞损伤起到保护作用。     

δ阿片受体激活对过氧化氢损伤的心肌细胞的保护作用

目的研究δ阿片受体激活剂D-丙(2)-D-亮-(5)-脑啡肽(DADLE)对过氧化氢(H2O2)损伤的心肌细胞的保护作用及其机制。方法分离乳大鼠心肌细胞,培养48h后分为正常对照、H2O2(200μmol.L-1)、H2O2+DADLE(1μmol.L-1)、H2O2+DADLE+纳曲吲哚(10μmol.L-1)和H2O2+DADLE+U0126(10nmol.L-1)组,继续培养48h。用[3H]TdR掺入法检测心肌细胞增殖反应,流式细胞仪检测心肌细胞凋亡百分率,乳酸脱氢酶(LDH)活性测定试剂盒测定培养上清LDH活性,硫代巴比妥酸显色法测定细胞内丙二醛(MDA)含量,黄嘌呤氧化酶法测定细胞内超氧化物歧化酶(SOD)活性,Western蛋白印迹法检测细胞外信号调节激酶磷酸化(p-ERK)水平。结果①与正常对照组比较,H2O2组心肌细胞[3H]TdR掺入值明显降低,细胞凋亡率升高;培养上清LDH活性和MDA含量明显增加,SOD活性和Ap-ERK/AERK的比值降低。②与H2O2组比较,DADLE可使心肌细胞[3H]TdR掺入值升高,细胞凋亡率下降;培养上清LDH活性和MDA含量降低,SOD活性和Ap-ERK/AERK的比值升高。③分别加入δ阿片受体拮抗剂纳曲吲哚和ERK拮抗剂U0126,DADLE对上述指标的逆转作用被抑制。结论δ阿片受体激活对H2O2损伤的心肌细胞具有保护作用,其机制可能与其增强心肌细胞的抗氧化功能及促进ERK磷酸化有关。     

阿魏酸预处理对阿霉素诱导H9c2心肌细胞损伤的保护作用

目的研究阿魏酸(FA)对阿霉素(DOX)诱导H9c2心肌细胞损伤的影响。方法 1μmol·L-1DOX处理H9c2细胞24 h,建立心肌损伤模型。FA预处理组,10、20、40μmol·L-1FA预处理2 h后,再与DOX共培养24 h。CCK-8比色法测定细胞生存率;相差显微镜观察细胞形态学改变;生化试剂盒检测LDH、CK、MDA、SOD;DCF-DA荧光染色流式细胞术检测细胞内活性氧;AO-EB染色、DNA琼脂糖凝胶电泳检测细胞凋亡;Western blot法测定caspase-3、Bax、Bcl-2表达。结果 DOX降低H9c2细胞生存率,诱导氧化应激损伤和细胞凋亡。FA预处理剂量依赖性提高细胞生存率,减轻LDH、CK外漏和损伤细胞形态学改变。FA减少ROS生成,降低细胞MDA水平,增加SOD酶活性,抑制DOX诱导的氧化应激。FA下调促凋亡蛋白caspase-3和Bax,上调凋亡抑制蛋白Bcl-2,减少心肌细胞凋亡。结论 FA可抑制DOX诱导的氧化应激和心肌细胞凋亡,减轻心肌损伤。     

吉马酮通过调控miR-297表达对H2O2诱导的小鼠海马神经元细胞凋亡和氧化应激的影响

摘要：目的:探讨吉马酮对H2O2诱导的小鼠海马神经元细胞凋亡和氧化应激的影响及分子机制。方法:分离培养小鼠海马神经元,将其分为对照组、H2O2组、H2O2+吉马酮低、中、高(50,100,200μmol·L-1)浓度组、H2O2+miR-NC组、H2O2+miR-297组、H2O2+吉马酮(200μmol·L-1)+anti-miR-NC组、H2O2+吉马酮(200μmol·L-1)+anti-miR-297组。流式细胞术检测细胞凋亡;蛋白质印迹（Western blot）法检测B细胞淋巴瘤/白血病-2（Bcl-2）、Bcl-2相关X（Bax）蛋白表达;丙二醛（MDA）和超氧化物歧化酶（SOD）试剂盒分别检测MDA含量和SOD活性;实时荧光定量PCR（RT-qPCR）检测miR-297表达水平。结果:吉马酮低、中、高浓度处理后,H2O2诱导的小鼠海马神经元中细胞凋亡率、Bax表达水平和MDA含量降低,Bcl-2表达水平、SOD活性和miR-297表达水平升高,且呈浓度依赖性（P<0.05）。miR-297过表达可降低H2O2诱导的海马神经元细胞凋亡率和MDA含量,提高SOD活性（P<0.05）。抑制miR-297表达逆转了吉马酮对H2O2诱导的海马神经元细胞凋亡和氧化应激的作用。结论:吉马酮可能通过上调miR-297表达抑制H2O2诱导的小鼠海马神经元细胞凋亡和氧化应激。     

吡格列酮对过氧化氢诱导的乳鼠心肌细胞凋亡的抑制作用

目的观察吡格列酮对乳鼠心肌细胞凋亡的抑制作用及其可能作用机制。方法采用培养的新生大鼠乳鼠心肌细胞制备H2O2200μmol·L-1氧化损伤模型。实验分为正常对照组、模型组、吡格列酮组(0.1,1及10μmol·L-1)、吡格列酮(10μmol.L-1)+GW9662(10μmol·L-1)组、维拉帕米1μmol.L-1组。MTT法测心肌细胞的活力;采用硫代巴比妥酸比色法、黄嘌呤氧化酶法分别测定心肌细胞中脂质过氧化产物丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性;流式细胞仪检测心肌细胞的凋亡率;采用Till阳离子测定系统,以Fura-2/AM为荧光探针,观察心肌细胞[Ca2+]i瞬间变化。结果H2O2200μmo.lL-1作用于心肌细胞12h能引起心肌细胞凋亡。吡格列酮浓度依赖性地增加受损心肌细胞的活力及SOD活性,降低MDA含量。吡格列酮10μmol·L-1抑制作用最明显,其与维拉帕米1μmol·L-1有相似的抑制作用,二者都可以减少心肌细胞的凋亡率,降低由H2O2诱导的升高的[Ca2+]i静息水平及频率。过氧化物酶体增殖物激活受体PPARγ受体特异性拮抗剂GW966210μmol.L-1能拮抗吡格列酮的部分抑制作用,但不影响吡格列酮对[Ca2+]i的瞬变作用。结论吡格列酮可以抑制H2O2诱导的乳鼠心肌细胞凋亡,其作用机制可能与其抗脂质氧化和减少细胞内钙超载有关,其抑制作用部分是通过PPARγ受体介导的。     

异丙酚对第三丁基过氧化氢诱导的心肌细胞凋亡的保护作用

目的观察异丙酚对第三丁基过氧化氢(t-BHP)诱导的心肌细胞凋亡的影响并探讨可能的机制。方法采用SD新生大鼠进行心肌细胞原代培养。实验分为5组:正常对照组、t-BHP组和异丙酚1、10、30μmol.L-1组。分光光度计法检测细胞内谷胱甘肽(GSH)、丙二醛(MDA)水平和超氧化物岐化酶(SOD)活性;四甲基偶氮唑盐比色法(MTT)检测细胞线粒体活性,罗丹明123(Rhodamine123)荧光染色、流式细胞仪检测细胞线粒体膜电位(ΔΨm),流式细胞术(FCM)测定细胞凋亡率,Western blot法检测caspase-3的表达。结果与正常对照组相比,100μmol.L-1的t-BHP处理心肌细胞4 h后,细胞内GSH水平明显下降(P<0.05),MDA含量增加(P<0.01),抗氧化酶SOD活性降低(P<0.01),细胞线粒体活性降低(P<0.01),线粒体膜电位ΔΨm明显下降(P<0.01),细胞凋亡率和caspase-3的表达明显升高(P<0.01);异丙酚10、30μmol.L-1能减少过氧化氢所致的细胞中MDA含量升高;提高SOD活性和GSH水平;提高线粒体活性、膜电位;抑制心肌细胞凋亡和caspase-3的表达升高(P(0.05,P(0.01)。结论异丙酚能减弱过氧化氢所致的心肌细胞凋亡,其作用机制可能是通过减少活性氧自由基导致的细胞膜氧化损伤、提高线粒体活性、维持线粒体的膜电位,从而抑制心肌细胞凋亡的发生。     

人参皂苷Rb1对H_2O_2诱导新生大鼠心肌细胞凋亡的保护作用

目的观察人参皂苷Rb1对H2O2诱导的新生大鼠心肌细胞凋亡的保护作用,并探讨其可能作用机制。方法在培养的新生大鼠心肌细胞上建立H2O2损伤模型,观察不同剂量组人参皂苷Rb1(20、40、80mg·L-1)对心肌细胞凋亡率、丙二醛(MDA)含量,超氧化物歧化酶(SOD)活性,以及细胞内钙离子变化的影响。结果不同剂量组人参皂苷Rb1可以减少心肌细胞凋亡率、降低MDA含量、增加SOD活性、减少细胞内钙超载。结论人参皂苷Rb1可以抑制H2O2引起的新生大鼠心肌细胞凋亡,其作用机制可能与其抗脂质氧化和减少细胞内钙超载有关。     

珠子参总皂苷对H2O2诱导新生大鼠心肌细胞凋亡的抑制作用

目的:观察珠子参总皂苷对H2O2诱导新生大鼠心肌细胞凋亡的抑制作用,并探讨其作用机制.方法:Wistar乳鼠心肌细胞原代培养,用H2O2建立氧化应激损伤模型,然后用珠子参总皂苷(100,200 μg/mL)孵育24 h后,MTT法检测珠子参总皂苷对细胞活力的影响,流式细胞仪和Hoechst33258染色检测珠子参总皂苷对氧化应激诱导细胞凋亡及胞内ROS含量的影响,比色法测定心肌细胞Caspase-3、Caspase-9的活性,荧光定量PCR测定心肌细胞Bcl-2和Bax mRNA表达,并计算Bcl-2与Bax的比值.结果:珠子参总皂苷(100,200 μg/mL)能显著改善心肌细胞活性,有效保护线粒体膜电位的稳定,抑制心肌细胞凋亡和改善细胞形态,降低细胞内活性氧(ROS)含量;下调Bax mRNA表达,上调Bcl-2mRNA表达及Bcl-2与Bax的比值;降低H2O2所致新生大鼠心肌细胞中Caspase-3、Caspase-9的活性.结论:珠子参总皂苷对H2O2诱导心肌细胞凋亡有显著的抑制作用,其机制可能与其稳定心肌细胞膜、清除ROS及调节心肌细胞Bcl-2、Bax和Caspase-3、Caspase 9表达有关.     

飞燕草素葡萄糖苷通过上调SOX2OT减轻高糖诱导的心肌细胞损伤

摘要：目的:探讨飞燕草素葡萄糖苷（DPg）对高糖（HG）诱导的心肌细胞H9C2损伤的影响及可能机制。方法:体外培养H9C2细胞,不同剂量(1,10,100μmol·L-1)的DPg作用于33.3 mmol·L-1葡萄糖诱导的H9C2细胞24 h,CCK-8法检测细胞增殖,流式细胞术检测细胞凋亡,Western Blot检测细胞中兔抗鼠B淋巴细胞瘤-2（Bcl-2）和Bcl-2相关蛋白（Bax）的蛋白表达,试剂盒检测细胞中丙二醛（MDA）和超氧化物歧化酶（SOD）水平,RT-qPCR检测SRY盒转录因子2重叠转录本（SOX2OT）表达。转染SOX2OT过表达载体、小干扰RNA至H9C2细胞,经33.3 mmol·L-1葡萄糖诱导24 h后,检测细胞增殖、凋亡、Bax和Bcl-2蛋白表达及MDA和SOD水平。结果:DPg可提高高糖诱导的H9C2细胞吸光度（A）值（P<0.05）,降低细胞凋亡率、Bax蛋白表达及MDA含量（P<0.05）,并促进Bcl-2蛋白表达和SOD活性（P<0.05）,且呈剂量依赖性。DPg可剂量依赖性促进高糖诱导的H9C2细胞中SOX2OT的表达,过表达SOX2OT可提高高糖诱导的H9C2细胞A值（P<0.05）,降低细胞凋亡率、Bax蛋白表达及MDA含量（P<0.05）,并促进Bcl-2蛋白表达和SOD活性（P<0.05）。与未敲减SOX2OT的H9C2细胞比较,敲减SOX2OT的H9C2细胞经100μmol·L-1DPg和高糖处理后A值、Bcl-2蛋白表达和SOD活性降低（P<0.05）,细胞凋亡率、Bax蛋白表达及MDA含量升高（P<0.05）,并抑制Bcl-2蛋白表达和SOD活性（P<0.05）。结论:DPg可能通过上调SOX2OT表达抑制HG诱导的H9C2细胞凋亡和氧化应激。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.