吡格列酮对大鼠缺血/再灌注心肌转化生长因子β1表达的影响

目的:观察吡格列酮对大鼠缺血/再灌注损伤心肌转化生长因子β1(TGFβ1)表达的影响。方法:30只SD大鼠随机分为5组(n=6):缺血/再灌注组、吡格列酮5 mg/(kg.d)组、吡格列酮10 mg/(kg.d)组、吡格列酮20 mg/(kg.d)组、吡格列酮20 mg/(kg.d)+过氧化物酶体增殖物激活受体γ(PPARγ)特异性阻断剂GW9662组,利用在体结扎左前降支的方法建立缺血/再灌注损伤模型,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL法)检测心肌细胞凋亡,RT-PCR方法检测心肌组织TGFβ1 mRNA的变化,Western blot方法检测心肌组织TGFβ1蛋白的变化。结果:TUNEL法显示吡格列酮抑制缺血/再灌注心肌细胞凋亡,吡格列酮上调TGFβ1表达,GW9662逆转吡格列酮对凋亡细胞的抑制作用,抑制吡格列酮促进TGFβ1表达上调的作用。结论:吡格列酮可抑制缺血/再灌注损伤诱导的心肌细胞凋亡,吡格列酮可促进TGFβ1上调,这两种作用是由PPARγ介导的。     

吡格列酮诱导心肌细胞凋亡和抑制心肌细胞肥大的作用及其机制研究

摘要 目的：探讨吡格列酮诱导心肌细胞凋亡和抑制心肌细胞肥大的作用及其机制研究。方法：选取1-3日龄的健康新生SD大鼠,采用酶学分离心肌细胞并培养。将心肌细胞分为4组：对照组、吡格列酮10 μM组、吡格列酮20 μM组、阿帕替尼2 μM组。采用自动细胞计数器（BioRad）计数吡格列酮和阿帕替尼对心肌细胞增殖的影响,流式细胞术检测心肌细胞凋亡率,[3H]-亮氨酸掺入法评估心肌细胞的肥厚,用相差显微镜检测心肌细胞直径。蛋白免疫印迹试验检测VEGFR-2,磷酸化VEGFR-2,蛋白激酶B （Akt）,磷酸化人体抑癌基因（P53）,兔抗人单克隆抗体（Bax）,B淋巴细胞瘤-2（Bcl-2）,哺乳动物雷帕霉素靶蛋白（mTOR）表达水平。免疫组织化学检测心肌细胞VEGFR-2、Bcl-2及Bax阳性指数。结果：吡格列酮和阿帕替尼均能抑制心肌细胞活力,其中吡格列酮以剂量依赖的方式抑制心肌细胞活力,即吡格列酮剂量越大,其抑制心肌细胞活力越强。吡格列酮或阿帕替尼治疗后,心肌细胞凋亡率显著增加,表明两者均诱导心肌细胞凋亡。吡格列酮或阿帕替尼治疗后,血管紧张素II（Ang II）诱导的[3H]-亮氨酸掺入量显著减少,心肌细胞直径减小,表明两者均抑制心肌细胞肥大。吡格列酮显著提高了新生大鼠心肌细胞中Bax和磷酸化-P53的蛋白表达,降低了mTOR、Akt、VEGFR-2、Bcl-2和磷酸化-VEGFR-2蛋白表达。与对照组比较,吡格列酮和阿帕替尼治疗后,VEGFR-2阳性指数和Bcl-2阳性指数显著降低,Bax阳性指数显著升高（P＜0.05）。结论：吡格列酮通过调节VEGFR-2信号通路诱导心肌细胞凋亡,抑制心肌细胞肥大。     

血红素氧合酶-1/一氧化碳通路参与辛伐他汀抗高血压诱发的大鼠心肌肥厚

为了探讨他汀类药物抑制心肌肥厚的作用机制,本研究应用一氧化氮合酶抑制剂左旋硝基精氨酸[N-nitro-L-arginine, L-NNA,15 mg／(kg·d)]制备大鼠高血压心肌肥厚模型,并分别给予不同剂量辛伐他汀[5或30 mg／(kg·d)进行干预。6周后测大鼠左心室功能、左心室重量指数(left ventricular mass index,LVMI)、心肌脑钠素(brain natriuretic peptide,BNP)含量、心肌羟脯氨酸含量和心肌血红素氧合酶(heme oxygenase,HO)活性。在体外培养的新生大鼠心肌细胞中,观察辛伐他汀对血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)引起的心肌细胞肥大的抑制作用与细胞血红素氧合酶-1(HO-1)表达、HO活性及CO生成间的关系。结果表明,辛伐他汀干预明显减轻L-NNA处理大鼠的心肌肥厚(LVMI值、心肌BNP和羟脯氨酸含量均显著低于单纯L-NNA处理组),改善左心室舒张功能,而且心肌HO活性显著升高。在离体培养的原代乳鼠心肌细胞,辛伐他汀浓度依赖性地抑制Ang Ⅱ引起的细胞肥大(3H-亮氨酸掺入),并相应增加HO-1 mRNA表达、HO活性和CO生成量。应用HO抑制剂锌卟啉能有效抑制辛伐他汀抗Ang Ⅱ诱导的心肌肥大作用。结果提示:辛伐他汀上调HO-1／CO通路是其抗高血压诱发的心肌肥厚的机制之一。     

非酒精性脂肪肝病大鼠肝脏SOCS-3 的表达与吡格列酮干预研究

目的:探讨SOCS-3在非酒精性脂肪肝病(NAFLD)发病中的作用以及吡格列酮的干预作用。方法:29只雄性SD大鼠随机分为正常对照组(8只),高脂饮食组(21只)。饲养8周后,从高质饮食组随机抽取5只大鼠证实造模成功后,将该组余下的16只大鼠继续以高脂饲料喂养,并随机分为NAFLD对照组(8只);吡格列酮干预组(8只),予以吡格列酮3mg·kg-·1d-1灌胃。16周末,处死所有大鼠,检测血糖、血胰岛素、血脂、肝脏SOCS-3 mRNA和SREBP-1c mRNA表达及肝脏病理学。结果:与正常对照组相比,NAFLD组血糖、血胰岛素、血脂、肝脏脂肪变水平及肝组织SOCS-3 mRNA、SREBP1c mRNA表达显著上调。吡格列酮干预组SOCS-3 mRNA、SREBP-1c mRNA表达较NAFLD组下调,且血糖、血胰岛素、血脂、肝脏脂肪变水平下降。SOCS-3 mRNA表达水平与胰岛素抵抗指数、SREBP-1c mRNA表达水平、肝脂肪变成显著正相关。结论:SOCS-3可能通过胰岛素抵抗及上调肝组织SREBP-1c mRNA表达参与NAFLD发病,吡格列酮能抑制肝脏SOCS-3的表达,对NAFLD有一定治疗作用。     

MLP参与心肌肥大发生过程与Calcineurin/NFAT信号通路有关

心肌肥大是心肌细胞面对多种病理刺激时的共同反应,以心肌细胞体积增大和胚胎期基因的重新表达为标志.心肌发育调控基因肌肉LIM蛋白(muscle LIM protein,MLP)的表达异常与心肌肥大有关.为研究MLP参与心肌肥大发生的分子机制,采用去氧肾上腺素（phenylephrine, PE）刺激大鼠原代培养心肌细胞,建立心肌细胞肥大模型,采用RNAi技术敲减MLP的表达,分析MLP与肥大信号通路钙调神经磷酸酶（calcineurin）/活化T细胞核因子（nuclear factor of activated T-cells, NFAT）的关系.结果显示, 原代培养的心肌细胞经一定浓度的PE刺激后细胞表面积增加,肥大标志蛋白ANP、BNP表达增高,并伴有MLP表达上调. RNAi方法敲减MLP的表达则明显抑制PE诱导的心肌细胞表面积增加和BNP表达增高,并且直接 影响NFAT的转录激活活性,提示MLP与心肌肥大的发生密切相关,并且可能是通过calcineurin/NFAT信号通路而参与心肌肥大的发生.     

钙调神经磷酸酶信号通路参与前列腺素F2α诱导的心肌肥厚

利用野百合碱（monocrotaline,MCT）诱导大鼠右心室肥厚模型和培养乳鼠心肌细胞,研究前列腺素F2α（prostaglandin F2α,PGF2α）在心肌肥厚中的作用及钙调神经磷酸酶（calcineurin,CaN）信号通路征其中的作用。在雄性Sprague-Dawley大鼠中,用MCT（60mg／kg）单次i．p．诱导右心室肥厚,同时用塞来旨布（20mg／kg）预防／治疗给药2周。用病理检测、电镜观察等方法观察心肌肥厚时组织病理改变;EIA试剂盒检测心肌组织PGF2α含量;RT-PCR检测心房钠尿肽（atrial natriuretic peptide,ANP）和CaNmRNA的表达;用蛋白免疫印迹法检测CaN及其下游因了NFAT3和GATA4蛋门质的表达。以心肌细胞直径、蛋白含量和ANP mRNA表达的变化为0．1μmol／L PGF2α诱导心肌细胞肥大的指标。以CaN mRNA表达作为该信号通路的主要指标,并观察CaN抑制剂环孢素A对PGF2α所致心肌细胞肥人和CaN mRNA表达的影响。结果显示：MCT注射2周（M2W组）,右心室肥厚指数（RVHI）、右心室／体重比及肺重／体重比分别增加了47％、53％和118％;注射后4周（M4W组）增加了64％、94％及156％。电镜观察发现右心室组织损伤。同时,右心室组织PGF2α含量在M2W和M4W组分别增加了44％和51％,与RVHI、ANP和CaN的mRNA表达,及CaN／NFAT3／GATA4的蛋白质表达均呈正相关。环氧酶抑制剂塞来昔布预防和治疗给药均明显改善MCT诱导的组织病理学改变。在高体细胞培养中,PGF2α（0．1μmol／L）明显使心肌细胞增大,蛋白质含量增加,ANP和CaN mRNA表达增强：同时,CaN抑制剂环孢素A明显抑制PGF2α诱导的心肌细胞肥大和CaN mRNA表达。上述结果提示：心肌组织局部PGF2α参与了MCT诱导的心肌肥厚过程,CaN信号通路是其细胞内信号转导通路之一。     

转化生长因子β1对大鼠心肌细胞肥大的影响

目的：研究转化生长因子β1（TGF-β1）及其下游Smad3信号蛋白在大鼠心肌细胞肥大中的作用。方法：TGF-β1干预培养新生大鼠心肌细胞,流式细胞仪检测心肌细胞总蛋白含量。结扎大鼠腹主动脉复制心肌肥厚模型,在不同时间点处死动物,检测左室质量指数（LVM1）,RT—PCR检测TGF-β1及Smad3的mRNA表达,Westernblot检测Smad3蛋白的表达。结果：不同剂量TGF-β1均能明显增加体外培养的心肌细胞总蛋白含量,TGF-β1（3ng／ml）还增加心肌细胞Smad3 mRNA和蛋白的表达,其表达量1h达高峰,持续至TGF-β1刺激后8h。大鼠腹主动脉结扎术后3d LVMI开始上升并持续至术后28d,心肌组织中TGF-β1、Smad3的mRNA表达水平以及Smad3蛋白表达术后3d也开始上升持续至术后28d,术后14d为表达高峰（P〈0．01）。结论：TGF-β1能诱导大鼠心肌细胞肥大,其信号蛋白Smad3参与了大鼠心肌肥大的病理过程。     

骨髓基质干细胞向心肌样细胞分化的实验研究

目的探讨大鼠骨髓基质干细胞向心肌样细胞分化后超微结构和细胞因子表达的变化,为下一步细胞移植治疗扩张型心肌病提供理论依据。方法在体外扩增、定向诱导骨髓基质干细胞向心肌样细胞分化的基础上,电镜下观察骨髓基质干细胞诱导前后超微结构的改变,RT-PCR检测心肌特异性因子ANP、BNP、α-MHC、β-MHC的表达,并与原代培养的心肌细胞比较,观察二者之间的生物学异同。结果免疫组化法证实诱导后的骨髓基质干细胞向心肌样细胞分化,电镜下胞浆内可见糖原颗粒,肌原纤维排列与原代培养的心肌细胞相似;RT-PCR证实诱导后的骨髓基质干细胞表达心肌特异性因子ANP、BNP、α-MHC、β-MHC。结论骨髓基质干细胞经5-氮胞苷定向诱导后在超微结构和细胞因子的表达上类似于心肌细胞,已向心肌样细胞分化。     

吡格列酮治疗STZ诱导糖尿病大鼠视网膜中TSP-1的变化

观察吡格列酮在治疗STZ诱导糖尿病大鼠视网膜中血小板反应素Ⅰ(thmmbospondin-1,TSP-1)的变化,探讨噻唑烷二酮类药物在糖尿病视网膜病变中的影响和可能作用机制。用链脲佐菌素(STZ)腹腔注射建立大鼠糖尿病模型,随机分正常对照组(C组),糖尿病安慰剂组(D组)和糖尿病吡格列酮治疗组(DP组),8周后取视网膜组织行免疫组织化学、RT-PCR半定量检测TSP- 1mRNA水平的变化,结果显示在糖尿病早期视网膜神经节细胞层、内核层中均有明显的TSP1表达,对比于糖尿病对照组,糖尿病治疗组视网膜单位面积表达TSP1的细胞数量要较二组对照组高;糖尿病治疗组、糖尿病对照组和正常对照组间TSP-1的荧光灰度值分别为528.2±47.78、493.4±39.36和417.7±28.74:内参照GAPDH的荧光灰度值分别为796.4±21.18、811.2±36.80和832.4±43.18,各组间TSP-1 mRNA表达有显著性差异,提示吡格列酮对糖尿病早期视网膜中TSP-1的表达变化有影响,吡格列酮可能在延缓糖尿病视网膜病变的发生发展中起一定的作用。     

非酒精性脂肪肝病大鼠肝脏SOCS-3的表达与吡格列酮干预研究

目的：探讨SOCS-3在非酒精性脂肪肝病（NAFLD）发病中的作用以及吡格列酮的干预作用。方法：29只雄性SD大鼠随机分为正常对照组（8只）,高脂饮食组（21只）。饲养8周后,从高质饮食组随机抽取5只大鼠证实造模成功后,将该组余下的16只大鼠继续以高脂饲料喂养,并随机分为NAFLD对照组（8只）;吡格酮干预组（8只）,予以吡格列酮3mg·kg^-1·d^-1灌胃。16周末,处死所有大鼠,检测血糖、血胰岛素、血脂、肝脏SOCS-3mRNA和SREBP-lcmRNA表达及肝脏病理学。结果：与正常对照组相比,NAFLD组血糖、血胰岛素、血脂、肝脏脂肪变水平及肝组织SOCS-3mRNA、SREBPlCmRNA表达显著上调。吡格列酮干预组sOCS．3mRNA、SREBP-1cmRNA表达较NAFLD组下调,且血糖、血胰岛素、血脂、肝脏脂肪变水平下降。SOCS-3mRNA表达水平与胰岛素抵抗指数、SREBP．1cmRNA表达水平、肝脂肪变成显著正相关。结论：SOCS-3可能通过胰岛素抵抗及上调肝组织SREBP-lcmRNA表达参与NAFLD发病,吡格列酮能抑制肝脏SOCS-3的表达,对NAFLD有一定治疗作用。     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

Scurvy in capybaras bred in captivity in Argentine

In order to determine if the absence of vitamin C in the diet of capybaras (Hydrochoerus hydrochaeris) causes scurvy, a group of seven young individuals were fed food pellets without ascorbic acid, while another group of eight individuals received the same food with 1 g of ascorbic acid per animal per day. Animals in the first group developed signs of scurvy-like gingivitis, breaking of the incisors and death of one animal. Clinical signs appeared between 25 and 104 days from the beginning of the trial in all individuals. Growth rates of individuals deprived of vitamin C was considerably less than those observed in the control group. Deficiency of ascorbic acid had a severe effect on reproduction of another population of captive capybaras. We found that the decrease in ascorbic acid content in the diet affected pregnancy, especially during the first stages. The results obtained suggest that it is necessary to supply a suitable quantity of vitamin C in the diet of this species in captivity.     

Changes in lactate dehydrogenase activity in chicken tissues in hyperthyreosis in ontogenesis

The lactate dehydrogenase activity in reactions of lactate oxidation and synthesis was studied in subfractions of the chicken brain, heart and liver at the embryonal, early postembryonal and adult stages of development after thyroxine administration. It has been shown that during embryogenesis thyroxine predominantly enhanced the rate of lactate oxidation in the mitochondrial tissues. A marked increase in the lactate synthesis was found in cytoplasm of the adult chicken tissues. Specificity of enzyme activity alterations was detected in the chicken brain during ontogenesis after thyroxine administration.     

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.