胸腺素β4对大鼠脑缺血再灌注后内质网应激的影响

目的研究胸腺素β4(Tβ4)对SD大鼠脑缺血再灌注损伤后内质网应激的影响。方法选取健康成年雄性SD大鼠48只,采用随机数字表法分为假手术组(Sham组)、脑缺血再灌注组(I/R组)和Tβ4干预组(Tβ4组),每组16只。采用线栓法制作大鼠大脑中动脉栓塞模型;Tβ4组在造模前、后1 h各腹腔注射1次Tβ4(8μg·kg~(-1)),Sham组和I/R组腹腔注射等量生理盐水。采用Longa评分法进行神经功能评分;采用TTC染色法测定脑梗死体积;采用TUNEL法检测神经细胞凋亡指数;采用HE染色法观察脑组织病理学改变;采用Western-blot法检测缺血脑组织葡萄糖调节蛋白78(GRP78)、C/EBP同源蛋白(CHOP)和Caspase 12表达水平。结果大鼠缺血再灌注24 h后,与Sham组相比,I/R组和Tβ4组大鼠神经功能评分、脑梗死体积及神经细胞凋亡指数均明显增加(P <0.05),GRP78、CHOP、Caspase 12蛋白表达增多(P <0.05);与I/R组相比,Tβ4组大鼠神经功能评分、脑梗死体积及神经细胞凋亡指数均有减少(P <0.05),GRP78表达增高,而CHOP和Caspase12表达明显降低(P <0.05)。结论胸腺素β4对大鼠脑缺血再灌注损伤具有一定的保护作用,该机制可能与其上调GRP78表达、下调CHOP和Caspase 12表达,从而减轻内质网应激有关。     

缺血预适应对局灶性脑缺血再灌注大鼠海马 CA1 区低氧诱导因子-1α、 血管内皮生长因子表达的影响#br#

目的 观察缺血预适应后局灶性脑缺血再灌注大鼠缺血侧海马 CA1 区低氧诱导因子（HIF） -1α、 血管内皮生长因子（VEGF）表达的变化, 探讨缺血预适应的脑保护机制。方法 雄性 SD 大鼠随机分为 3 组： 假手术（SO）组、 脑缺血（MCAO）组和缺血预适应（BIP）组, 后两组按缺血后再灌注时间不同分为再灌注 2、 6、 12、 24、 48、 72 h 6 个亚组。制备大鼠局灶性脑缺血预适应模型, 采用免疫组织化学法与 Western blot 法观察脑缺血后再灌注各个时间点海马 CA1 区 HIF-1α和 VEGF 的表达变化。结果 SO 组未见神经功能缺损症状, 与 MCAO 组相比, BIP 组再灌注各时间点神经功能缺损评分低 （P＜0.05）。MCAO 组、 BIP 组 HIF-1α、 VEGF 阳性细胞及蛋白表达均于再灌注 2 h 开始增多, 6～12 h 表达递增, 24 h 表达至高峰, 随后表达减少, 72 h 表达仍高于 SO 组。两组各时间点 HIF-1α和 VEGF阳性细胞及蛋白表达均高于 SO 组 （P＜0.05）。除再灌注 2 h、 6 h MCAO 组与 BIP 组 HIF-1α的阳性细胞表达差异无统计学意义外, 2 组各时间点 VEGF 阳性细胞表达、 HIF-1α、 VEGF 蛋白表达均是 BIP 组高于 MCAO 组（P＜0.05）。结论 脑缺血预适应可能通过上调 HIF-1α、 VEGF 的表达, 发挥脑保护作用。     

后适应对大鼠脑缺血神经细胞凋亡和内皮型一氧化氮合酶与诱导型一氧化氮合酶的影响

目的：研究缺血耐受（后适应和预适应）对脑缺血再灌注神经细胞凋亡及内皮型一氧化氮合酶（eNOS）和诱导型一氧化氮合酶（iNOS）蛋白表达的影响。方法：50只雄性SD大鼠随机分为假手术组（sham）、脑缺血再灌注模型组（MCAO）、预适应组（MCAO＋preconditioning）、后适应组（MCAO＋postconditioning）和尼莫地平组（MCAO＋nimodipine），每组10只大鼠。预适应组大鼠在MCAO前24h，双侧颈总动脉夹闭2min，再灌注20min，循环两次。后适应组大鼠在脑缺血再灌注开始时，再灌注20s，栓塞20s，循环3次。大鼠大脑中动脉阻断1．5h，再灌注24h。用TUNEL法和免疫组化染色法分别检测缺血半暗带凋亡细胞和iNOS、eNOS蛋白表达。结果：与MCAO组比较，后适应组大鼠脑缺血半暗带的凋亡细胞显著减少，eNOS蛋白表达显著增加，iNOS蛋白表达显著减少，差异有统计学意义（P〈0．05）。结论：缺血后适应能诱导脑缺血耐受，对脑缺血／再灌注损伤产生保护作用，其保护作用与促进eNOS蛋白的表达，抑制iNOS蛋白的表达有关。     

硫化氢对大鼠局灶性脑缺血/再灌注损伤的保护作用及其机制

目的探讨硫化氢(H2S)对大鼠局灶性脑缺血/再灌注损伤的保护作用及其机制。方法♂SD大鼠随机分成3组:假手术组、脑缺血/再灌注(I/R)组和硫氢化钠(NaHS)+I/R。线栓法建立大鼠左侧大脑中动脉栓塞(MCAO)模型,缺血2 h,再灌注24 h后,计算各组死亡率、Longa评分标准进行神经功能缺陷评分,2,3,5-三苯基氯化四氮唑(TTC)染色测量脑梗死体积,免疫荧光法检测大脑皮质和海马组织中P2X7受体蛋白表达。结果 NaHS+I/R组大鼠死亡率(27.27%)明显低于I/R组(42.86%),该组大鼠神经功能缺陷评分也明显低于I/R组(P<0.05),且脑梗死体积(21.88%±3.53%)明显低于I/R组(36.71%±3.73%)(P<0.01)。免疫荧光结果显示,与假手术组相比,I/R组大脑皮质、海马CA1区P2X7阳性表达细胞数明显增多(P<0.01);与I/R组相比,NaHS+I/R组大脑皮质、海马CA1区P2X7阳性表达细胞数明显减少(P<0.01)。结论 H2S可对局灶性脑缺血/再灌注损伤大鼠发挥脑保护作用,其机制可能与下调P2X7受体蛋白表达有关。     

Nrf2调控AIM2炎症小体在大鼠脑缺血再灌注 损伤中的作用

目的 探讨核因子E2相关因子2（Nrf2）对脑缺血再灌注损伤的影响及对黑色素瘤缺乏因子2（AIM2）炎症 小体的调控作用。方法 将108只雄性SD大鼠按随机数字表法分为假手术组（Sham组）、脑缺血再灌注模型组（I/R 组）、Nrf2抑制剂鸦胆子苦醇（Bru）干预组（Bru组），每组再按随机数字表法进一步分为脑缺血再灌注后8、24、72 h组， 共9组，每组12只。I/R组和Bru组采用线栓法建立大脑中动脉栓塞（MCAO）模型。于相应时间点对各组行神经功能 缺损评分；采用2，3，5-氯化三苯基四氮唑（TTC）染色测定脑梗死面积；HE染色检测脑组织病理损伤；荧光定量PCR 法检测缺血侧脑组织中AIM2 mRNA表达；Western blot法检测缺血区脑组织中AIM2、Nrf2蛋白的表达；酶联免疫吸 附试验法检测缺血区脑组织中凋亡相关斑点样蛋白（ASC）、半胱氨酸蛋白酶（Caspase）-1、白细胞介素（IL）-1β和IL-18 的表达。结果 与Sham组比较，I/R组各时间点神经功能缺损评分升高，脑组织病理损伤显著加重，AIM2 mRNA以 及Nrf2、AIM2、ASC、Caspase-1、IL-1β、IL-18蛋白表达水平升高（P＜0.05）。与I/R组比较，Bru组各时间点神经功能 缺损评分升高，脑组织病理损伤更重，Nrf2蛋白表达水平降低，AIM2 mRNA和蛋白，ASC、Caspase-1、IL-1β、IL-18蛋 白表达水平升高（P＜0.05）。结论 Nrf2在脑缺血再灌注损伤中具有内源性神经保护作用，其机制可能与Nrf2可负 性调控AIM2炎症小体，减少炎性细胞因子的释放有关。     

脂质胞壁酸诱导的延迟预适应对大鼠局灶性脑缺血/再灌注损伤的保护作用

目的 探讨LTA诱导的延迟预适应对大鼠局灶性脑缺血／再灌注损（I／R）损伤的作用。方法 采用改良Longa法制作大鼠右大脑中动脉（MCA）闭塞2h造成局灶性脑缺血模型，恢复血液灌流24h。大鼠在施行脑缺血前24h腹腔注射脂质胞壁酸（LTA，1mg／kg）诱导延迟预适应，检测脑组织再灌注24h后组织中超氧化物歧化酶（SOD）、丙二醛（MDA）和一氧化氮（NO）含量以及大鼠神经症状，并用透射电镜观测大鼠大脑皮层神经细胞的超微结构改变。结果 LTA预适应能明显减少脑I／R后组织中MDA的含量（P〈0、01），提高SOD的水平（P〈0．01），减轻神经细胞的超微结构损伤和保护细胞膜结构完整性，LTA预适应还能明显改善脑I／R后的神经功能，减少神经缺欠评分值（P〈0．01）。LTA预适应亦能明显降低I／R导致脑组织中NO含量的升高（P〈0．01）。结论 LTA诱导的延迟预适应能显著减少大鼠脑组织再灌注损伤，减少脑组织坏死，其作用机制与减少脑I／R后自由基和NO毒性作用有关。     

无创性延迟肢体缺血预适应对大鼠脑缺血再灌注皮层的保护作用

目的：研究无创性延迟肢体缺血预适应（NDLIP）对大鼠脑缺血再灌注损伤的保护作用。方法：通过连续3d,每天1次3个循环左后肢无创性5min缺血、5rain再灌注,建立NDLIP模型。实验分4组：假手术组（sham）、缺血再灌注组（I／R）、早期脑缺血预适应＋I／R组（ECIP＋I／R）、NDLIP＋I／R组。观察其对脑缺血／再灌注的神经缺损症状,脑梗死范围,超氧化物歧化酶（SOD）,谷胱甘肽过氧化物酶（GSH—PX）活力,黄嘌呤氧化酶活力（XOD）,丙二醛（MDA）含量影响。结果：与I/R组相比,ECIP＋I／R组及NDLIP＋I／R组缺血1h,再灌注24h后评分有非常显著的降低;脑梗死范围显著减小。与I／R组比较,ECIP＋I／R组和NDLIP＋I／R组的T-SOD和Mn—SOD活力、GSH—PX活力均升高;XOD活力明显降低,MAD含量明显减少;ECIP＋I／R组和NDLIP＋I／R组间差异无统计学意义。结论：NDLIP可降低I／R对神经的损伤、减小脑梗死范围、提高脑组织抗氧化能力,减少I／R对脑组织的损伤。     

丙泊酚在脑缺血再灌注中对凋亡诱导因子核转位的影响

目的研究丙泊酚是否能降低凋亡诱导因子(AIF)线粒体核转位,阻止Caspase非依赖性神经元凋亡,从而起到脑保护作用。方法 Wistar大鼠60只随机分为假手术组(S组)、缺血再灌注组(I/R组)、抑制剂组(I组)、丙泊酚组(P组)。S组大鼠仅接受手术而不遭受全脑缺血再灌注损伤打击;I/R组大鼠采用二血管夹闭法制造缺血模型10 min,然后再灌注;I组大鼠在缺血前2 min经静脉注入Caspase抑制剂;P组大鼠在全脑缺血再灌注前1 h,经股静脉持续泵注丙泊酚持续1 h,之后注入抑制剂,2 min后建立全脑缺血再灌注损伤模型。缺血再灌注后6、24、48 h,取海马组织用流式细胞仪测细胞凋亡率、Western blot法分析AIF线粒体核转位、凋亡相关基因Bax、Bcl-2的表达情况。结果与I/R、S、I组相比,P组神经元凋亡率显著降低,Bax/Bcl-2比值显著降低,AIF蛋白表达水平明显减少。结论丙泊酚对脑缺血再灌注损伤的脑保护作用可能与抑制海马神经元中AIF线粒体核转位有关。     

氟伐他汀预处理对缺血再灌注的大鼠心肌细胞Fas蛋白表达及心室重塑的影响

目的研究氟伐他汀预处理对缺血-再灌注损伤大鼠心肌细胞Fas蛋白表达及心室重塑的影响。方法将30只SD大鼠随机分成氟伐他汀组（Flu组）、缺血-再灌注组（I/R组）及缺血-预适应组（IPC组）,每组10只。分别用20mg/kg.d氟伐他汀、生理盐水灌胃给药。2周后均经历缺血-再灌注损伤。观察心室重塑和心肌细胞形态,并测定Fas蛋白阳性细胞OD值和心肌细胞凋亡指数。结果 IPC组及Flu组心肌细胞损伤及心室重塑程度明显减轻。Flu组、IPC组Fas蛋白阳性细胞OD值分别为0.20±0.07、0.27±0.11,与I/R组Fas蛋白阳性细胞OD值0.36±0.15比较,P〈0.05;Flu组、I/R组细胞凋亡指数分别为（26.66±9.94）%、（34.45±16.27）%,与I/R组（65.52±20.39）%比较,P〈0.01;而Flu组、IPC组Fas蛋白阳性细胞OD值、细胞凋亡指数比较,P〉0.05。结论氟伐他汀预处理能有效的通过Fas/FasL系统抑制缺血-再灌注损伤的细胞凋亡过程从而保护心肌细胞。     

转导血红素氧合酶-1蛋白减轻脑缺血/再灌注沙土鼠海马神经元的损伤

目的评价血红素氧合酶-1(HO-1)蛋白转导对脑缺血/再灌注损伤沙土鼠海马神经元的影响。方法构建11R(11个精氨酸残基)-HO-1蛋白,50只沙土鼠随机分为5组(n=10):脑缺血/再灌注组(C组),沙土鼠行脑缺血/再灌注损伤。脑缺血/再灌注+生理盐水组(S组)、脑缺血/再灌注+11R组(R组)、脑缺血/再灌注+5 mg·kg-111R-HO-1组(H1组)和脑缺血/再灌注+25 mg·kg-111R-HO-1组(H2组),各组沙土鼠腹腔分别注射5 mg·kg-1生理盐水、5mg·kg-111R蛋白、5 mg·kg-111R-HO-1蛋白或25 mg·kg-111R-HO-1蛋白,3 h后行脑缺血/再灌注损伤。24 h后取沙土鼠海马,电镜下观察海马组织线粒体的变化,检测海马神经元凋亡、Caspase-3和HO-1蛋白表达、cAMP水平。结果C组、S组和R组3组间海马神经元线粒体变性率、神经元凋亡率、Caspase-3和HO-1蛋白表达、cAMP水平变化无差异(P>0.05);H1组海马神经元线粒体变性率降低、神经元凋亡率降低、Caspase-3蛋白表达下调、HO-1蛋白表达上调、cAMP水平升高(vs C组、S组和R组,P<0.01);H2组海马神经元线粒体变性率降低、神经元凋亡率降低、Caspase-3蛋白表达下调、HO-1蛋白表达上调、cAMP水平升高(vs H1组,P<0.01)。结论 HO-1蛋白转导减轻了脑缺血/再灌注沙土鼠海马神经元的损伤。     

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.     

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.     

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.