κ受体激动剂对异丙肾上腺素诱导大鼠乳鼠心肌肥厚的作用机制研究

目的：利用体外培养的大鼠乳鼠心肌细胞,研究异丙肾上腺素（ISO）诱导肥大心肌细胞中κ阿片受体（κ-OR）的作用及其机制。方法：新生大鼠心肌细胞培养,Lowry’s法测心肌细胞的蛋白含量;用消化分离法,利用计算机图像分析系统测心肌细胞的体积;[3H]leucine掺入法测定心肌细胞蛋白的合成,Western blot法测细胞外信号调节激酶（ERK）磷酸化水平。结果：在给予ISO诱导心肌肥大的情况下,ERK通路抑制剂U0126可减少肥大心肌细胞的蛋白质含量、体积、蛋白合成和ERK磷酸化程度;κ-OR激动剂U50,488H（U50）能够减少肥大心肌细胞的蛋白含量、体积和蛋白合成,但与U0126共同作用时（U0126先孵育30min）,其抑制心肌肥大作用一定程度上减小。结论：ISO激活引起ERK磷酸化增加,抑制ERK可以阻断ISO的致肥大作用;κ-OR激活能够抑制ISO所诱导的心肌细胞肥大,其作用机制与ERK相关。     

κ-阿片受体激动对异丙肾上腺素诱导的乳大鼠心肌细胞肥厚的抑制作用

目的通过观察选择性κ-阿片受体(κ-OR)激动剂U50488H抑制异丙肾上腺素(Iso)诱导乳大鼠心肌细胞肥厚的作用及其对细胞内游离钙离子浓度([Ca2+]i)瞬变及钙调素依赖蛋白激酶Ⅱ(CaMKⅡ)表达的影响,研究κ-OR激动抑制Iso诱导的大鼠心肌细胞肥厚的信号传导机制。方法以体外培养的乳大鼠心肌细胞为模型,应用β肾上腺素受体激动剂Iso10μmol.L-1诱导心肌肥大,观察U50488H1μmo.lL-1的作用,并进一步探讨在CaMKⅡ特异性抑制剂KN930.2μmol.L-1,普萘洛尔2μmol.L-1及L-钙通道阻滞剂维拉帕米1μmol.L-1存在情况下,κ-OR的激活对心肌肥厚的作用。用Lowry法检测心肌细胞蛋白含量;消化分离法及计算机图像分析系统检测心肌细胞体积;[3H]亮氨酸掺入法测定心肌细胞蛋白的合成;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;用Western蛋白印迹法测定CaMKⅡδB表达。结果Iso10μmol.L-1使心肌细胞总蛋白含量、体积和蛋白合成明显增加,U50488H1μmol.L-1抑制Iso诱导的心肌肥大,且抑制程度与KN930.2μmol.L-1,普萘洛尔2μmol.L-1及维拉帕米1μmol.L-1相似,在KN93存在的情况下,U50488H抑制Iso诱导的心肌肥大作用增强;U50488H能降低Iso引起的心肌细胞[Ca2+]i瞬间变化升高;Iso能明显增强心肌细胞内CaMKⅡδB的表达,U50488H能降低其表达。结论κ-OR激动剂U50488H可能通过降低心肌细胞[Ca2+]i瞬间变化和减少心肌细胞内CaMKⅡδB的表达,抑制Iso诱导的乳大鼠心肌细胞肥厚。     

RISK信号通路在β_2-肾上腺素受体激动剂Clenbuterol减轻心肌细胞缺氧/复氧损伤中的作用

目的研究β2-肾上腺素受体激动剂clenbuterol对原代培养的心肌细胞缺氧/复氧损伤的作用及其是否与激活再灌注损伤挽救激酶(reperfusion injury salvage kinase,RISK)信号通路有关。方法将原代培养的新生Wistar大鼠乳鼠心肌细胞分为8组,1正常培养组;2缺氧/复氧(A/R)组;3clenbuterol(1μmol·L-1)+A/R;4ICI118,551(10μmol·L-1)+clenbuterol(1μmol·L-1)+A/R组;5美托洛尔metoprolol(10μmol·L-1)+clenbuterol(1μmol·L-1)+A/R组;6 metoprolol(10μmol·L-1)+A/R组;7 PD98059(20μmol·L-1)+clenbuterol(1μmol·L-1)+A/R组;8LY294002(10μmol·L-1)+clenbuterol(1μmol·L-1)+A/R组。采用MTT法测定各组细胞存活率;比色法检测心肌细胞培养液的乳酸脱氢酶(LDH)含量;Hoechst 33342荧光染色法检测细胞凋亡率;分子探针DCFH-DA检测细胞内活性氧的水平;Western blot检测心肌细胞缺氧/复氧后ERK及p-ERK1/2蛋白的表达水平。结果与A/R组比较,clenbuterol+A/R组明显增高细胞存活率,降低LDH含量,降低细胞凋亡率,ROS产生减少,p-ERK1/2蛋白表达水平增高,而选择性β2受体阻断剂ICI 118,551可取消clenbuterol的上述作用,β1受体阻断剂Metoprolol对clenbuterol的作用无影响,PI3K抑制剂LY294002和ERK1/2抑制剂PD98059可阻断clenbuterol对心肌细胞缺氧/复氧损伤的保护作用。结论clenbuterol能够减轻心肌细胞缺氧/复氧损伤,加入选择性β2受体阻断剂ICI 118,551,PI3K抑制剂LY294002和ERK抑制剂PD98059均使clenbuterol的保护作用取消,表明clenbuterol可通过激动β2肾上腺素受体,激活RISK信号通路发挥抗心肌细胞缺氧/复氧损伤的作用。     

κ受体激动对去甲肾上腺素诱导的心肌肥大的抑制作用

目的　利用体外培养的乳鼠心肌细胞 ,观测κ阿片肽受体激动对去甲肾上腺素 (norepinephrine ,NE)诱导的心肌肥大的抑制作用 ,并探讨作用机制。方法　对培养乳鼠心肌细胞分组给药 48h后 ,用Lowrys法测心肌细胞的蛋白质含量 ;用消化分离法 ,通过目镜标尺测心肌细胞体积 ;用镜下计数法测心肌细胞的搏动频率。结果　κ阿片肽受体激动剂U5 0 ,488H(简称U5 0 ) (0 1～ 10 μmol·L-1)对培养心肌细胞的蛋白质含量具有抑制作用并呈剂量依赖性 ;对NE诱导的心肌细胞蛋白含量增加、体积增大具有抑制作用 ;同时观察到U5 0 ,488H(1μmol·L-1)具有抑制心肌细胞搏动频率的作用。U5 0 ,488H的上述作用均可被κ阿片肽受体拮抗剂Nor BNI(1μmol·L-1)拮抗。结论　U5 0 ,488H对NE诱导的心肌肥大具有抑制作用 ,这种作用是通过激动κ阿片肽受体发挥的。     

钙调磷酸酶信号通路参与κ-阿片受体激动对异丙肾上腺素诱导的乳大鼠心肌细胞肥大的抑制作用

目的研究钙调磷酸酶(CaN)信号通路在κ-阿片受体(κ-OR)激动对异丙肾上腺素(Iso)诱导的乳大鼠心肌细胞肥大中的抑制作用。方法利用体外培养模型,以Iso 10μmol.L-1诱导心肌肥大,观察U50488H1μmo.lL-1的作用,并探讨在环孢素A(CsA)1μmol.L-1、cAMP三乙胺盐(Rp-cAMPS)及百日咳毒素(PTX)5 mg.L-1存在的情况下,κ-OR的激活对心肌肥大的影响。用Lowry等法测定心肌细胞蛋白质含量;用消化分离法及计算机图像分析系统测定细胞体积;采用Till阳离子测定系统,以Fura-2/AM为荧光探针,观察胞内[Ca2 +]i瞬间变化,用Western印迹法测定CaN的表达。结果与正常对照组相比,Iso 10μmo.lL-1使心肌细胞蛋白质含量增加了42.1%,体积增大了86.7%,[Ca2 +]i瞬变增加了57.5%,CaN表达增加了101.7%;加入κ-OR激动剂U50488H1μmol.L-1后,与Iso模型组相比,心肌细胞总蛋白质含量和细胞体积分别降低了24.7%和46.7%,[Ca2 +]i瞬变降低了43.8%,CaN表达降低了48.8%;CsA和Rp-cAMPS对Iso诱导的上述指标的抑制程度与U50488H相似;PTX预处理的情况下U50488H对Iso诱导的上述指标的抑制作用消失。结论κ-OR激动通过降低胞内[Ca2 +]i瞬变和CaN表达抑制Iso诱导的乳大鼠心肌细胞肥大。     

β肾上腺素受体激动对人脐静脉内皮细胞一氧化氮合酶磷酸化的影响

目的：观察血管内皮β肾上腺素受体激动对内皮一氧化氮合酶（eNOS）蛋白表达及磷酸化水平的影响,阐明β受体激动后eNOS调节的分子机制。方法：异丙肾上腺素（ISO）1μmol/L与培养的人脐静脉内皮细胞（HUVEC）孵育30min后,裂解细胞并用免疫沉淀法分离eNOS蛋白,运用同位素两步色谱法（L-[^3H]精氨酸转化法）检测eNOS活性;蛋白免疫印迹增强化学发光法检测eNOS表达水平和eNOS蛋白丝氨酸磷酸化水平,并观察高选择性β1或β2肾上腺素受体阻断剂对上述作用的影响。结果：ISO与内皮细胞孵育30min引起eNOS活性增高;不影响eNOS蛋白表达,但丝氨酸磷酸化水平明显增加;选择性β2受体阻断剂ICI 118551可完全阻断ISO的作用,而选择性β1受体阻断剂CGP 20712A无影响。结论：ISO通过提高eNOS丝氨酸磷酸化水平增加eNOS活性,这种作用是通过β2肾上腺素受体介导的。     

ATP敏感性钾通道在U50488H抑制去氧肾上腺素诱导的乳大鼠心肌细胞肥大中的作用

目的研究ATP敏感性钾通道(KATP)在κ-阿片受体激动剂U50488H抑制乳大鼠心肌细胞肥大中的作用。方法以原代培养的新生大鼠心肌细胞为模型,应用去氧肾上腺素(PE)10μmol.L-1诱导心肌细胞肥大。细胞处理分为正常对照组、PE10μmol.L-1模型组、5-羟基癸酸(5-HD)100μmol.L-1组,格列本脲(Gli)50μmol.L-1组、PE10μmol.L-1+U50488H1μmol.L-1组、PE10μmol.L-1+Gli50μmo.lL-1+U50488H1μmo.lL-1组和PE10μmo.lL-1+5-HD100μmo.lL-1+U50488H1μmol.L-1组,细胞培养液中先加入Gli50μmol.L-1或者5-HD100μmol.L-1,30min后再加入U50448H1μmol.L-1,30min后最后加入PE10μmol.L-1,48h后进行指标检测,Lowry等法检测心肌细胞蛋白质含量;消化分离法及计算机图像分析系统检测心肌细胞体积;Western印迹法测定KATP通道Kir6.2亚基的表达。结果与正常对照组相比,PE10μmol.L-1模型组使心肌细胞总蛋白质含量比正常细胞增加了52.2%,细胞体积增加了95.0%,而Kir6.2的表达没有明显变化。与PE10μmol.L-1模型组相比,细胞加入U50488H1μmol.L-1后,心肌细胞总蛋白质含量和细胞体积分别降低了42.3%和47.9%,但是Kir6.2表达增加了39.2%。与PE10μmo.lL-1+U50488H1μmol.L-1组相比,在非选择性KATP通道阻滞剂Gli50μmol.L-1或线粒体KATP通道阻滞剂5-HD100μmol.L-1存在的情况下,Kir6.2表达分别减少了49.3%和52.1%,U50488H抑制PE诱导的心肌细胞肥大作用减弱,并且两组之间没有显著差异。结论 U50488H可能是通过开放KATP通道,主要是线粒体KATP通道来抑制PE诱导的乳大鼠心肌细胞肥大。     

к阿片受体可通过抑制β肾上腺素受体进而调节心肌细胞Cx43发挥抗缺血/再灌注性心律失常

目的观察κ阿片受体选择性激动剂(U50488H)及β肾上腺素受体激动剂(异丙肾上腺素,ISO)对大鼠心脏缺血/再灌注(ischemia and reperfusion,I/R)引起的心律失常的影响,初步探讨U50488H对心肌细胞缝隙连接蛋白Cx43的调节机制。方法实验大鼠随机分为5组即对照组、I/R组、ISO+I/R组、U50488H+ISO+I/R组、Nor-BNI(κ阿片受体阻断剂)+U50488H+ISO+I/R组。观察每组心律失常的发生情况并计算心律失常评分,RT-PCR检测Cx43 mRNA表达及用免疫组化方法显示大鼠心肌Cx43的分布特征,半定量统计分析。结果①心律失常评分:ISO+I/R组高于I/R组(P<0.05),在ISO前给予U50488H则可以降低ISO引起的心律失常(P<0.05),其效应可被Nor-BNI阻断。②Cx43 mRNA水平:ISO+I/R组比I/R组略增高,在ISO前给予U50488H则可使基因表达水平降低(P<0.05),其效应可被Nor-BNI阻断。③Cx43蛋白表达:ISO+I/R组Total-Cx43高于I/R组(P<0.05),P-Cx43降低但与I/R组比较差异无统计学意义,给予U50488H后,可提高总Cx43和P-Cx43表达量(P<0.05),其效应可被Nor-BNI阻断。结论κ阿片受体激动剂可通过抑制β肾上腺素受体对Cx43调节从而发挥抗缺血/再灌注性心律失常的作用。     

埃他卡林对异丙肾上腺素诱导心肌细胞肥大的影响

目的在原代培养的新生大鼠心肌细胞上观察埃他卡林(iptakalim,IPT)对异丙肾上腺素诱导心肌细胞肥大的影响。方法采用SD大鼠乳鼠原代心肌细胞培养法,Lowry法测定心肌细胞总蛋白含量;激光共聚焦显微镜观察心肌细胞内游离Ca2+浓度。结果①剂量为1×10-5mol.L-1的异丙肾上腺素(isoprenaline,ISO)能有效地诱导心肌细胞肥大;②IPT能剂量依赖性抑制ISO诱导心肌细胞总蛋白含量和心肌细胞游离Ca2+浓度增加。结论IPT能明显抑制ISO诱导的心肌细胞总蛋白含量的增加,其机制可能与抑制Ca2+内流有关。     

腺苷A1受体激活在钙调磷酸酶信号通路上对异丙肾上腺素诱导的心肌细胞肥大的抑制作用

目的观察腺苷A1受体激活在钙调磷酸酶(CaN)通路上对异丙肾上腺素(Iso)诱导的心肌细胞肥大的抑制作用及机制。方法体外培养大鼠乳鼠心肌细胞,以Iso 10μmol.L-1诱导心肌细胞肥大,观察腺苷A1受体激动剂R(-)-N6-(2-phenylIsopropyl)adenosine(R-PIA)1μmol.L-1对其作用,进一步探讨钙调神经磷酸酶特异性抑制剂环孢菌素A(CSA)1μmol.L-1、PKA抑制剂cAMP三乙胺盐(RP-cAMPS)1μmol.L-1、百日咳毒素(PTX)5 mg.L-1存在时,腺苷A1受体的激活对心肌细胞肥大的影响。通过Lowry法测心肌细胞蛋白含量;RT-PCR法检测心肌细胞心钠素(ANP)的mRNA表达;Western blot法测心肌细胞CaN的相对表达水平;以Fluo-3/AM为荧光探针,共聚焦显微镜下测量心肌细胞[Ca2+]i瞬变。结果 10μmol.L-1 Iso可以诱导心肌细胞肥大,腺苷A1受体激动剂R-PIA可以使其蛋白含量降低、ANP的mRNA表达减少、CaN相对表达降低、[Ca2+]i荧光强度减小,CSA、RP-cAMPS有类似抑制作用,PTX预处理的情况下,R-PIA对Iso诱导的心肌肥大的抑制作用消失。结论腺苷A1受体可以通过钙调磷酸酶通路抑制Iso诱导的心肌肥大,其机制与降低细胞内[Ca2+]i浓度及CaN表达有关。     

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.     

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.     

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.     

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.