预防性应用吡格列酮改善胰岛素抵抗的脂肪内分泌机制

目的探讨吡格列酮改善胰岛素抵抗过程中脂肪内分泌功能的改变。方法45只W istar雄性大鼠随机分为对照组、胰岛素抵抗组和吡格列酮组。胰岛素抵抗动物模型成功建立后,分析各组抵抗素、脂联素和脂联素受体的表达。结果与胰岛素抵抗组比较,吡格列酮组大鼠的体质量、空腹血糖、游离脂肪酸差异不明显,但胰岛素、胰岛素抵抗指数、葡萄糖耐量和胰岛素耐量明显改善（P〈0.01）,脂联素表达明显增高（P〈0.05）。结论脂肪内分泌功能紊乱在高脂诱导胰岛素抵抗的发生中起着重要的作用,预防性应用吡格列酮可明显延缓大鼠胰岛素抵抗的发生,改善脂肪内分泌功能。     

胰岛素样生长因子-1对大鼠结肠平滑肌细胞增殖、凋亡及MAPK通路的影响

目的 探讨胰岛素样生长因子-1(IGF-1)对大鼠结肠平滑肌细胞(SMCs)增殖、凋亡及丝裂原活化蛋白激酶(MAPK)信号转导通路的影响.方法 利用酶解法分离培养Sprague-Dawley大鼠结肠SMCs,进行免疫组化染色鉴定后,将其分为对照组、IGF-1组和IGF-1+ PD98059[细胞外信号调节激酶(ERK)抑制剂]组,分别采用噻唑蓝(MTT)法检测SMCs增殖,流式细胞术AnnexinV-FITC/PI检测SMCs凋亡,Western印迹检测磷酸化ERK、ERK、磷酸化p38MAPK、p38MAPK和磷酸化c-Jun氨基末端激酶(JNK)、JNK的表达.结果 分离培养的细胞经免疫组化鉴定为结肠SMCs,IGF-1组较对照组细胞增殖增强[(1.786 ±0.271)比(0.998±0.057),P＜0.01],凋亡率降低[(2.59±0.28)％比(20.68±2.48)％,P＜0.01],磷酸化ERK表达增强,磷酸化ERK/ERK比值升高[(42.71±3.74)％比(23.88±2.52％),P均＜0.01];磷酸化p38MAPK、p38MAPK、磷酸化JNK、JNK表达无差异(P均＞0.05).IGF-1+ PD98059组较对照组细胞增殖下降[(0.154±0.021)比(0.998±0.057),P＜0.01],凋亡率升高[(84.31±7.54)％比(20.68±2.48)％,P＜0.01],磷酸化ERK表达减弱,磷酸化ERK/ERK比值降低[(10.47±1.22)％比(23.88±2.52)％,P均＜0.01].结论 IGF-1可能通过激活结肠SMCs MAPK通路中的ERK途径,促进细胞增殖,抑制凋亡,可能与p38MAPK途径和JNK途径无关.     

吡格列酮对谷氨酸引起皮质神经元损伤的保护作用及其机制

目的观察吡格列酮对谷氨酸引起皮质神经元损伤的保护作用及机制。方法初生大鼠乳鼠的皮质神经元体外培养7 d后用于实验,建立谷氨酸损伤模型,用Hoechst33258核染色观察细胞凋亡的形态学改变,MTT法测定细胞活力,W estern印迹法检测磷酸化p38MAP激酶蛋白水平。结果谷氨酸组细胞存活率较对照组明显降低(P0.01),吡格列酮、SB203580和一氧化氮合酶抑制剂(SMT)提高细胞存活百分率(P0.01)。谷氨酸组细胞凋亡百分比较对照组增加(P0.01),吡格列酮SB203580和SMT均能降低细胞凋亡百分比(P0.01)。谷氨酸组磷酸化p38MAP激酶蛋白水平较对照组明显升高(P0.01),吡格列酮明显降低磷酸化p38MAP激酶蛋白水平(P0.01)。结论吡格列酮对谷氨酸引起体外培养皮质神经元损伤具有保护作用,此作用与降低磷酸化p38MAP激酶蛋白水平有关。     

PPAR-γ依赖性ERK磷酸化介导吡格列酮引起的钠和碳酸氢根在肾近曲小管的重吸收

目的噻唑烷二酮类药物是PPAR-γ的选择性配体,用于改善胰岛素抵抗,但因其可引起浮肿、钠潴留而使用受限。本研究探讨该类药物引起浮肿的机制及其传导通路。方法兔肾近曲小管在吡格列酮（0．03、0．3、3μmol／L）及PPAR-γ拮抗剂（5μmol／LGW9662）或MAPK抑制剂（10μmol／L PD98059）作用下,观察Na^＋／HCO3^-转运活动度及HCO3重吸收率的变化,Western blot法测定ERK的磷酸化。结果（1）0．3μmol／L吡格列酮刺激兔肾近曲小管Na^＋和HCO3^-的转运和重吸收,该刺激作用被MAPK抑制剂或PPAR-γ拮抗剂阻断。（2）0．3μmol／L吡格列酮引起ERK磷酸化,被MAPK抑制剂或PPAR-γ拮抗剂阻滞。结论在兔肾近曲小管,通过PPAR-γ依赖的ERK磷酸化介导吡格列酮引起的肾近曲小管Na^＋和HCO3^-的重吸收。     

吡格列酮和非诺贝特对高脂饮食大鼠胰岛细胞内信号分子的影响

目的 观察吡格列酮和非诺贝特对高脂饮食大鼠胰岛内PPAR-α、-γ和细胞内信号分子的影响.方法 40只雄性SD大鼠随机分为4组:空白对照组(NC)、单纯高脂饮食组(HF)、高脂+非诺贝特组(FF)、高脂+吡格列酮组(FP).HE染色测定胰岛面积;免疫组织化学方法检测胰岛中各种蛋白的表达.结果 HF组胰岛面积、胰岛素、PPAR-γ、PPAR-α、NF-кB、p38丝裂原活化的蛋白激酶(MAPK)、ERKl蛋白水平与对照组相比,差别有统计学意义(P<0.05);吡格列酮明显增加PPAR-γ蛋白水平,降低NF-кB、p38 MAPK、ERKl的表达水平;非诺贝特能使PPAR-α.表达水平明显升高,也能够降低NF-кB、p38MAPK的水平.结论 非诺贝特和吡格列酮在一定程度上可以纠正胰岛功能的异常和细胞内信号转导的紊乱,保护胰岛细胞.     

吡格列酮对载脂蛋白E~(-/-)小鼠动脉粥样硬化中Toll样受体4/丝裂原活化蛋白激酶信号通路的影响

目的探讨吡格列酮对载脂蛋白E(Apo E)-/-小鼠主动脉粥样硬化病变中Toll样受体4/丝裂原活化蛋白激酶(TLR4/MAPKs)信号通路的影响。方法取8只10周龄雄性C3H小鼠作为对照组,用普通饲料喂养;另取24只同周龄雄性Apo E-/-小鼠用高脂饲料喂养,复制动脉粥样硬化模型成功后按随机数表法分成3组:模型组、吡格列酮低剂量(5 mg·kg-1·d-1)组和高剂量(10 mg·kg-1·d-1)组各8只。干预4周后,HE染色观察主动脉粥样硬化病变情况。应用Western Blot技术检测对照组和干预组动脉粥样硬化中丝裂原活化蛋白激酶ERK1/2、JNK1/2及p38MAPK磷酸化水平。结果与对照组比较,各组Apo E-/-小鼠动脉粥样硬化斑块中TLR4表达增强,其中ERK1/2与JNK磷酸化水平明显升高(均为P<0.05),但p38MAPK水平变化不明显(均为P>0.05);与模型组比较,吡咯列酮低剂量组和高剂量组小鼠动脉粥样硬化斑块中ERK1/2与JNK磷酸化水平均降低(ERK1/2:吡咯列酮低剂量组比模型组:0.5037±0.0438比0.8800±0.0436,吡咯列酮高剂量组比模型组:0.3843±0.0236比0.8800±0.0436;JNK:吡咯列酮低剂量组比模型组:0.5037±0.0437比0.7900±0.0308,吡咯列酮高剂量组比模型组:0.3843±0.0237比0.7900±0.0308,均为P<0.05)。结论吡格列酮可能通过影响TLR4/MAPKs/ERK1/2与TLR4/MAPKs/JNK信号通路,延缓动脉粥样硬化的发生与发展。     

吡格列酮对大鼠缺血再灌注诱导的内质网应激相关的心肌保护作用

目的观察吡格列酮对大鼠心肌缺血再灌注损伤（MIRI）时JNK、p-JNK及caspasc-12蛋白表达的影响,探讨吡格列酮通过JNK通路对内质网应激途径的心肌保护作用。方法Wistar大鼠40只随机分为假手术组（sham组）、缺血再灌注组（I／R组）、I／R＋Pio（吡格列酮）组及I／R＋Pi0＋sP600125组各10只。制作大鼠MIRI模型;TUNEL检测心肌细胞凋亡,免疫组织化学检测各组caspase-12表达变化,westernBlot法检测各组JNK、P-JNK的表达。结果吡格列酮预处理组大鼠心肌细胞凋亡、JNK磷酸化率及caspase-12蛋白表达水平明显比I／R组降低（P〈0．05）,加用JNK抑制剂（SP600125）后上述指标进一步下降,与吡格列酮组比较差异有统计学意义（P〈0．05）。结论缺血再灌注损伤可激活JNK通路,诱导过度的ERS,增加ER凋亡信号介导的细胞凋亡。吡格列酮预处理可减少ER凋亡信号介导的细胞凋亡,JNK信号途径在吡格列酮预处理抑制ER凋亡信号分子活化的机制中发挥重要作用。     

吡格列酮对2型糖尿病大鼠骨骼肌脂联素受体1表达的影响

目的观察吡格列酮对2型糖尿病大鼠血清脂联素以及骨骼肌脂联素受体1(AdipoR1)表达的影响,探讨吡格列酮对2型糖尿病胰岛素抵抗的改善作用及机制。方法 40只8周龄健康雌性SD大鼠,随机分为正常对照组(n=10)、糖尿病组(n=15)及吡格列酮组(n=15)。用高糖高脂饲料加小剂量链脲佐菌素建立2型糖尿病大鼠模型,成模后吡格列酮组给予10 mg/(kg.d)吡格列酮灌胃,正常对照组和糖尿病组给予同体积生理盐水灌胃,共12周。3个月后股静脉取血,酶联免疫吸附法(ELISA)测定血清脂联素水平,留取大鼠骨骼肌,光、电镜观察骨骼肌结构,免疫组织化学染色法测定骨骼肌AdipoR1蛋白的表达。结果与正常对照组(1.73±0.32 mg/L)比较,糖尿病组血清脂联素(1.01±0.27 mg/L)水平显著降低,而吡格列酮组(1.34±0.43 mg/L)较糖尿病组显著升高,差异有统计学意义(P<0.05)。骨骼肌AdipoR1免疫组织化学染色正常对照组着色深且广泛,糖尿病组较正常对照组染色浅,吡格列酮组较糖尿病组染色深,但较正常对照组浅。光镜及电镜结果显示大鼠骨骼肌结构未见明显异常。结论 2型糖尿病大鼠血清脂联素水平及骨骼肌AdipoR1表达降低并导致糖脂代谢紊乱及胰岛素抵抗。吡格列酮可上调血清脂联素及骨骼肌AdipoR1的表达,从而调节糖脂代谢,改善胰岛素抵抗。     

罗格列酮上调高脂饮食老年大鼠骨骼肌GLUT4和PKB的表达

目的 观察高脂饮食对老年大鼠骨骼肌葡萄糖转运蛋白4(GLUT4)和蛋白激酶B(PKB)的表达变化及罗格列酮的干预效果.方法 老年大鼠随机分为对照组、高脂组(HF)、高脂+罗格列酮干预组(RSG),每组20只.应用清醒状态下正常葡萄糖高胰岛素钳夹技术的葡萄糖输注率评价胰岛素抵抗,用荧光定量PCR法和Western印迹技术检测骨骼肌GLUT4和PKB的表达.结果 高脂组骨骼肌长链脂酰辅酶A(LCACoA)升高而葡萄糖输注率明显下降(P＜0.01),骨骼肌GLUT4和PKB的表达明显降低(P＜0.05,P＜0.01),罗格列酮干预组显著缓解高脂组上述变化(P＜0.05,P＜0.01).结论 罗格列酮上调高脂饮食老年大鼠骨骼肌GLUT4和PKB的表达,是改善老年胰岛素抵抗的机制之一.     

脂联素调控人成骨细胞OPG/RANKL表达的信号转导机制

目的 研究脂联素调控人成骨细胞护骨素(OPG)和NF-кB受体活化凶子配体(RANKL)表达的作用机制.方法 人成骨细胞OPG和RANKL mRNA的表达以实时PCR检测,p38丝裂原活化蛋白激酶(p38 MAPK)、细胞外信号调节激酶(ERK1/2)、c-Jun氨基端激酶(JNK)的磷酸化水平用Western印迹法检测.廊用小分子RNA干扰技术(siRNA)阻断脂联素受体(AdR)表达,并以p38 MAPK抑制剂(SB203580)和JNK抑制剂(SP600125)干预,以观察脂联素对人成骨细胞OPG/RANKL作用的调节机制.结果 用siRNA沉默AdRl的表达可消除脂联素促进人成骨细胞RANKL表达和抑制OPG表达的作用;脂联素干预前予SB203580阻断p38 MAPK后,也可消除脂联素对成骨细胞RANKL和OPG的作用,而SP600125并无作用.结论 在人成骨细胞中,脂联素通过AdR1/p38 MARK途径促进RANKL表达和抑制OPG的表达.     

Constitutive Activation of Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase in B-Cell Lymphoproliferative Disorders

Signaling molecules such as p21(ras) (Ras), mitogen-activated protein kinase (MAPK), and Akt kinase play pivotal roles in the proliferation and survival of lymphoid cells in response to many kinds of stimulation. It is not fully understood, however, how these molecules participate in the growth of malignant lymphoid cells. We determined whether Ras, MAPKs such as extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38 MAPK, and Akt kinase are activated in B-cell tumors, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, Burkitt-like lymphoma, diffuse large B-cell lymphoma, and plasma cell leukemia. We found that Lyn protein tyrosine kinase was constitutively phosphorylated on tyrosine, and that ERK and p38 MAPK were constitutively active in all cases of the B-cell tumor. In contrast, activation of Ras and Akt kinase was found in limited cases, and JNK kinase activity was not observed in any case. These results suggest that ERK and p38 play roles in the oncogenesis of B-cell tumors.     

Chemical-genetic analysis of cyclin dependent kinase 2 function reveals an important role in cellular transformation by multiple oncogenic pathways

A family of conserved serine/threonine kinases known as cyclin-dependent kinases (CDKs) drives orderly cell cycle progression in mammalian cells. Prior studies have suggested that CDK2 regulates S-phase entry and progression, and frequently shows increased activity in a wide spectrum of human tumors. Genetic KO/knockdown approaches, however, have suggested that lack of CDK2 protein does not prevent cellular proliferation, both during somatic development in mice as well as in human cancer cell lines. Here, we use an alternative, chemical-genetic approach to achieve specific inhibition of CDK2 kinase activity in cells. We directly compare small-molecule inhibition of CDK2 kinase activity with siRNA knockdown and show that small-molecule inhibition results in marked defects in proliferation of nontransformed cells, whereas siRNA knockdown does not, highlighting the differences between these two approaches. In addition, CDK2 inhibition drastically diminishes anchorage-independent growth of human cancer cells and cells transformed with various oncogenes. Our results establish that CDK2 activity is necessary for normal mammalian cell cycle progression and suggest that it might be a useful therapeutic target for treating cancer.     

血清肌钙蛋白T及I对急性心肌梗死早期诊断的临床价值

目的 :观察急性心肌梗死 (AMI)患者入院前后血清肌钙蛋白T(cTnT)、肌钙蛋白I(cTnI)的变化 ,探讨cTnT、cTnI对早期诊断AMI的价值。方法 :对 10 0例患者 (AMI 5 0例、不稳定型心绞痛 5 0例 )和 2 0例健康人进行了血清cTnT、cTnI、肌酸激酶 (CK)和肌酸激酶同工酶 (CK MB)检测。结果 :①血清cTnT、cTnI、CK和CK MB检测AMI的敏感度和特异度分别为 80 %和 90 %、85 %和 92 %、92 %和 6 1%、75 %和 75 %。②AMI患者血清cTnT、cTnI浓度平均升高 (17.5± 5 .7)和 (16 .2± 4 .8)倍 ,显著高于CK(7.6± 3.1)倍、CK MB (6 .5± 2 .9)倍 (P <0 .0 1) ;③AMI后 3h内cTnT、cTnI阳性检出率为 5 0 .0 %、4 0 .0 % ,明显高于CK MB(2 4 .0 % )和CK(2 0 .0 % ) ;AMI 5d后cTnT、cTnI阳性检出率为 70 .0 %、6 6 .0 % ,而CK MB和CK仅为 4 .0 %和 8.0 %。结论 :血清cTnT、cTnI能早期确切诊断AMI ,具有较宽的时间诊断窗口 ,是心肌梗死早期诊断较敏感和特异的血清标志物     

IkappaB kinase epsilon and TANK-binding kinase 1 activate AKT by direct phosphorylation

AKT activation requires phosphorylation of the activation loop (T308) by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and the hydrophobic motif (S473) by the mammalian target of rapamycin complex 2 (mTORC2). We recently observed that phosphorylation of the AKT hydrophobic motif was dramatically elevated, rather than decreased, in mTOR knockout heart tissues, indicating the existence of other kinase(s) contributing to AKT phosphorylation. Here we show that the atypical IκB kinase ε and TANK-binding kinase 1 (IKKε/TBK1) phosphorylate AKT on both the hydrophobic motif and the activation loop in a manner dependent on PI3K signaling. This dual phosphorylation results in a robust AKT activation in vitro. Consistently, we found that growth factors can induce AKT (S473) phosphorylation in Rictor(-/-) cells, and this effect is insensitive to mTOR inhibitor Torin1. In IKKε/TBK1 double-knockout cells, AKT activation by growth factors is compromised. We also observed that TBK1 expression is elevated in the mTOR knockout heart tissues, and that TBK1 is required for Ras-induced mouse embryonic fibroblast transformation. Our observations suggest a physiological function of IKKε/TBK1 in AKT regulation and a possible mechanism of IKKε/TBK1 in oncogenesis by activating AKT.     

AT9283, a potent inhibitor of the Aurora kinases and Jak2, has therapeutic potential in myeloproliferative disorders

Constitutive activation of Janus kinase (Jak) 2 is the most prevalent pathogenic event observed in the myeloproliferative disorders (MPD), suggesting that inhibitors of Jak2 may prove valuable in their management. Inhibition of the Aurora kinases has also proven to be an effective therapeutic strategy in a number of haematological malignancies. AT9283 is a multi‐targeted kinase inhibitor with potent activity against Jak2 and Aurora kinases A and B, and is currently being evaluated in clinical trials. To investigate the therapeutic potential of AT9283 in the MPD we studied its activity in a number of Jak2‐dependent systems. AT9283 potently inhibited proliferation and Jak2‐related signalling in Jak2‐dependent cell lines as well as inhibiting the formation of erythroid colonies from haematopoietic progenitors isolated from MPD patients with Jak2 mutations. The compound also demonstrated significant therapeutic potential in vivo in an ETV6‐JAK2 (TEL‐JAK2) murine leukaemia model. Inhibition of both Jak2 and Aurora B was observed in the model systems used, indicating a dual mechanism of action. Our results suggest that AT9283 may be a valuable therapy in patients with MPD and that the dual inhibition of Jak2 and the Aurora kinases may potentially offer combinatorial efficacy in the treatment of these diseases.     

高糖对血管内皮细胞蛋白激酶Cα和δ表达及功能的影响

以高糖培养的人脐静脉内皮细胞（HUVECs）研究显示,高糖可诱导血管内皮细胞（EC）功能紊乱,凋亡增加,NO浓度降低,可溶性细胞间黏附分子1水平增加;高糖可诱导HUVECs的蛋白激酶C（PKC）α及PKCδ表达位置转移,PKCδ表达增强,但对PKCα表达影响不明显。高糖所致EC功能异常,可能部分是通过PKCα及PKCδ途径来实现的。     

Clinical values of creatine kinase and its isoenzymes in children and adolescents with vasovagal syncope

Background and aimVasovagal syncope (VVS) in children and adolescents is a common disorder. There may be an internal relationship between creatine kinase (CK) and its isoenzymes (CKMB) and syncope. The aim of this study was to evaluate the changes of CK and CKMB in children and adolescents with VVS.Methods and resultsThe VVS group included 218 patients (93 male and 125 female). The control group included 129 healthy children (78 male and 51 female). Serum CK and CKMB levels were estimated. We founded ①Serum CK and CK-MB levels decreased in VVS group than that in control group (P < 0.05). ②The CK levels of female were significantly lower than those of male in VVS group (P < 0.05). ③Serum level of CK-MB were in negative correlation with age, height, weight, BMI whereas in positively correlation with HR. ④CK was effected by CK-MB (β = 0.147, P = 0.037) while CK-MB was independently influenced by age (β = −0.203, P = 0.002) and DBP (β = 0.171, P = 0.011). ⑤Both CK and CK-MB significantly influenced on VVS occurrence after adjusting for the effects of gender, age, height, weight, BMI and HR.ConclusionThe serum CK and CKMB levels decrease in children and adolescents with VVS. CK and CK-MB are the independent protective factors with VVS.     

Sphingolipids in cardiovascular and cerebrovascular systems: Pathological implications and potential therapeutic targets

The sphingolipid metabolites ceramide, sphingosine, and sphingosine-1-phosphate (S1P) and its enzyme sphingosine kinase (SphK) play an important role in the regulation of cell proliferation, survival, inflammation, and cell death. Ceramide and sphingosine usually inhibit proliferation and promote apoptosis, while its metabolite S1P phosphorylated by SphK stimulates growth and suppresses apoptosis. Because these metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determine cell fate. The relevance of this “sphingolipid rheostat” and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations. A central finding of these studies is that SphK is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S1P, but also decreases levels of pro-apoptotic ceramide and sphingosine. Activation of bioactive sphingolipid S1P signaling has emerged as a critical protective pathway in response to acute ischemic injury in both cardiac and cerebrovascular disease, and these observations have considerable relevance for future potential therapeutic targets.     

Protein kinase C and downstream signaling pathways in a three-dimensional model of phorbol ester-induced angiogenesis

Angiogenesis, a critical process in both health and disease, is mediated by a number of signaling pathways. Although proangiogenic stimuli, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and the phorbol ester phorbol-12 myristate-13 acetate (PMA) are known to promote blood vessel formation, their downstream targets are ill defined. We sought to investigate the signaling pathways required for vessel assembly by utilizing a three-dimensional collagen matrix in which human umbilical vein endothelial cells (HUVECs) form tubular structures. Our data show that PMA is sufficient for the induction of angiogenesis, and that protein kinase C (PKC) is necessary for this process. Evaluation of PKC isoforms and revealed that these proteins are uniquely regulated. Characterization of an additional PMA target, protein kinase D (PKD) demonstrated that this enzyme becomes phosphorylated in HUVECs, and may therefore be involved in proangiogenic signaling. Further examination of downstream effectors of PKC showed that extracellular signal-regulated kinase (ERK) is critical for angiogenesis, and is accordingly phosphorylated in response to PMA. Surprisingly however, phosphorylation of ERK is independent of PKC activity. In addition, we show that the PKC target sphingosine kinase (SPK) is required for vessel formation. These findings illustrate the complexities of blood vessel formation, and suggest that activators utilize multiple independent pathways to invoke a complete angiogenic response.