羟基红花黄色素A通过激活Kv通道舒张大鼠冠状动脉

目的:研究羟基红花黄色素A (hydroxysafflor yellow A,HSYA)对离体大鼠冠状动脉(rat coronary artery,RCA)的舒张作用,并探讨该作用与电压门控性钾通道(Kv通道)的关系。方法:采用DMT离体血管环技术明确HSYA对RCA的肌源性作用及血管内皮对该作用的影响;采用全细胞膜片钳技术观察HSYA对RCA平滑肌细胞Kv电流的影响;采用Western blot技术探讨HSYA对RCA平滑肌细胞Kv1.2和Kv1.5通道蛋白表达的影响。结果:10μmol/L和30μmol/L HSYA能够舒张60 mmol/L KCl或0.1 mmol/L U46619预收缩的RCA(P<0.05),但其对预收缩的内皮完整组和去内皮组RCA舒张作用的差异无统计学显著性(P>0.05);HSYA(3μmol/L、10μmol/L和30μmol/L)可使RCA平滑肌细胞Kv电流显著增加(P<0.05);10μmol/L和30μmol/L HSYA能够增加RCA平滑肌细胞Kv1.2和Kv1.5通道蛋白的表达。结论:HSYA可舒张预收缩的RCA,该作用与血...     

高钾溶液对猪冠状动脉内皮细胞功能影响的实验研究

目的 :探讨高钾溶液对猪冠状动脉内皮细胞功能的影响及其机制。方法 :取新鲜猪心外膜下冠状动脉前降支下三分之一 ,切成 3mm长的血管环。采用器官槽法 ,分别用Krebs Henseleit重碳酸盐缓冲液 (KH)、2 0mmol/L的高钾溶液、50mmol/L的高钾溶液浸泡血管环 1h后 ,检测在 7μmol/L环加氧酶阻断剂消炎痛、3 0 0 μmol/L一氧化氮合成酶阻断剂N 硝基 L 精氨酸、1mmol/L钙激动性钾通道阻断剂四乙胺、或 3μmol/LATP敏感性钾通道阻断剂优降糖的作用下 ,3 0nmol/L前列腺素F2α引发的预收缩强度和非受体介导钙离子载体 ( 10 -1 0 ～ 10 -6mol/L)引发的内皮源性舒张反应。结果 :与单纯浸泡于KH的冠状动脉相比 ,内皮源性超极化因子 (EDHF)介导的内皮源性舒张反应 ,经 2 0mmol/L、50mmol/L高钾溶液及四乙胺作用后显著降低 ,但加入优降糖后改变不明显。结论 :EDHF在冠状动脉内皮源性舒张中起重要的作用 ,高浓度钾离子抑制EDHF的作用、EDHF介导血管舒张的作用过程主要与钙激动性钾通道有关     

心肌肽素抑制大鼠心室肌细胞瞬时外向钾电流

目的研究新药心肌肽素对大鼠心室肌细胞瞬时外向钾电流(Ito)的影响及其在离子通道水平的作用机制。方法用急性酶解法分离大鼠心室肌细胞,用标准的全细胞膜片钳技术,观察不同浓度的心肌肽素对Ito的影响。结果心肌肽素呈浓度依赖性抑制大鼠心室肌细胞Ito,心肌肽素浓度(mg/L)为10、50、100、250和500时分别使Ito降低(%)4、13、22、32和38。心肌肽素50 mg/L使Ito电流密度-电压曲线下移,但不改变曲线的形状,也不改变Ito稳态激活曲线。结论心肌肽素抑制大鼠心室肌细胞Ito,可能是其抗心律失常作用的机制之一。     

肌肽对高糖诱导的大鼠H9C2心肌细胞损伤的保护作用及其机制

目的 研究肌肽对高糖诱导受损的大鼠心肌细胞（H9C2）的保护作用及其机制。 方法 用含50 mmol/L葡萄糖的高糖培养液建立高糖损伤模型,实验观察组给予5、15 mmol/L肌肽进行保护。MTT检测各组H9C2心肌细胞活力;ELISA测定培养液中IL-1β、IL-6和TNF-α炎症因子浓度含量;试剂盒检测细胞中SOD、MDA、AST和LDH活性;二氢乙锭测定细胞中活性氧含量;免疫印迹测定细胞中bax、bcl-2、p65和IκB-α的蛋白表达。 结果 肌肽预处理组细胞内活性氧含量下降,SOD活性升高,AST和LDH活性减弱,MDA含量降低;其培养液中炎症因子含量明显降低,细胞核中p65蛋白水平降低,胞浆中IκB-α蛋白水平升高,bax和bax/bcl-2比值降低。 结论 肌肽能够减轻高糖诱导的H9C2心肌细胞损伤,降低细胞炎症反应,其机制可能与ROS/NF-κB信号通路有关。     

大鼠肺动脉平滑肌细胞的分离及电压门控性钾电流的记录方法

目的介绍一种大鼠肺动脉平滑肌细胞(PASMCs)的急性分离方法并观察电压门控性钾电流电生理特性。方法应用胶原酶和木瓜蛋白酶联合消化法获得大鼠PASMCs,利用全细胞膜片钳技术记录PASMCs膜上的电压门控性钾通道(Kv)电流。结果在相差显微镜下观察大鼠PASMCs呈舒展梭形,边界清晰,有完整的细胞膜,胞浆均匀,数量多,活性好。结论用酶急性分离的大鼠PASMCs,容易进行全细胞膜片钳记录,方法简单、稳定、可靠。     

RXR激动剂通过抑制PKC激活对抗高糖诱导的大鼠血管平滑肌细胞增殖

 目的:探讨视黄醇类X受体 (RXR)激动剂对高糖诱导的大鼠主动脉平滑肌细胞(RASMCs)增殖的影响及其作用机制。方法:体外组织块干涸法培养RASMCs,以25 mmol/L葡萄糖干预,模拟糖尿病患者体内环境,通过WST-1法检测细胞增殖活性,BrdU插入法测定细胞DNA合成,流式细胞术检测细胞周期进程。用免疫印迹杂交方法检测细胞周期蛋白依赖激酶2(CDK2)、细胞周期蛋白依赖激酶抑制物p27Kip1的蛋白表达及蛋白激酶C (PKC)的磷酸化水平。结果:(1) 在高糖环境(葡萄糖终浓度为25 mmol/L)下,RASMCs的增殖活性、DNA合成速率及其在细胞周期S期的分布比例均显著增加;(2) 高糖显著增加RASMCs内CDK2蛋白的表达,但明显降低p27Kip1的蛋白表达水平;(3) RXR天然配体9-顺式维甲酸(9-cis-RA)可显著抑制高糖诱导的RASMCs增殖活性增强、DNA合成加速及RASMCs在细胞周期S期分布比例的增加幅度,且具有浓度依赖性;10-7mol/L浓度的SR11237(RXR特异性配体)与等浓度的9-cis-RA具有相似的抑制效应;(4) 9-cis-RA 和SR11237均可显著抑制高糖诱导的CDK2蛋白表达水平的增加幅度,同时上调高糖环境下p27Kip1蛋白的表达;(5) PKC抑制剂(PKC inhibitor peptide, 20 μmol/L)显著抑制高糖环境下RASMCs的增殖活性和CDK2蛋白的表达,但明显增加高糖条件下p27Kip1蛋白的表达;(6) 9-cis-RA 和SR11237可抑制高糖诱导的PKC蛋白磷酸化。结论: PKC的活化参与了高糖诱导下RASMCs的增殖过程。RXR激动剂通过抑制PKC活化对抗高糖诱导的血管平滑肌细胞增殖。     

缺氧缺糖/复氧复糖对大鼠皮质及海马神经元ClC-3氯离子通道表达的影响

目的:研究原代培养大鼠前额叶皮质和海马神经元缺氧缺糖/复氧复糖后电压门控氯离子通道3(voltage-gated chloride channel 3,ClC-3)在mRNA和蛋白水平的表达。方法:取原代培养8 d的大鼠皮质和海马神经元,随机分为对照组和模型组。模型组缺氧缺糖30 min后再复氧复糖,于复氧复糖后6、24、48、72 h采用反转录酶-聚合酶链锁反应(RT-PCR)、Western blot技术检测ClC-3 mRNA及蛋白水平的表达。结果:原代培养大鼠皮质和海马神经元ClC-3 mRNA及蛋白水平呈低水平表达。缺氧缺糖/复氧复糖24 h后培养皮质神经元ClC-3mRNA及蛋白水平开始上调,48 h仍然高表达,72 h后下降(P<0.05)。海马神经元ClC-3 mRNA在缺氧缺糖/复氧复糖6 h后即开始升高,高峰持续从24～48 h(P<0.05),72 h下降至略高于正常水平(P<0.05)。海马神经元ClC-3蛋白在24 h后表达开始逐渐升高,至72 h仍高表达(P<0.05)。结论:C1C-3通道表达上调可能增强复氧复糖后细胞对氧化应激、炎性反应的同时也促进细胞凋亡。     

阿托伐他汀通过抑制PKC的激活对抗高糖诱导的人脐静脉内皮细胞氧化应激反应

目的:探讨阿托伐他汀对高糖诱导的人脐静脉血管内皮细胞(HUVECs)产生氧化应激的影响及其作用机制。方法:体外培养HUVECs,以25 mmol/L葡萄糖干预,模拟糖尿病患者体内环境,通过流式细胞术和共聚焦显微镜检测细胞内的活性氧(ROS)水平,采用Lucigenin分析方法测定还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性,分别应用实时荧光定量PCR和免疫印迹杂交的方法检测 NADPH氧化酶亚基Nox4和Nox2/gp91^phox的表达水平,用免疫印迹杂交方法检测蛋白激酶C(PKC)蛋白的磷酸化水平。结果:(1)在高糖环境(终浓度为25 mmol/L)下,HUVECs内ROS生成显著增加,NADPH氧化酶的活性显著增强,NADPH 氧化酶Nox4和Nox2/gp91^phox亚基的mRNA和蛋白表达水平显著上调;(2)阿托伐他汀可显著抑制高糖诱导的ROS 生成、NADPH氧化酶活性的增强及NADPH 氧化酶Nox4和Nox2/gp91^phox亚基表达水平的增加幅度,且具有浓度依赖性;(3)PKC抑制剂(PKC inhibitor peptide, 20 μmol/L)可显著抑制高糖环境下ROS的生成、NADPH氧化酶活性的增强及NADPH 氧化酶Nox4和Nox2/gp91^phox亚基表达水平的增加幅度;(4)阿托伐他汀可抑制高糖诱导的PKC蛋白的磷酸化。结论:PKC的活化参与了高糖诱导的HUVECs产生的氧化应激反应。阿托伐他汀通过抑制PKC蛋白的活化对抗高糖诱导的内皮细胞产生的氧化应激反应。     

活性氧与丝裂原激活蛋白激酶通路的相互作用介导高糖引起的心肌细胞损伤

目的探讨活性氧(ROS)与丝裂原激活蛋白激酶(MAPK)通路的相互作用在高糖损伤H9c2心肌细胞中的作用。方法应用细胞计数盒(CCK-8)检测细胞存活率;Hoechst 33258核染色检测凋亡细胞形态及数量的改变;双氯荧光素(DCFH-DA)染色荧光显微镜照相检测细胞内ROS水平;Western blot测定蛋白质表达水平。结果高糖(35 mmol/L葡萄糖)处理H9c2心肌细胞24 h可引起明显的损伤,表现为细胞存活率下降,凋亡细胞数量及ROS水平明显升高。另方面,高糖可明显地上调磷酸化(p)p38MAPK、细胞外信号调节蛋白激酶1/2(ERK1/2)及c-Jun N端激酶(JNK)(为MAPK家族的3个成员)的表达水平。N-乙酰半胱氨酸(NAC,为ROS清除剂)能抑制高糖引起的心肌细胞毒性和细胞凋亡,也能阻断高糖对p-p38MAPK、p-ERK1/2及p-JNK表达的上调作用。此外,p38MAPK、ERK1/2和JNK的选择性抑制剂均能抑制高糖引起的心肌损伤,并能抑制ROS生成增多。结论在高糖损伤H9c2心肌细胞中,存在ROS与MAPK通路的正相互作用,这种相互作用可能在高糖引起的心肌细胞损伤中起着重要的作用。     

线粒体KATP通道开放对培养成年大鼠心肌细胞PKC epsilon转位激活的影响

目的：探讨MitoKATP通道特异性开放剂二氮嗪(DZ)预处理对PKCε的转位激活作用及其与活性氧生成的关系。 方法： 采用免疫荧光和Western blotting等技术检测培养成年大鼠心室肌细胞PKCε的表达。 结果： ①MitoKATP通道特异性开放剂DZ预处理能引起PKCε向心肌细胞肌丝样结构转位；②活性氧清除剂2-巯基丙酰氨基乙酸(MPG)能抑制DZ预处理引起的PKCε转位；③PKC特性抑制剂氯化白屈菜赤碱(CH)能完全消除DZ预处理引起的PKCε转位。 结论： MitoKATP通道开放能够激活PKCε向肌丝样结构转位。MitoKATP通道开放过程中生成的ROS是引起PKCε转位激活的重要原因。     

酸中毒致大鼠离体冠状动脉收缩的机制

目的:探讨细胞外酸中毒(pH_(ex)6.8)致大鼠离体冠状动脉(RCA)收缩的机制。方法:采用离体微血管张力法,通过观察Na~+-H~+交换体1(NHE-1)选择性抑制剂cariporide(HOE-642)或Na~+-HCO_3~-共同转运体(NBC)抑制剂S0859预孵对pH_(ex)6.8所致RCA收缩的影响,探讨酸碱转运体在酸中毒收缩RCA中的作用;通过观察氯通道抑制剂NPPB和尼氟酸(NFA)预孵及细胞外去除氯离子对pH_(ex)6.8所致RCA收缩的影响,探讨氯离子通道在酸中毒收缩RCA中的作用。结果:pH_(ex)6.8引起RCA静息张力升高,最大张力为(3.90±0.95)mN。30μmol/L HOE-642和100μmol/L S0859均抑制pH_(ex)6.8引起的RCA收缩(P0.01)。NPPB和NFA均呈浓度依赖性地抑制pH_(ex)6.8引起的RCA收缩和KCl(60 mmol/L)引起的收缩。100μmol/L的NPPB和NFA均抑制U46619(1μmol/L)引起的RCA收缩(P0.01)。等摩尔NaBr代替细胞外液中NaCl后,几乎完全抑制pH_(ex)6.8引起的RCA收缩(P0.01),但是对KCl(60 mmol/L)或U46619(1μmol/L)引起的RCA收缩无显著影响。结论:细胞外液酸化所致的RCA收缩与激活NHE-1和NBC及促进氯离子跨细胞膜运动有关。     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

Extracellular Mg2+ blocks endothelin-1-induced contraction through the inhibition of non-selective cation channels in coronary smooth muscle

This study investigated the effects of changing the extracellular [Mg²⁺] ([Mg²⁺]_o) on endothelin-1 (ET-1)-induced contraction of rabbit coronary artery smooth muscle and the involvement of non-selective cation (NSC) channels in this response. Increased [Mg²⁺]_o shifted the concentration/contraction relationship curve of ET-1 to the right. In whole-cell patch clamp recordings, ET-1 (10^–7 M) induced a long-lasting inwards current (94.7±7.2 pA) that was inhibited by 8 mM [Mg²⁺]_o (45.3±4.4%) and NSC channel blockers (10^–3 M streptomycin and 10^–3 M La³⁺), but not by the voltage-dependent Ca²⁺ channel blocker nicardipine. The current/voltage (I/V) curve was linear. Furthermore, in pressurized arteries, the ET-1-induced contraction was also inhibited by La³⁺ and streptomycin, but not by nicardipine. U-73122, a selective phospholipase C (PLC) inhibitor and staurosporine and GF 109203X, which block protein kinase C (PKC), reduced ET-1-activated NSC currents by 54.2±5.1%, 60.3±5.5% and 48.5±2.9%, respectively. The inwards current was increased by 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol 12,13-dibutyrate (PDBu), which activate PKC selectively. Like transient receptor potential channel (TRPC3) currents, ET-1-activated NSC currents had a linear I/V relationship, were blocked by flufenamate and activated by a diacylglycerol analogue. These results suggest that [Mg²⁺]_o blocks ET-1-induced contraction of coronary arteries by inhibiting NSC channels. Activation of PLC and PKC might be involved in activation of NSC channels.     

高糖及高胰岛素对肺泡巨噬细胞氧化应激的影响

目的： 探讨高糖、高糖＋高胰岛素环境对大鼠肺泡巨噬细胞（AM）氧化应激损伤的影响。方法: 采用支气管肺泡灌洗的方法分离大鼠AM,高糖及高糖＋高胰岛素环境下培养,用卡介苗（BCG）、干扰素α-2b（IFNα-2b）或两者联合活化,检测其丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性和乳酸脱氢酶（LDH）活性。结果: BCG和IFNα-2b＋BCG活化AM时,其SOD活性均低于对照组(P<0.05),而MDA含量和LDH活性均高于对照组(P<0.05);IFNα-2b活化AM时,其MDA含量、LDH活性和SOD活性与对照组无明显差别（P>0.05）。结论: 在高糖、高糖＋高胰岛素环境可诱导AM氧化应激损伤,从而使糖尿病个体易发生肺部感染。     

糖皮质激素诱导的蛋白激酶3种亚型在高糖诱导的人肾小管上皮细胞中的表达

目的：研究高糖环境下人近端肾小管上皮细胞（HKC）中血清和糖皮质激素诱导的蛋白激酶（SGK）3种亚型SGK1、SGK2和SGK3的表达，探讨SGK 3种亚型在介导高糖致肾小管上皮细胞过度合成细胞外基质（ECM）中的作用。 方法: 将细胞分为对照组、高糖组和渗透压对照组。分别采用RT-PCR方法和Western blotting方法检测SGK1、SGK2和SGK3 mRNA水平和SGK1蛋白水平的表达，ELISA方法和间接免疫荧光方法检测培养液中和HKC胞内纤连蛋白（FN）含量。 结果: HKC细胞中存在SGK1、SGK2和SGK3的表达。高糖刺激下， SGK1、SGK2和SGK3 mRNA和SGK1蛋白表达明显升高（P<0.01）；同时伴有HKC FN合成和分泌的增加，这与SGK上调存在一定联系。 结论: 高糖能促进近端肾小管上皮细胞SGK1、SGK2和SGK3的表达，并可能通过SGK1、SGK2和SGK3介导的信号转导途径促进细胞外基质积聚，可能在糖尿病肾病的发生和发展中发挥致病作用。     

Short-term response of metabolic hormones to coronary artery bypass surgery

PurposeTo explore the response pattern of plasma adipokine and ghrelin levels to coronary artery bypass graft (CABG) surgery in patients with (on-pump) and without (off-pump) cardiopulmonary bypass (CPB).Material/methodsSixteen consecutive patients (age: 62 ± 10 years, male: 10) with obstructive coronary artery disease (CAD) who underwent elective CABG surgery with CPB and intraoperative GIK infusion were selected for on-pump group and 19 CAD patients (age: 63 ± 10 years, male: 16) were included in the off-pump group. Blood samples were taken before, during and after surgery. Intraoperative samples were withdrawn simultaneously for peripheral vein and sinus coronarius (SC). Plasma adipokine concentrations were measured by ELISA, those of ghrelin by RIA kits.ResultsIn response to surgical intervention there was an early, transient fall in plasma levels of adiponectin (p < 0.0001) and resistin (p = 0.002) followed by an increase to approach their initial values. Plasma ghrelin also increased (p = 0.045), this increase, however, was confined to the period of GIK supported CPB. Plasma insulin (p = 0.003) and resistin (p = 0.009) was significantly higher in the peripheral vein than in SC. The perioperative hormone profile of patients without CPB (off-pump) proved to be comparable to that of on-pump patients in spite of the insulin administration and greater oxidative and inflammatory stress.ConclusionsAdipose tissue-derived factors appear to mediate the metabolic and vascular changes that occur in patients with CABG surgery. Epicardial adipose tissue is unlikely to have major contribution to the development of CAD as adipokines are not elevated in SC independent of the mode of intervention.     

Vasoconstriction in rat isolated mesentery and small intestine in response to various activators of protein kinase C

The vasculature of rat isolated mesentery and small intestine was perfused with a gelatin-containing physiological salt solution (GPSS). When 5-hydroxytryptamine (5HT, 1×10^–4 M), or the calcium ionophore A23187 (1×10^–4 M), or 12-deoxyphorbol 13-phenylacetate (DOPPA, 1×10^–6 M), or 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPAA, 1×10^–6 M) or thymeleatoxin (TMX, 1×10^–6 M) was added to the GPSS for 5 min there was a gradual rise in perfusion pressure, whereas resiniferatoxin (RFX, 1×10^–6 M) was without effect. Pre-treatment of the tissue with the protein kinase C (PKC) inhibitor Ro 31-8220 (1×10^–6 M) significantly reduced the rise in perfusion pressure in response to 5HT, DOPPA, DOPPAA and TMX, but not that to A23187. Platelet-activating factor (PAF, 5×10^–6 M) caused an almost immediate but transient rise in perfusion pressure, followed by a more gradual rise, neither response being blocked by Ro 31-8220. When blood vessels of the mesentery alone were perfused with gelatin-free PSS, PAF caused a transient rise in perfusion pressure, but with no subsequent gradual rise over 5 min. After Ca²⁺-depletion this transient response was also absent. In contrast, when blood vessels were perfused with gelatin-free PSS, DOPPA and TMX still caused gradual rises in perfusion pressure, which were totally abolished by Ro 31-8220. TMX had no effect at all when the tissue was depleted of Ca²⁺, whereas the response to DOPPA was only partially reduced. Phenylephrine (PE, 1×10^–6 M) produced a fairly rapid but non-sustained rise in perfusion pressure, which was reduced by Ro 31-8220, and more or less abolished by Ca²⁺-depletion. It is suggested that the pressor response to DOPPA involved activation of both Ca²⁺-dependent and Ca²⁺-independent PKC isoenzymes. TMX and PE, however, appear to activate only a Ca²⁺-dependent isoenzyme.