Triapine通过ROS/GSH/GPX4轴诱导A549细胞铁死亡北大核心CSCD

目的探究Triapine诱导非小细胞肺癌细胞A549发生铁死亡的作用与机制。方法MTT实验和克隆形成实验评价Triapine对A549细胞增殖的作用;使用DCFH-DA探针分析Triapine对A549细胞内ROS水平的影响;试剂盒检测Triapine处理后A549细胞内谷胱甘肽(glutathione,GSH)和脂质过氧化物(lipid peroxides,LPO)水平变化;Western blot分析Triapine对A549细胞内谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白表达的影响;用ROS抑制剂进行干预,检测GPX4蛋白的表达和细胞活性的变化。结果Triapine能够抑制A549细胞增殖,且IC_(50)为(3.7±0.08)mg·L^(-1);Triapine能诱导A549细胞ROS累积,降低GSH水平,提升LPO含量,下调GPX4的表达;ROS抑制剂干预能恢复Triapine引起的GPX4表达降低及细胞活性下降。结论Triapine可能通过提升A549细胞内ROS水平消耗GSH,导致GPX4表达下降,诱导铁死亡发生。     

苍术素调节Nrf2/SLC7A11/GPX4信号通路对脑胶质瘤细胞铁死亡的影响

目的 探讨苍术素调节Nrf2/SLC7A11/GPX4信号通路对脑胶质瘤细胞铁死亡的影响及其机制。方法 体外培养脑胶质瘤U251细胞,将U251细胞分为对照组、苍术素低剂量组(5 mol/L)、中剂量组(10 mol/L)、高剂量组(20 mol/L)、ML385组(Nrf2抑制剂1 mol/L)。MTT和Edu实验检测细胞增殖;流式细胞术检测细胞凋亡率;试剂盒检测亚铁离子(Fe²⁺)、乳酸脱氢酶(LDH)、丙二醛(MDA)、谷胱甘肽(GSH)、活性氧(ROS)水平;Western blot检测Ki-67、cleaved-caspase3、Nrf2、SLC7A11、GPX4蛋白表达;建立脑胶质瘤裸鼠模型,观察肿瘤生长情况,检测肿瘤组织中Ki-67、cleaved-caspase3、Nrf2、SLC7A11、GPX4蛋白水平。结果 细胞实验结果显示,与对照组比较,苍术素低、中、高剂量组U251细胞Fe²⁺、MDA、LDH、ROS水平、细胞凋亡率、cleaved-caspase3水平显著性升高,GSH水平、OD490(24 h、48 h)值和细胞...     

下调SPTBN2通过抑制Nrf2/GPX4信号诱导肺腺癌细胞A549铁死亡

目的 研究下调SPTBN 2对肺腺癌细胞A549铁死亡的影响和机制。方法 肺腺癌细胞A549分成Control、si-NC(转染siRNA control)、si-SPTBN 2(转染SPTBN 2 siRNA)、si-Nrf 2(转染Nrf 2 siRNA)、si-SPTBN 2+Vector(转染SPTBN 2 siRNA、阴性对照载体)、si-SPTBN 2+Nrf两组(转染SPTBN 2 siRNA、Nrf 2过表达载体),Western blot检测ACSL4、SLC7A11、Nrf 2、GPX4蛋白表达,CCK-8实验检测增殖,DCFH-DA法检测ROS,比色法检测Fe ²⁺、MDA、4-HNE,微量法检测GSH。结果 与Control、si-NC组比较,si-SPTBN 2、si-Nrf两组肺腺癌细胞增殖活性降低,细胞内Fe ²⁺含量升高,细胞中ACSL4、SLC7A11、Nrf 2、GPX4蛋白表达减少,ROS、MDA、4-HNE含量升高,GSH含量降低。与si-SPTBN 2+Vector组比较,si-SPTBN 2+Nrf...     

淫羊藿苷调节Nrf2/SLC7A11/GPX4信号通路对高糖诱导的小胶质细胞铁死亡的影响

摘要：目的:探讨淫羊藿苷调节核转录E2相关因子2（Nrf2）/溶质载体蛋白7家族成员11（SLC7A11）/谷胱甘肽过氧化物酶4（GPX4）通路对高糖诱导的小胶质细胞铁死亡的影响。方法:将小胶质细胞（BV2）分为对照组(5.5 mmol·L-1葡萄糖)、高糖组(35 mmol·L-1葡萄糖)、淫羊藿苷低(35 mmol·L-1葡萄糖+0.37μmol·L-1淫羊藿苷)、高(35 mmol·L-1葡萄糖+1.48μmol·L-1淫羊藿苷)剂量组、抑制剂组(35 mmol·L-1葡萄糖+1.48μmol·L-1淫羊藿苷+1μmol·L-1的Nrf2抑制剂ML385)。采用CCK-8法检测细胞增殖,进行细胞形态学观察,用超氧化物阴离子荧光探针（DHE）、氟硼二吡咯(BODIPYTM)检测细胞内活性氧（ROS）、脂质过氧化（LPO）水平,特异性荧光探针检测细胞内活性铁含量,Western Blot检测细胞Nrf2、SLC7A11、GPX4、血红素加氧酶1（HO-1）、环氧合酶2（COX2）、酰基辅酶A合成酶长链家族成员4（ACSL4）蛋白的表达。结果:与对照组比较,高糖组的BV2细胞形态不规则,出现细胞碎片,细胞光密度(OD450)值、Nrf2、SLC7A11、GPX4、HO-1表达明显降低（P＜0.05）,ROS和LPO水平、活性铁含量、COX2、ACSL4表达显著升高（P＜0.05）;与高糖组比较,低、高剂量淫羊藿苷组的BV2细胞损伤减少,细胞OD450值、Nrf2、SLC7A11、GPX4、HO-1表达升高（P＜0.05）,ROS和LPO水平、活性铁含量、COX2、ACSL4表达降低（P＜0.05）;与高剂量淫羊藿苷组比较,抑制剂组减弱了淫羊藿苷对高糖诱导的小胶质细胞铁死亡的抑制作用。结论:淫羊藿苷通过激活Nrf2/SLC7A11/GPX4通路从而抑制高糖诱导的小胶质细胞铁死亡。     

蛇莓提取物通过GPX4诱导铁死亡抑制肾癌细胞OS-RC-2增殖

目的 探讨蛇莓醇提物通过铁死亡抑制肾癌细胞OS-RC-2增殖的作用机制。方法 蛇莓醇提物给药,CCK-8试验检测细胞增殖;活性氧(reactive oxygen species, ROS)检测试剂盒、GSH试剂盒、MDA试剂盒、细胞凋亡试剂盒分别检测细胞ROS、GSH、MDA含量及凋亡水平;网络药理学分析蛇莓成分-靶点;分子对接检测蛇莓成分与铁死亡核心靶点GPX4的结合能力;Western blot对铁死亡信号通路关键蛋白进行检测;利用铁死亡的抑制剂Fer-1和ROS抑制剂NAC进行回复试验,检测细胞增殖及铁死亡蛋白的变化;蛇莓醇提物中的Dulcitol (卫矛醇)给药,检测细胞增殖及GPX4蛋白的变化。结果 蛇莓可在一定程度上呈时间-浓度依赖性抑制细胞增殖,显著增加ROS、MDA水平而降低GSH水平;流式细胞术显示蛇莓可显著促进细胞凋亡,但并没有呈浓度依赖方式;网络药理学和分子对接结果显示蛇莓中的卫矛醇与GPX4结合稳定(-6.5 kJ·mol^-1);Western blot结果显示蛇莓明显降低GPX4、SLC7A11并升高HO-1及Transferrin蛋白水...     

人参皂苷Rg1通过激活Nrf2/xCT/GPX4通路抑制神经元铁死亡改善缺血性脑卒中损伤北大核心CSCD

目的研究人参皂苷Rg1对缺血性脑卒中后神经元铁死亡的抑制作用及其机制。方法细胞水平建立HT22细胞氧糖剥夺/复氧(oxygen glucose deprivation/reoxygenation,OGD/R)模型,CCK-8法检测Rg1对OGD/R损伤后HT22细胞活力的影响,试剂盒检测细胞铁死亡标志物GSH/GSSG、SOD、MDA和Fe 2+含量的影响;Western blot检测细胞GPX4、xCT和Nrf2蛋白的变化;ML385干预Nrf2验证其在Rg1调节OGD/R引起的细胞铁死亡中的作用。动物水平建立大鼠短暂性大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)再灌注模型,分为假手术组、模型组、Rg1治疗组(40 mg·kg^(-1))和Fer-1铁死亡抑制剂组(0.2 mg·kg^(-1))。缺血90 min后拔栓并给药,再灌注24 h后进行Zea Longa和Garcia Test评分评估神经受损程度;透射电镜观察皮质神经元线粒体形态变化;Western blot检测GPX4和xCT蛋白水平。结果Rg1能明显提高OGD/R后HT22细胞存活率,上调GSH/GSSG比值,恢复SOD活性,降低MDA和Fe 2+含量,并促进GPX4,xCT和Nrf2蛋白表达,而ML385干预明显抑制了Rg1对OGD/R后HT22细胞存活率及GPX4、xCT蛋白的上调作用。在体动物实验结果表明,Rg1可上调MCAO大鼠皮质组织GPX4和xCT的表达,缓解神经元线粒体的形态损伤,改善神经功能。结论人参皂苷Rg1可通过促进Nrf2/xCT/GPX4通路抑制缺血性脑卒中神经元铁死亡。     

黄芪皂苷Ⅱ对人肝癌多药耐药细胞BEL-7402/FU 的逆转耐药作用

摘要目的：探讨黄芪皂苷II（AstragalosideII,ASⅡ）对肝癌多药耐药性的逆转作用。方法：MTT法检测人肝癌多药耐药细胞BEL-7402／FU对化疗药物的敏感性和逆转耐药倍数,RT-PCR法检测多药耐药蛋白-1（MDRl）基因表达,免疫细胞化学法检测P一糖蛋白（P-gP）蛋白表达水平,流式细胞仪检测AS1I对细胞内Rhodaminel23的蓄积影响。结果：BEL-7402／FU细胞对5-氟尿嘧啶（5-FU）、阿霉素、丝裂霉素的耐药倍数分别为19．64、1．98、1．92。0．04、0．08mg／mL的ASⅡ能增强5-FU对BEL-7402／FU的细胞毒作用,逆转耐药倍数分别为1．49,1．81。0．08、0．16mg／mL的ASⅡ作用BEL-7402／FU细胞24h后,能下调MDRlmRNA和P-gP蛋白表达水平,提高细胞内Rhodaminel23的荧光表达率。结论：ASⅡ能部分逆转肝癌多药耐药性,其机制可能与下调MDRl1TIRNA表达及抑制P-gP的功能与表达有关。     

野木瓜果实总皂苷对BEL-7402/5 FU细胞多药耐药性的逆转作用

目的 研究野木瓜果实总皂苷对人肝癌耐药细胞BEL-7402/5 FU多药耐药(MDR)的逆转作用,并初步探讨其耐药逆转机制.方法 采用MTT法测定5-氟尿嘧啶(5-FU)、阿霉素(ADM)、丝裂霉素(MMC)和野木瓜果实总皂苷对BEL-7402细胞、BEL-7402/5 FU细胞的毒性及野木瓜果实总皂苷逆转MDR的效果;利用蛋白质印迹法检测P-糖蛋白(P-gp)的表达情况.结果 BEL-7402/5 FU细胞对5-FU、ADM、MMC的耐药指数分别为21.71,2.73,2.11;野木瓜果实总皂苷能有效抑制BEL-7402/5 FU细胞增殖,无细胞毒性剂量为5,10 μg/mL,逆转耐药倍数分别为1.36、1.93;野木瓜果实总皂苷联合5-FU作用BEL-7402/5 FU细胞,可降低P-gp蛋白表达.结论 野木瓜果实总皂苷能部分逆转BEL-7402/5 FU细胞MDR,其机制可能是通过下调P-gp的表达而实现的.     

欧前胡素阻断Noxs-ROS通路抑制MKN-45及HT-29细胞增殖活性

目的 探讨欧前胡素对人胃癌细胞(MKN-45)和人结肠癌细胞(HT-29)中烟酰胺腺嘌呤二核苷酸磷酸氧化酶(Noxs)-活性氧(ROS)氧化应激通路的作用及对肿瘤细胞增殖活性的影响。方法 MKN-45和HT-29细胞经过不同浓度(3、10、30、100 μmol/L)的欧前胡素及阳性对照二苯基氯化碘盐(DPI)处理后,使用CCK8法检测细胞增殖活性,使用DHE荧光探针检测细胞内ROS水平,使用Western blotting检测细胞内Nox1、Nox2、Nox3、Nox4、Nox5家族蛋白表达。结果 CCK8法结果显示,欧前胡素可浓度相关性地抑制MKN-45和HT-29细胞增殖活性;DHE荧光探针法结果显示,欧前胡素干预可使MKN-45和HT-29细胞荧光强度明显减弱,ROS水平降低;Western blotting结果显示,给予欧前胡素可使MKN-45细胞Nox1、Nox2、Nox3、Nox4、Nox5蛋白表达水平显著降低(P<0.05、0.01、0.001),使HT-29细胞Nox1、Nox2蛋白表达水平显著降低(P<0.05、0.01)。结论 欧前胡素可通过抑制MKN-45和HT-29细胞中Noxs-ROS通路,诱导细胞增殖活性下降,发挥抗肿瘤作用。     

姜黄素逆转人结肠癌细胞株HCT-8/VCR多药耐药的研究

目的探讨姜黄素对人结肠癌耐药细胞株HCT-8/VCR多药耐药性的逆转作用及可能机制。方法运用MTT法进行药敏试验,流式细胞仪检测细胞内罗丹明123(Rh123)的表达,RT-PCR法检测多药耐药基因mdr1 mR-NA水平的变化,Western blot法检测mdr1基因蛋白产物P糖蛋白(P-gp)表达水平的变化。结果经25μmol/L姜黄素处理后,增强了HCT-8/VCR细胞对VCR的敏感性,其逆转倍数为4.58倍,增加了细胞内Rh123的蓄积(P<0.05),明显抑制了多药耐药基因mdr1 mRNA及其蛋白的表达(P<0.05)。结论本研究结果提示姜黄素能抑制耐药基因的表达及功能,提高细胞对化疗药物的敏感性,从而逆转结肠癌细胞的多药耐药性。     

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.     

Pharmacological treatment of COVID-19: an opinion paper

The precocity and efficacy of the vaccines developed so far against COVID-19 has been the most significant and saving advance against the pandemic. The development of vaccines has not prevented, during the whole period of the pandemic, the constant search for therapeutic medicines, both among existing drugs with different indications and in the development of new drugs. The Scientific Committee of the COVID-19 of the Illustrious College of Physicians of Madrid wanted to offer an early, simplified and critical approach to these new drugs, to new developments in immunotherapy and to what has been learned from the immune response modulators already known and which have proven effective against the virus, in order to help understand the current situation.     

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.     

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.