冬虫夏草菌丝体水提醇沉物体外抗肿瘤活性研究

目的 研究冬虫夏草菌丝体水提醇沉物的体外抗肿瘤活性及其机制。方法 采用热水浸提-醇沉法得到水提物,高效液相色谱仪测定水提醇沉物中腺苷的量;采用MTT法测定其抗肿瘤活性,利用流式细胞仪结合碘化丙锭染色法检测其对细胞周期的抑制。结果 实验表明水提醇沉物能抑制人肝癌HepG2细胞株及大细胞肺癌NCI-H460细胞株的增殖,并呈浓度相关性;半数抑制浓度（IC₅₀）值分别为（1.49±0.19）和（1.67±0.27）mg/mL。细胞周期分析表明,水提醇沉物分别阻滞HepG2及NCI-H460细胞周期于G₂/M期、S期,并可诱导上述两种细胞发生凋亡。结论 冬虫夏草水提醇沉物通过阻滞HepG2及NCI-H460细胞周期循环,诱导其凋亡,从而表现出良好的增殖抑制活性。为深入研究冬虫夏草菌丝体水提醇沉物抗肿瘤的机制提供了实验证据。     

猫爪草总皂苷体外抗人非小细胞肺癌NCI-H460细胞活性研究

目的 观察猫爪草总皂苷体外对人非小细胞肺癌NCI-H460细胞活性的影响。方法 采用系统溶剂法提取猫爪草总皂苷,3H-TdR掺入法观察猫爪草总皂苷对NCI-H460细胞增殖的影响;流式细胞仪检测其对NCI-H460细胞凋亡和周期的影响。结果 猫爪草总皂苷对NCI-H460细胞增殖有较好的抑制作用,呈现较好的量效关系,可促进NCI-H460细胞的早期凋亡,细胞周期出现G0/G1期的阻滞。结论 猫爪草总皂苷能较好的抑制NCI-H460细胞增殖,其机制可能与诱导凋亡、G0/G1期阻滞有关。     

不可逆性泛HER抑制剂HM781-36B在埃罗替尼耐药型非小细胞肺癌中的抗肿瘤活性

目的 研究不可逆性泛人表皮生长因子受体（HER）抑制剂HM781-36B对埃罗替尼耐药型非小细胞肺癌细胞株NCI-H1975的体外抗肿瘤活性。方法 采用水溶性甲臢化合物（MTS）法检测HM781-36B抑制NCI-H1975细胞的体外增殖能力;利用流式细胞检测技术探讨其可能的作用机制;Western blot检测HM781-36B对HER家族（pEGFR和pHER2）和下游TEC通路（pERK和pAKT）的抑制作用。结果 HM781-36B（0.001~10 μM）能够剂量依赖性地抑制NCI-H1975细胞的体外增殖;HM781-36B处理NCI-H1975细胞后,诱导其细胞凋亡和G1细胞周期停滞;并抑制HER家族（pEGFR和pHER2）的表达和阻止下游信号级联放大的关键成分（pERK和pAKT）。结论 HM781-36B可能成为治疗第一代EGFR-TKI耐药非小细胞肺癌的有效药物。     

Wentilactone A抑制小细胞肺癌系NCI-H1688细胞的迁移研究

目的 探讨小分子化合物Wentilactone A (WA)抑制小细胞肺癌(small cell lung cancer,SCLC)细胞系NCI-H1688细胞迁移的机制。方法 采用划痕实验、噻唑蓝[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT]实验检测小分子化合物WA对细胞迁移和增殖能力的影响。免疫荧光实验检测化合物WA作用后SCLC细胞系NCI-H1688细胞中ATF3蛋白的表达。Western blot验证ATF3/Nrf2/AKR1C1信号通路的关键蛋白。结果 小分子化合物WA抑制SCLC细胞系NCI-H1688细胞的迁移和增殖,加入化合物WA 24 h组与48 h组的IC₅₀分别为(1.03±0.30)和(0.46±0.18) μmol/L。WA作用组NCI-H1688细胞的相对迁移距离为(8.73±1.06) mm,低于对照组的(15.63±3.11) mm,过表达AKR1C1基因后NCI-H1688细胞迁移距离为(24.37±0.90) mm,过表达AKR1C1基因并且WA作用后NCI-H1688细胞的迁移距离为(14.17±1.31) mm,差异有统计学意义(P<0.05)。ATF3是AKR1C1基因的负性调节因子,化合物WA作用后,ATF3蛋白表达水平升高,抑制Nrf2与ARE结合,从而抑制AKR1C1蛋白的表达。结论 WA通过ATF3/Nrf2/AKR1C1信号通路抑制SCLC细胞系NCI-H1688细胞的迁移和增殖。     

Jatamanvaltrate P对乳腺癌细胞T47D增殖的影响及机制研究

目的 考察蜘蛛香中环烯醚萜类化合物Jatamanvaltrate P对乳腺癌细胞T47D的增殖影响及作用机制。方法 MTT法检测Jatamanvaltrate P对乳腺癌细胞T47D和BT-20增殖的影响;DAPI染色和流式细胞术检测Jatamanvaltrate P对T47D细胞凋亡和周期的影响;Western blot检测与细胞周期、凋亡相关蛋白的变化。结果 Jatamanvaltrate P能明显抑制乳腺癌细胞增殖,并且呈时间-剂量依赖性;Jatamanvaltrate P能明显将T47D细胞阻滞于G₂/M期,下调周期相关蛋白cdc-2和Cyclin B1,上调p-cdc-2;Jatamanvaltrate P能诱导T47D细胞产生凋亡小体,显著性增加T47D细胞凋亡比例,呈现剂量依赖性;Jatamanvaltrate P处理后的蛋白PARP和Caspase 3/8/9出现了活化形式。结论 Jatamanvaltrate P通过阻滞细胞周期、诱导细胞凋亡来抑制乳腺癌细胞T47D的增殖,有望成为一种潜在的治疗乳腺癌的药物。     

淫羊藿苷对H2O2诱导的软骨细胞氧化损伤的保护作用及其机制

目的 探究淫羊藿苷（icariin，ICA）对H₂O₂诱导的软骨细胞氧化损伤的保护作用及相关机制。方法 分离SD新生大鼠软骨细胞，随机分为对照组、H₂O₂模型组、ICA低剂量组、ICA中剂量组、ICA高剂量组；采用CCK8法检测各组细胞增殖能力的变化；采用ELISA试剂盒检测各组细胞中活性氧（reactive oxygen，ROS）、超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malondialdehyde，MDA）、过氧化氢酶（catalase，CAT）以及谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）的表达情况；流式细胞术检测各组细胞周期情况，并计算增殖指数（proliferation index，PI）；Hoechst染色观察各组细胞核凋亡情况；分别采用荧光定量PCR （qRT-PCR）和Western blotting检测凋亡相关因子及Nrf2/HO-1通路的表达情况。结果 与对照组相比，H₂O₂模型组细胞增殖能力降低，ROS、MDA含量升高，SOD、CAT及GSH-Px含量下降，细胞凋亡情况加重；经ICA干预后，软骨细胞的增殖能力上升，ROS、MDA含量下降，SOD、CAT及GSH-Px含量增加，并且ICA能够有效抑制软骨细胞凋亡，上调Nrf2和HO-1蛋白的表达。结论 ICA对H₂O₂诱导的软骨细胞氧化损伤具有保护作用，能够抑制软骨细胞凋亡，其机制跟Nrf2/HO-1信号通路有关。     

乌骨藤中C21甾体苷诱导SACC-83和SACC-LM细胞凋亡的作用及其机制研究

目的 研究乌骨藤提取物C₂₁甾体苷对唾液腺腺样囊性癌（salivary adenoid cystic carcinomacv,SACC）低侵袭细胞株（salivary adenoid cystic carcinoma-83,SACC-83）和肺高转移细胞株（SACC-LM）增殖抑制和诱导凋亡的作用及其机制。方法 用不同浓度（5,10,20,40,60,80,100 μmol·L^-1） C₂₁甾体苷处理SACC-83和SACC-LM细胞48 h后,MTT法检测细胞活力,并计算药物的IC₂₀和IC₅₀;细胞克隆形成实验检测细胞增殖能力;流式细胞术检测SACC-83及SACC-LM细胞凋亡情况;实时荧光定量PCR和Western blot检测SACC-83和SACC-LM细胞Bcl-2、Bax、caspase 3的mRNA和蛋白的表达。结果 不同浓度的C₂₁甾体苷降低SACC-83和SACC-LM细胞活力,抑制细胞增殖,并且作用于SACC-83细胞C₂₁甾体苷的IC₂₀浓度为7.49 μmol·L^-1,IC₅₀浓度为38.34 μmol·L^-1;作用于SACC-LM细胞的C₂₁甾体苷IC₂₀浓度为9.30 μmol·L^-1,IC₅₀浓度为46.04 μmol·L^-1;细胞克隆集落形成明显减少。C₂₁甾体苷IC₂₀浓度分别促进SACC-83及SACC-LM细胞凋亡,且随着给药时间延长,凋亡率增加,具有显著性差异（P<0.05,P<0.01）。经7.49,9.30 μmol·L^-1 C₂₁甾体苷分别处理SACC-83及SACC-LM细胞后,Bcl-2的mRNA及蛋白水平显著降低（P<0.01）,而Bax、Caspase 3的mRNA和蛋白水平显著升高（P<0.05）。结论 乌骨藤C₂₁甾体苷抑制SACC-83及SACC-LM细胞增殖、促进凋亡,其作用机制可能与调控Bcl-2、Bax和Caspase 3表达有关。     

基于代谢组学的柠檬烯抗非小细胞肺癌作用机制研究

目的 采用非靶向代谢组学等方法，探究柠檬烯抑制非小细胞肺癌增殖的作用机制。方法 以肺癌A549细胞为研究对象，通过CCK-8法测定柠檬烯抑制A549细胞活力及IC₅₀；通过集落形成、流式细胞检测、铁含量测定及线粒体染色等实验，分别评价柠檬烯的体外抗肺癌及诱导铁死亡作用；代谢组学分析发现柠檬烯的潜在作用通路；最后采用Western blotting对相关通路主要蛋白进行验证。结果 与对照组相比，柠檬烯给药组可以显著抑制A549细胞的增殖及集落的形成，且呈剂量依赖性；光学显微镜观察发现，柠檬烯给药后A549细胞出现脱落现象，并可显著改变其形态；同时柠檬烯具有诱导A549细胞凋亡作用，并阻滞在G₀-G₁期；共聚焦显微镜发现柠檬烯作用后，A549细胞线粒体荧光减弱，同时细胞内铁含量亦显著增加，呈现典型的铁死亡表现；代谢组学研究发现谷胱甘肽(glutathione，GSH)代谢、精氨酸生物合成、D-谷氨酰胺和D-谷氨酸代谢及半胱氨酸和蛋氨酸代谢等多条差异代谢通路，这些通路与细胞内GSH合成密切相关；Western blotting实验发现，柠檬烯给药后细胞中SLC40A1、SLC7A11(xCT)及GPX4蛋白含量显著减少。结论 柠檬烯抗肺癌作用机制可能与降低肺癌细胞中GSH合成及增加Fe²⁺含量诱导其铁死亡有关。     

丹参多酚酸盐对胶质瘤U251细胞增殖及凋亡的影响

目的 研究丹参多酚酸盐对胶质瘤U251细胞增殖及凋亡的影响,并探讨其发生机制。方法 应用CCK-8试剂盒检测丹参多酚酸盐对U251细胞增殖的影响,流式细胞仪观察丹参多酚酸盐对U251细胞周期及细胞凋亡的影响,qRT-PCR检测自噬基因Beclin-1的表达。结果 丹参多酚酸盐可明显抑制胶质瘤U251细胞增殖,使细胞阻滞在G₀/G₁期,促进细胞凋亡,上调自噬基因Beclin-1 mRNA的表达。结论 丹参多酚酸盐可抑制胶质瘤U251细胞生长,诱导细胞发生凋亡,促凋亡效应可能与自噬基因Beclin-1 mRNA表达增多有关。     

冬虫夏草菌丝体水提醇沉物体外抗肿瘤活性研究

目的研究冬虫夏草菌丝体水提醇沉物的体外抗肿瘤活性及其机制。方法采用热水浸提一醇沉法得到水提物,高效液相色谱仪测定水提醇沉物中腺苷的量;采用MTT法测定其抗肿瘤活性,利用流式细胞仪结合碘化丙锭染色法检测其对细胞周期的抑制。结果实验表明水提醇沉物能抑制人肝癌HepG2细胞株及大细胞肺癌NCI．H460细胞株的增殖,并呈浓度相关性;半数抑制浓度（IC50）值分别为（1．49±0．19）和（1．67±0．27）mg／mL。细胞周期分析表明,水提醇沉物分别阻滞HepG2及NCI．H460细胞周期于G,／M期、S期,并可诱导上述两种细胞发生凋亡。结论冬虫夏草水提醇沉物通过阻滞HepG2及NCI-H460细胞周期循环,诱导其凋亡,从而表现出良好的增殖抑制活性。为深入研究冬虫夏草菌丝体水提醇沉物抗肿瘤的机制提供了实验证据。     

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.