二氮嗪预处理对大鼠心肌线粒体呼吸功能与呼吸酶活性的影响

目的： 探讨特异性线粒体三磷酸腺苷敏感性钾通道开放剂二氮嗪预处理对离体大鼠缺血再灌注心肌线粒体呼吸功能和酶活性的影响。方法: 采用Langendorff装置建立大鼠离体心肌缺血再灌注模型，将72只SD大鼠随机分为正常组（NOR）、缺血再灌注组（IR）、二氮嗪预处理组（DIA）、5－羟葵酸拮抗二氮嗪组（5HD-DIA）。NOR组在平衡灌注20 min后续灌100 min，IR组在平衡20 min后续灌30 min，继后全心缺血40 min，复灌30 min。DIA组在缺血前给予含二氮嗪50 μmol/L的K-H液10 min后，全心缺血40 min，复灌30 min。5HD-DIA组在二氮嗪预处理之前先给予含5－羟葵酸100 μmol/L K-H液10 min，其余同二氮嗪预处理组。分别于平衡末、缺血前及再灌注末取心肌并分离、制备线粒体，测定各组线粒体的呼吸功能、呼吸酶活性。结果: 再灌注末DIA组的线粒体呼吸功能（呼吸控制率、磷氧比、3态呼吸速率）和呼吸酶活性（NADH氧化酶、琥珀酸氧化酶、细胞色素C 氧化酶）明显优于IR组和5HD-DIA组（P<0.05）但次于NOR组（P<0.01）；而IR组和5HD-DIA组比较无显著差异(P>0.05)。结论: 线粒体钾通道开放剂二氮嗪预处理能够保护缺血/再灌注损伤心肌的线粒体，其机制与保护线粒体的呼吸功能及呼吸链的酶活性有关。     

5-羟基癸酸盐对慢性低氧大鼠肺动脉Kv1.5通道表达的影响

目的: 以大鼠为研究对象,研究线粒体ATP敏感性钾通道(mitoK_ATP)的抑制剂5-羟基癸酸盐(5-HD)对慢性低氧肺动脉高压大鼠的影响及其潜在机制。方法: 24只SD雄性大鼠随机分成对照组、低氧组、低氧+5-羟基癸酸盐干预组,每组8只。将低氧组和5-HD干预组大鼠放入常压低氧舱内 以建立低氧肺动脉高压模型。 4周后测定平均肺动脉压(mPAP)及右心室与左心室及室间隔的重量比 ,并采用RT-PCR及Western blotting技术,分析各组肺动脉Kv1.5 mRNA及蛋白表达。结果: (1) 慢性低氧组大鼠的mPAP及RV/(LV+S)显著高于正常对照组(P<0.05),5-HD干预组mPAP及RV/(LV+S)显著低于低氧组,均P<0.05。(2) 低氧组Kv1.5通道mRNA及蛋白表达显著低于正常组,5-HD组Kv1.5通道表达显著高于低氧组, 均P<0.05。结论: mitoK_ATP通道的抑制剂5-HD通过降低mPAP及RV/(LV+S),在慢性低氧肺动脉高压中起保护作用。mitoK_ATP通道的抑制及Kv1.5通道表达的上调可能与该保护作用有关。     

K_(ATP)通道开放剂对氧糖剥夺诱导的SH-SY5Y细胞凋亡及AIF信号通路的影响

目的观察KA TP通道开放剂对氧糖剥夺诱导SH-SY5Y细胞凋亡的影响,并进一步探讨K ATP通道开放剂对凋亡诱导因子(apoptosis inducing factor,AIF)信号通路的影响。方法取传代后3d的SH-SY5Y细胞,分为A组(正常对照组)、B组(氧糖剥夺组)、C组(二氮嗪处理组)、D组(二氮嗪+5-羟葵酸处理组)。采用MTT方法检测细胞活力,应用流式检测细胞凋亡,应用Western-blot及免疫荧光染色检测AIF蛋白表达变化及移位。结果与正常对照组细胞相比,氧糖剥夺后细胞活力明显降低,100及200μmol/L的二氮嗪处理以后细胞活力明显升高,200μmol/L二氮嗪升高细胞活力更明显。与正常对照组细胞相比,氧糖剥夺后细胞凋亡率明显增高,二氮嗪处理以后细胞凋亡率明显下降。A,B,C,D四组AIF蛋白总量表达无显著性的差异(P0.05);在正常对照组,AIF蛋白以胞浆表达为主,氧糖剥夺导致AIF发生了明显的细胞核转移,二氮嗪处理显著减少氧糖剥夺诱导的AIF发生的细胞核转移。结论 KA TP通道开放剂二氮嗪能明显增加氧糖剥夺后SH-SY5Y细胞活力,减少细胞凋亡,减少氧糖剥夺诱导的AIF发生的细胞核转移。     

成年大鼠脑内ATP敏感性钾通道亚型mRNA表达的研究

为探讨ATP敏感性钾通道(KATP)亚型在大鼠脑组织中的表达,本研究采用半定量逆转录聚合酶链反应 (RT PCR)检测了大鼠小脑、大脑皮质、海马、纹状体、黑质的KATP亚型mRNA的表达。结果显示Kir6. 1、Kir6. 2、Sur1、Sur2B在小脑、大脑皮质、海马、纹状体、黑质中均有表达,Sur2A在脑组织中未见表达。Kir6. 1mRNA在海马和黑质的相对表达水平明显高于小脑、大脑皮质和纹状体(P<0.01);Kir6. 2和Sur1mRNA在黑质的相对表达水平明显高于小脑、大脑皮质、海马和纹状体 (P<0.01 );Sur2BmRNA在黑质、海马和纹状体的相对表达水平明显高于小脑和大脑皮质(P<0.01 )。以上结果提示KATP在脑内具有广泛表达,其表达水平在不同部位存在着差异性。     

ATP敏感性钾通道对腺苷受体诱导的心脏预处理延迟效应的影向

目的研究以腺苷A受体激动剂预处理的延迟保护效应与心肌三磷酸腺苷(ATP)敏感性钾通道的关系.方法Wistar大鼠随机分4组,其中PS、PG组以CCPA预处理,IC组仅注射CCPA溶剂,GC组不作CCPA预处理.24h后取出心脏,以改良Langendorff灌注装置制成离体心脏模型.缺血前30min PG、GC组先经主动脉灌注优降糖.平衡灌注后4℃改良St.Thomas心麻液诱导心脏停搏,维持12～15℃缺血180min..之后复灌37℃氧合平衡液60min.观察心功能、ATP等指标.结果在复灌60min时左室压力上升和下降最大速率恢复率(±dp/dtmax,%)及心肌ATP含量均高于PG、GC:、IC三组,差异都具有显著性(P＜0.01);PG、GC、IC组间无显著性差异(P＞0.05).结论以腺苷A受体激动剂预处理诱发大鼠心脏产生的延迟保护效应与心肌ATP敏感性钾通道开放有关.     

二氮嗪后处理对离体大鼠心功能及线粒体心磷脂的影响

目的:建立离体大鼠心肌缺血/再灌注损伤模型,观察二氮嗪(diazoxide,D)后处理对缺血/再灌注损伤离体大鼠心功能及线粒体心磷脂的影响,并探讨ATP敏感性钾通道在二氮嗪后处理心肌保护中的作用。方法:采用Langendorff装置建立离体大鼠心肌缺血/再灌注损伤模型,将SD大鼠随机分为对照组(control)、缺血再灌注模型组(I/R)、二氮嗪后处理组(I/R+D)、5-羟葵酸拮抗二氮嗪后处理组(I/R+5-HD+D),每组8只,均先灌注平衡20 min。Control组:灌注平衡后续灌70 min;I/R组:缺血前灌注4℃ST.Thomas停跳液,全心缺血40 min,再灌30 min;I/R+D组:全心缺血40 min,缺血后给予含二氮嗪(50μmol/L)的K-H液灌注5 min后,再灌25 min;I/R+5-HD+D组:二氮嗪后处理前给予含5-羟葵酸(100μmol/L)的K-H液灌注5 min,再灌20 min。观察各组续(再)灌注末心率、冠脉流出液量、心功能、心肌酶学及心肌线粒体心磷脂的变化。结果:各组续(再)灌注末比较,I/R组较control组及I/R+D组心率减慢、冠脉流出液量降低,心功能明显受损,心肌酶增加,心磷酯含量减少,但与I/R+5-HD+D无明显差异。结论:二氮嗪后处理通过增加线粒体心磷脂含量,减少心肌酶的释放,改善心脏功能,减轻心肌的再灌注损伤,产生心肌保护作用。5-羟葵酸能够完全阻断二氮嗪的心肌保护作用。     

Diazoxide对缺氧大鼠肺动脉平滑肌细胞内氧自由基的变化及细胞增殖的作用

目的： 研究线粒体膜上ATP敏感钾通道（MitoK_ATP）开放剂diazoxide和线粒体膜电位（ΔΨm）在缺氧导致的大鼠肺动脉平滑肌细胞内氧自由基的变化及细胞增殖/凋亡失衡中的作用，探索缺氧性肺动脉重建和肺动脉高压形成的发生机制。方法： 取大鼠正常肺组织，分离出肺动脉平滑肌细胞（PASMCs）进行常氧或慢性缺氧培养。将样本分为6组：① 正常对照组；② 线粒体膜上ATP敏感钾通道（MitoK_ATP）开放剂diazoxide组；③ MitoK_ATP 阻断剂5-HD组；④ 慢性缺氧(CH)组；⑤ CH+diazoxide组；⑥ CH+5-HD组。利用激光共焦显微镜（Leica SP-1 USA）成像检测线粒体膜电位，荧光染色检测细胞内氧自由基含量，流式细胞仪检测细胞周期和MTT法检测细胞增殖情况。结果： ① Diazoxide作用24 h后，R-123荧光强度明显强于正常对照组，线粒体膜电位去极化，细胞内氧自由基含量明显高于正常对照组，细胞增殖明显多于正常对照组、凋亡少于正常对照组，P<0.05；而5-HD作用24 h后，上述指标与正常对照组相比较，均无显著差异,P>0.05；②慢性缺氧24 h，结果与diazoxide组相似，R-123荧光强度明显强于正常对照组，线粒体膜电位去极化，细胞内氧自由基含量明显高于正常对照组，细胞增殖明显多于正常对照组、凋亡少于正常对照组，P<0.05；CH+diazoxide组，R-123荧光强度和线粒体膜电位去极化明显强于缺氧组，细胞内氧自由基含量明显高于缺氧组，细胞增殖明显多于缺氧组、凋亡少于缺氧组，P<0.05；CH+5-HD组，R-123荧光强度和线粒体膜电位去极化明显弱于缺氧组，细胞内氧自由基含量明显低于缺氧组，细胞增殖明显少于缺氧组、凋亡多于缺氧组，P<0.05;R-123荧光强度、MTT的A值与细胞内氧自由基含量均呈显著正相关；凋亡细胞百分比与细胞内氧自由基含量呈显著负相关。结论： 本实验结果显示，diazoxide能够通过开放线粒体膜上ATP敏感的钾通道，引起ΔΨm去极化，ΔΨm的变化影响细胞内氧自由基的含量，氧自由基可能作为信号分子通过影响肺动脉平滑肌细胞的增殖/凋亡的平衡，参与了缺氧性肺动脉重建和肺动脉高压的发生和发展过程。     

KATP通道开放剂对新生大鼠缺氧缺血后大脑 μ-calpain活化、c-Fos和c-Jun表达的影响

目的:探讨ATP敏感钾通道(K_ATP)开放剂二氮嗪(diazoxide)对新生大鼠缺氧缺血性脑损伤(HIBI)后皮层和海马μ-calpain活化、c-fos和c-jun蛋白(c-Fos,c-Jun)表达的影响。方法:采用新生7 d龄SD大鼠复制HIBI模型,分别在缺氧缺血(HI)前、后侧脑室注射diazoxide 5 μL(1 g/L)。采用Western blot法检测皮层和海马HI后4 h c-Fos和c-Jun蛋白条带的积分光度值(ID)及24 h μ-calpain两个活性片段(76/80 kD)的ID比值。结果:HI对照组皮层和海马c-Fos和c-Jun的ID值显著高于正常对照组;HI前给药组显著低于HI对照组(P<0.01);HI后给药组也较HI对照组低(P<0.05)。HI前、后给药均能抑制HI后μ-calpain的裂解,降低两个活性片段的比值。结论:K_ATP通道开放剂diazoxide可能通过降低c-Fos和c-Jun的表达、抑制μ-calpain的活化,起到对HIBI的拮抗及治疗作用。     

缬草单萜氧化物对兔心室肌线粒体ATP敏感性钾通道的影响

目的 :研究缬草单萜氧化物 (VMO)对兔单个心室肌细胞线粒体ATP敏感性钾通道 (mitoKATP)的影响。方法 :采用酶解法分离单个兔心室肌细胞。实验分为 30 μg/LVMO组、6 0 μg/LVMO组、12 0 μg/LVMO组、5 羟癸酸 ( 5 HD)组和 5 HD +12 0 μg/LVMO组。用罗丹明 (Rhodamine 12 3)染色 ,激光扫描共聚焦显微镜 (多光子模式 )分别观察各组线粒体荧光强度变化。结果 :① 30 μg/LVMO组、6 0 μg/LVMO组、12 0 μg/LVMO组均可见用药后线粒体荧光强度明显增加 ,分别增加 13.90± 1.2 0 %、2 1.2 0± 2 .30 %和 2 6 .4 0± 2 .50 % ;② 50 0 μmol/L5 HD不影响线粒体荧光强度 ,但可以阻断VMO对线粒体荧光的增强效应。结论 :VMO对兔心室肌细胞mitoKATP有开放作用。     

正常和缺血大鼠脑线粒体对环孢菌素A的反应

目的：研究正常和缺血脑线粒体对环孢菌素A（CsA）的反应，并观察线粒体ATP敏感性钾通道与线粒体渗透性转换孔的关系。方法：本实验采用分光光度法，在分离线粒体上观察线粒体渗透性转换孔抑制剂和线粒体ATP敏感性钾通道开放剂对正常与缺血脑线粒体肿胀的影响。结果：在正常脑线粒体，0.5 μmol/L和 1 μmol/L CsA以及30 μmol/L 二氮嗪（DE）均可明显减轻Ca²⁺诱导的线粒体肿胀程度，苍术苷（Atr）能取消此作用，而5 μmol/L CsA不能减轻线粒体的肿胀。在全脑缺血5 min后的线粒体，0.5 μmol/L CsA可减轻Ca²⁺诱导的线粒体肿胀，该作用可被Atr取消，但1 μmol/L CsA不能减轻肿胀；30 μmol/L DE也可明显减轻Ca2+诱导的缺血脑线粒体肿胀程度，100 μmol/L和200 μmol/L 5-羟基癸酸盐（5-HD）和Atr均可取消其作用。结论：缺血脑线粒体对线粒体渗透性转换孔开放的抑制剂比正常脑线粒体更为敏感，脑线粒体ATP敏感性钾通道激活可能是抑制线粒体渗透性转换孔开放的调控机制之一。     

Immunohistochemical analysis of Smac/DIABLO expression in human carcinomas and sarcomas

Second mitochondria-derived activator of caspases (Smac/DIABLO) is released from mitochondria into the cytosol during apoptosis, promoting caspase activation by neutralizing the inhibition of inhibitor of apoptosis proteins (IAPs) on caspases. Alteration of apoptosis is essential for cancer development, and cancer cell death by radiation and chemotherapy is largely dependent upon apoptosis. In this study, archival tissues of 100 carcinomas and 50 sarcomas from various origins were analyzed by immunohistochemistry for the expression of Smac/DIABLO. Smac/DIABLO immunoreactivity was seen in 62 of 100 (62%) carcinomas, including 42 of 60 stomach carcinomas, 7 of 10 colorectal carcinomas, 4 of 10 lung carcinomas, 7 of 10 ovarian carcinomas, and 2 of 10 prostate carcinomas. Smac/DIABLO is expressed in 11 of 50 (22%) sarcomas, including 2 of 8 malignant schwannomas, 5 of 11 rhabdomyosarcomas, 2 of 7 malignant fibrous histiocytomas, 1 of 6 leiomyosarcomas, 0 of 8 angiosarcomas, 0 of 8 liposarcomas, and 1 of 2 Ewing's sarcomas. These data demonstrated that Smac/DIABLO expression levels vary depending on the individual cancer types. Furthermore, the present study showed that many human cancers do not express Smac/DIABLO, and suggest that lack of Smac/DIABLO expression in the cancer cells may inhibit apoptosis, thereby promoting their survival.     

Expression of Smac/DIABLO in mouse preimplantation embryos and its correlation to apoptosis and fragmentation

Regulation of early embryonal development during fertilization and implantation is crucial for mammalian reproduction. Several studies have described cell death during preimplantation embryogenesis in a range of mammalian species, both in vivo and in vitro. Therefore, apoptosis may be involved in early embryonic arrest and the characteristic cytoplasmic fragments are the equivalents of apoptotic bodies, the end-product of apoptosis. Although apoptosis is expected to associate with fragmentation in early preimplantation embryos, the mechanism through which this fragmentation occurs has not been elucidated. Recently, second mitochondria-derived activator of caspase/Direct IAP Binding Protein with Low pI (Smac/DIABLO) was identified as a mitochondrial protein that is released into the cytosol during apoptosis. Once released, the Smac/DIABLO blocks the anti-apoptotic activity of inhibitor of apoptosis proteins (IAPs). We hypothesized that the Smac/DIABLO may be involved in the fragmentation of mouse preimplantation embryos. Therefore, we investigated the expression of Smac/DIABLO mRNA and protein and its localization in mouse oocytes and preimplantation embryos. Smac/DIABLO mRNA was detected by RT-PCR in the oocytes and the preimplantation embryos. Immunohistochemistry studies showed that the Smac/DIABLO protein localized in mitochondria and was released into the cytosol in both fragmented embryos and embryos in which apoptosis was induced by staurosporine. These observations indicate that the Smac/DIABLO is involved in the fragmentation and apoptosis of preimplantation embryos.     

Expression of Smac/DIABLO in B-cell non-Hodgkin and Hodgkin lymphomas

Inhibitor of apoptosis proteins (IAPs) are upregulated in cancers and suppress cell death, in part, through their ability to directly inhibit caspases. Inhibitor of apoptosis proteins are differentially expressed in B-cell lymphomas. The functions of some IAPs are counteracted by the cell death inducer, second mitochondrial-derived activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO). In this study, we investigated the expression levels of Smac/DIABLO in 14 lymphoma cell lines by Western blot analysis. We also assessed 247 B-cell non-Hodgkin's lymphoma (NHL) and 40 Hodgkin's lymphoma (HL) tumors using immunohistochemical methods. Smac/DIABLO was expressed in most NHL and all HL cell lines. In NHL, Smac/DIABLO was expressed in 117 (47%) tumors and was differentially expressed in various NHL types. In most NHLs, from 29% to 68% of tumors were positive; however, Smac/DIABLO was not detected in small lymphocytic lymphoma/chronic lymphocytic leukemia and Burkitt lymphoma, and was rare in extranodal marginal zone B-cell lymphoma. In HL, Smac/DIABLO was positive in 25 (63%) tumors. Unlike NHL, all types of HL were positive for Smac/DIABLO, although nodular sclerosis was least often positive. The differential expression of Smac/DIABLO in NHLs suggests that apoptotic mechanisms are differentially involved in their pathogenesis. These results may also have implications for using Smac/DIABLO or its agonists as therapeutic agents.     

Rapid induction of IAP family proteins and Smac/DIABLO expression after proapoptotic stimulation with doxorubicin in RPMI 8226 multiple myeloma cells

We studied the expression dynamics of inhibitor of apoptosis protein (IAP) family members and Smac/DIABLO after treatment with doxorubicin in human multiple myeloma cell line RPMI 8226 and its doxorubicin-resistant variant DRR. Proapoptotic stimulation with doxorubicin rapidly induced the overexpression of mRNA as well as protein for IAPs in RPMI 8226 cells followed by a gradual decrease of their expression. Smac/DIABLO, which is known to neutralize IAPs, showed increased expression at the mRNA level after treatment; however, Western blot analysis revealed a slight decrease of the amount of protein. Immunoprecipitation analysis revealed the association of Smac/DIABLO with cIAP1 or XIAP after treatment with doxorubicin. In contrast to the RPMI 8226 cells, DRR cells did not undergo apoptosis in response to doxorubicin treatment. The DRR cells had higher levels of IAPs expression at the mRNA level and did not show a remarkable peak or decrease in the expression of mRNAs for cIAP1, cIAP2, XIAP, and survivin after treatment with doxorubicin. Furthermore, the expression of Smac/DIABLO mRNA was not up-regulated after treatment. These findings indicate that the suppression of IAPs expression by Smac/DIABLO shortly after proapoptotic stimulation might play a role in the mechanisms of apoptotic induction, and that the maintenance of high IAPs expression and low Smac/DIABLO expression after treatment might lead to the doxorubicin-resistance of multiple myeloma cells.     

The role of NF-κB and Smac/DIABLO proteins in the treatment response and survival of acute myeloid leukemia patients

IntroductionThe misbalance between a family of inhibitor of apoptosis proteins (IAP), regulated by the nuclear factor kappa B (NF-κB) and their natural antagonist second mitochondrial-derived activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO) are important to biology of acute myeloid leukemia (AML).Material and methodsThe aim of the study was to assess NF-κB and Smac/DIABLO proteins expression in blasts of 109 newly diagnosed AML patients using the multicolor flow cytometry and evaluate their influence on AML patients outcome.ResultsExpression of NF-κB and of Smac/DIABLO proteins were found in 95% and 98% of the patients, respectively. A negative correlation between Smac/DIABLO and NF-κB was observed. Age < 60 years old as well as higher Smac/DIABLO expression were associated with a higher probability of complete response achievement in the multivariate analysis. Longer overall survival (OS) in the univariate and multivariate analyses was influenced by age < 60 years old, a favorable or intermediate-risk karyotype and high Smac/DIABLO expression. Additionally, in the survival analysis of the subgroups, the patients aged < 60 years old, with high Smac/DIABLO expression, lower NF-κB expression and < 50% of bone marrow blasts who were treated with standard treatment had better OS.ConclusionsLower NF-κB and higher Smac/DIABLO expression may influence AML patients outcome.     

外源性硫化氢及K_(ATP)通道对大鼠慢性应激结肠高动力的调节

目的:研究外源性硫化氢(hydrogen sulfide,H2S)及ATP敏感性钾通道(ATP-sensitive potassium channels,KATP)在慢性应激结肠高动力中的作用。方法:制作慢性避水应激(water avoidance stress,WAS)和假避水应激(sham water avoidance stress,SWAS)大鼠模型,观察2组大鼠结肠肌条的收缩活性以及硫氢化钠(Na HS)和格列本脲预处理后对2组大鼠结肠肌条收缩影响并计算Na HS的半数抑制浓度(half maximal inhibitory concentration,IC50),使用免疫荧光及Western blotting法观察KATP通道各亚基在结肠中的分布及表达。结果:WAS组结肠肌条收缩活性明显高于SWAS组;Na HS浓度依赖性抑制2组大鼠纵行肌(longitudinal muscle,LM)和环形肌(circular muscle,CM)的收缩;WAS组LM和CM的Na HS IC50分别为0.2033 mmol/L和0.1438 mmol/L,均明显低于SWAS组(P0.01);格列本脲明显增加2组大鼠肌条Na HS IC50(P0.01);Kir6.1、Kir6.2和SUR-2B在2组大鼠结肠固有肌细胞膜均有分布;WAS组(去除黏膜及黏膜下层后)Kir6.1和SUR2B蛋白表达高于SWAS组(P0.01)。结论:H2S外源性供体Na HS对慢性应激结肠高动力具有潜在的治疗作用。KATP通道亚基Kir6.1/SUR2B表达增加可能是慢性应激结肠动力紊乱的一种适应性反应。