Effect of Sodium Tanshinone ⅡA Sulfonate on Phosphorylation of Extracellular Signal-regulated Kinasel/2 in Angiotensin Ⅱ-induced Hypertrophy of Myocardial Cells

Objective： To observe the effects of sodium tanshinone ⅡA sulfonate （STS） on angiotensin Ⅱ （Ang Ⅱ）-induced hypertrophy of myocardial cells through the expression of phosphorylated extracellular signal-regulated kinase （p-ERK1/2）. Methods： In the primary culture of neonatal rat myocardial cells, the total protein content in myocardial cells was determined by coomassie brilliant blue and the protein synthesis rate was measured by [3H]-Leucine incorporation as indexes for hypertrophy of myocardial cells. The expression of p-ERK1/2 was determined using Western blot and immunofluorescence labeling. Results： （1） The total protein and protein synthesis rate increased significantly in contrast to the control group after the myocardial cells were stimulated by Ang Ⅱ （1 μ mol/L） for 24 h; STS markedly inhibited the increment of the total protein level induced by Ang Ⅱ and the syntheses of protein. （2） After pretreatment of myocardial cells with Ang Ⅱ （1 μmol/L） for 5 min, the p-ERK1/2 protein expression was increased, with the most obvious effect shown at about 10 min; pretreatment of myocardial cells with STS at different doses （2, 10, 50μmol/L） for 30 min resulted in obvious inhibition of the expression of p-ERK1/2 stimulated by Ang Ⅱ in a dose-dependent manner. （3） After the myocardial cells were stimulated by AngⅡ （1 μ mol/L）, the immunofluorescence of ERK1/2 rapidly appeared in the nucleus. The activation and translocation process of ERK1/2 induced by Ang Ⅱ was blocked distinctly by STS. （Conclusion： STS inhibited the myocardial cell hypertrophy induced by Ang Ⅱ, and the mechanism may be associated with the inhibition of p-ERK1/2 expression.     

替米沙坦对大鼠心肌肥厚组织中RASSF1A表达的影响

目的:观察替米沙坦对心肌肥厚大鼠左室心肌组织中RASSF1A表达的影响。方法:SD大鼠30只,随机分为对照组、模型组和干预组。模型组和干预组给予异丙肾上腺素3mg/(kg·d)皮下注射,干预组在建模同时灌胃替米沙坦8.33mg/(kg·d)。10d后,进行血流动力学和心肌肥厚指数测定;取大鼠左心室心肌组织,分别采用RT-PCR和免疫组化法检测RASSF1AmRNA和蛋白的表达,同时观察病理学变化。结果:血流动力学、心室肥厚指数及病理观察结果提示,模型组大鼠发生心肌肥厚,干预组上述变化较模型组减轻。3组大鼠心肌组织中RASSF1AmRNA和蛋白的表达差异均有统计学意义(F=38.968,284.445,P<0.001)。与对照组相比,模型组、干预组RASSF1AmRNA和蛋白的表达降低(P<0.05);与模型组相比,干预组RASSF1AmRNA和蛋白的表达升高(P<0.05)。结论:替米沙坦可通过调节RASSF1A的表达抑制心肌肥厚的发生。     

丝裂素活化蛋白激酶在心肌细胞肥大中的作用

目的：研究神经肽Ｙ（ＮＰＹ）在高血压心肌细胞肥大中的作用。方法：复制腹主动脉狭窄和高盐摄入性高血压大鼠模型。测定对照组和高血压血浆、心肌ＮＰＹ水平和心肌丝裂素活化蛋白激酶（ＭＡＰＫ）活性。结果：高血压大鼠心重、ＮＰＹ水平和心肌ＭＡＰＫ活性均较显著高于对照组（Ｐ〈０．０５或Ｐ〈０．０１）。心肌ＭＡＰＫ活性与左心室肥大程度呈正相关（ｒ＝０．８０９，Ｐ〈０．０１）。结论：ＭＡＰＫ系统可能在ＮＰＹ致心肌细     

丹参酮ⅡA对高糖培养的大鼠血管平滑肌细胞p38MAPK信号通路的影响

目的：观察丹参酮ⅡA对高糖诱导的血管平滑肌细胞增殖的影响，分析该作用与氧化应激以及丝裂原活化蛋白激酶p38（p38 mitogen－activated protein kinases，p38 MAPK）信号通路的关系。方法：取第3代原代培养的大鼠血管平滑肌细胞建立高糖诱导细胞增殖模型，实验分为正常对照组、高糖组和丹参酮ⅡA组，BrdU掺入法测定血管平滑肌细胞的DNA合成水平；黄嘌呤氧化酶法、硫代巴比妥酸法分别测定细胞培养上清中超氧化物歧化酶（SOD）、丙二醛（MDA）含量；免疫印迹法（Western blot）检测磷酸化p38 MAPK蛋白表达。结果：高糖诱导的血管平滑肌细胞呈快速增殖状态，在10～25mmol·L^-1间增殖效应呈剂量依赖性；丹参酮ⅡA能抑制高糖诱导的血管平滑肌细胞增殖，提高高糖培养的细胞中SOD水平，降低MDA含量，减少P-p38MAPK蛋白的表达。结论：丹参酮ⅡA抑制高糖诱导的血管平滑肌细胞增殖，这可能与其能减少血管平滑肌细胞中的氧自由基，抑制p38MAPK信号通路有关。     

异丙肾上腺素致大鼠心肌肥厚及对相关因子的影响

目的 探讨异丙肾上腺素(isoproterenol,Iso) 致大鼠心肌肥厚及对相关因子的影响.方法 Wistar大鼠30只,随机分成对照组和Iso组.Iso组背部皮下注射5 mg/(kg·d),连续7 d后,测量大鼠心脏重量指数(HW/BW)、左心室重量指数(LVW/ BW)及左心室游离壁厚度;分光光度法检测左心室心肌组织及血清羟脯氨酸含量并计算胶原含量;分光光度法检测左心室心肌组织中丙二醛(malondialdehyde, MDA)含量和超氧化物歧化酶(superoxide dismutase, SOD)活性.结果 与对照组比较,Iso组大鼠心重指数和左心室重量指数明显增大,左心室游离壁厚明显增厚,左心室心肌组织及血清中胶原含量增高,心肌组织中MDA含量增高、SOD活性下降.结论 Iso能促进大鼠心肌肥厚和相关因子的变化.     

Role of β-adrenergic signal transduction pathway on myocardial ischemic preconditioning of rats

Summary To study the changes in every part of the β-adrenergic signal transduction pathway and their effects on ischemic preconditioning of rat myocardiumin vivo. SD rats were divided into three groups: IP group, I/R group and CON group. The IP group was further divided into PC1−, 2−, 3−, and PC1+, 2+, 3+ groups according to preconditioning procedure. The rats received surgical procedure and underwent left coronary artery occlusion and reperfusion. We analyzed the infarct size by TTC staining, measured serum myocardial enzymes, studied the β-AR Bmax and Kd by radioligand binding assay of receptors, checked the activity of AC and PKA by the method of biochemistry and examined the content of cAMP by radioimmunoassay. The infarct area was much smaller in the IP group than in the I/R group (P<0.001), while the enzymes were significantly higher in I/R (P<0.001). The Bmax of β-AR in IP was much higher than that in I/R (P<0.001), but no difference in Kd could be seen between IP and I/R groups. In IP, the activity of AC and PKA and the content of cAMP were higher than those in I/R (P<0.05, 0.002 and 0.001, respectively). In the procedure of preconditioning, the content of cAMP and the activity of PKA showed the characteristic of cyclic fluctuation. Ischemic preconditioning can protect the heart from necrosis and reduce endo-enzyme leakage. The system of β-adrenergic signal transduction pathway probably takes part in the protection effect of the IP, which might be clicited by the PKA. LAN Xiaoli, female, born in 1973, M. D., Ph. D.     

Effects of calmodulin-dependent protein kinase II inhibitor, KN-93, on electrophysiological features of rabbit hypertrophic cardiac myocytes

Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related.The aim of this study was to determine the effects of the calmodulin-dependent protein kinase(CaMK) Ⅱ inhibitor,KN-93,on L-type calcium current(I Ca,L) and early after-depolarizations(EADs) in hypertrophic cardiomyocytes.A rabbit model of myocardial hypertrophy was constructed through abdominal aortic coarctation(LVH group).The control group(sham group) received a sham operation,in which the abdominal aortic was dissected but not coarcted.Eight weeks later,the degree of left ventricular hypertrophy(LVH) was evaluated using echocardiography.Individual cardiomyocyte was isolated through collagenase digestion.Action potentials(APs) and I Ca,L were recorded using the perforated patch clamp technique.APs were recorded under current clamp conditions and I Ca,L was recorded under voltage clamp conditions.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were observed under the conditions of low potassium(2 mmol/L),low magnesium(0.25 mmol/L) Tyrode’s solution perfusion,and slow frequency(0.25-0.5 Hz) electrical stimulation.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were also evaluated after treatment with different concentrations of KN-92(KN-92 group) and KN-93(KN-93 group).Eight weeks later,the model was successfully established.Under the conditions of low potassium,low magnesium Tyrode’s solution perfusion,and slow frequency electrical stimulation,the incidence of EADs was 0/12,11/12,10/12,and 5/12 in sham group,LVH group,KN-92 group(0.5 μmol/L),and KN-93 group(0.5 μmol/L),respectively.When the drug concentration was increased to 1 μmol/L in KN-92 group and KN-93 group,the incidence of EADs was 10/12 and 2/12,respectively.At 0 mV,the current density was 6.7±1.0 and 6.3±0.7 PA·PF-1 in LVH group and sham group,respectively(P>0.05,n=12).When the drug concentration was 0.5 μmol/L i     

阿霉素大鼠心肌重塑中PKC的表达

目的探讨蛋白激酶C（PKC）在阿霉素诱导大鼠心肌胶原重塑中的作用。方法20只8周龄雄性SD大鼠随机分为2组：对照组腹腔注射生理盐水2ml／kg,连续8周;模型组腹腔注射阿霉素（ADM）2mg／kg,连续8周;末次注射后无菌取心脏。光镜下观察心肌的病理改变;免疫组化法检测大鼠心肌组织I、Ⅲ型胶原的表达并计算二者比值;Western blotting方法检测大鼠心肌组织PKC的表达。结果光镜下心肌病理改变：对照组心肌细胞排列整齐,心肌纤维结构清晰,未见心肌纤维破坏;模型组心肌细胞肥大、伸长、灶状坏死,肌质变性,核大、深染,问质纤维化、胶原增生。心肌I、Ⅲ型胶原表达：与对照组相比,模型组I、Ⅲ型心肌胶原的表达及I／Ⅲ比值均升高（P〈0．01）。心肌组织PKC的表达：模型组PKC表达较对照组明显增加（P〈0．01）。结论PKC参与了阿霉素诱导的SD大鼠心肌纤维化。     

蛋白激酶C不同亚型对丙泊酚血管舒张作用的影响

目的探讨丙泊酚的血管舒张作用与蛋白激酶C(PKC)不同亚型间的关系。方法将SD大鼠胸主动脉环随机分为内皮完整组(n=36)和去内皮组(n=36),每组各分6个亚组:①10 nmol/L Go6976+1×10-6mol/L去甲肾上腺素(NA)+丙泊酚处理组(n=6);②10μmol/L Rottlerin+1×10-6mol/L NA+丙泊酚处理组(n=6);③2μmol/L PKCε-Pseudo(假底物)+1×10-6mol/L NA+丙泊酚处理组(n=6);④2μmol/L PKCθ-Pseudo+1×10-6mol/L NA+丙泊酚处理组(n=6);⑤2μmol/L PKCζ-Pseudo+1×10-6mol/L NA+丙泊酚处理组(n=6);⑥1×10-6mol/L NA+丙泊酚处理组(对照组,n=6)。PKCα抑制剂Go6976,PKCδ抑制剂Rottlerin,PKCζ、θ和ε假底物孵育血管环30 min后,加1×10-6mol/L NA收缩血管环达峰值,每15 min加递增浓度的丙泊酚(1×10-6、5×10-6、1×10-5、5×10-5、1×10-4mol/L),观察血管张力的变化。结果内...     

The cAMP-mediated protein kinase signal transduction pathway is involved in the pyrogenic effect of CRH in rats

Objective To determine whether the cyclic adenosine monophosphate (cAMP) mediated protei n kinase signal transduction pathway is involved in the pyrogenic action of cort icotropin releasing hormone (CRH) in rats.Methods Corticotropin releasing hormone, 2’,3’-dideoxy-adenosine (DDA) and adenosi ne-3’,5’-(cyclic) monophosphorothionate, Rp-Isomer (Rp-cAMPS), were administ ered intracerebroventricularly (i.c.v.). The colonic temperature was measure d using a thermistor, and the content of cAMP in the hypothalamus was determined by radioimmunoassay. Hypothalamic incubation was used to assess the effects of CRH on the content of cAMP in the hypothalamus in vitro. Results Microinjection (i.c.v.) of CRH (2.5 μg, 5.0 μg and 10 μg) caused incr eases in colonic temperature and the hypothalamus cAMP level in conscious rats. CRH increased hypothalamus cAMP level in vitro. The pyrogenic effects of CRH w ere abolished or markedly inhibited by prior injection (i.c.v.) of an adenyla te cyclase inhibitor, DDA (30 μg), or an inhibitor of cAMP-dependent protein kinase, Rp-cAMPS (15 μg). Conclusion cAMP mediates the pyrogenic action of centrally administered of CRH in rats, and protein kinase A may play an important role in the central CRH-induced fever. The cAMP-dependent protein kinase signal transduction pathway may be involved in the central mechanisms of the pyrogenic action of CRH in rats.     

丹参酮对高血压大鼠心脏保护作用

目的：通过建立肾性高血压大鼠模型,观察高血压大鼠心脏形态及NOS活性和NO产量变化,来探讨丹参酮ⅡA （Tanshinone ⅡA,TSN）对心脏的保护机制。方法：应用经典的双肾双狭复制肾血管性高血压大鼠模型（two～kidney, two～clip re-novascular hypertension model ,2K2C RH model）,设立假手术组、高血压组、丹参酮治疗组。采用鼠尾测压法测定大鼠血压;分别应用化学比色法法和硝酸还原酶法测定心肌组织 NOS活性测定及一氧化氮（ NO）的浓度;结果：丹参酮可促进心肌组织NOS活性及NO产量增加,并可明显改善RH大鼠心肌肥大和间质纤维化。结论：丹参酮可明显改善RH大鼠心肌肥大和心肌间质纤维化,此作用可能与TSN促进心肌组织eNOS蛋白表达、上调NOS活性和NO产量有关。     

Effects of transforming growth factor-β1 and signal protein Smad3 on rat cardiomyocyte hypertrophy

Background SMAD proteins have recently been identified as the first family of putative transforming growth factor-β1 (TGF-β1) signal transducers. This study was to investigate the effects of TGF-β1 and signal protein Smad3 on rat cardiac hypertrophy.Methods The incorporation of [3H]-leucine was measured to determine the hypertrophy of cardiomyocyte incubated with different doses of TGF-β1 in cultured neonatal cardiomyocytes. The model of rat cardiac hypertrophy was produced with constriction of the abdominal aorta. At different times after the operation, rats were killed, and their left ventricular mass index (LVMI) determined.The mRNA expression of TGF-β1 and Smad3 of cultured cells and hypertrophic left ventricles were assessed by RT-PCR. The protein expression of Smad3 was assessed by Western blot.Results In cultured neonatal cardiomyocytes, TGF-β1 significantly promoted incorporation of [3H]-leucine. With the concentration of 3 pg/L, it increased the expression of Smad3 in mRNA and protein levels after 15 minutes, and continued for up to 8 hours of cultured cardiomyocytes. The LVMI and theexpression of TGF-β1 (mRNA) and Smad3 (mRNA and protein) of hypertrophic left ventricle were increased by day 3 after the operation and continued to the 4th week. The peak expression of these was in the second week after operation.Conclusion TGF-β1 has positive effects on could be related to the pathologic progressionrat cardiomyocyte hypertrophy. Signal protein Smad3 of rat cardiac hypertrophy.     

大鼠神经干细胞中PI3-K/AKT信号转导通路的研究

目的 应用PI3-K特异性阻滞剂LY294002孵育大鼠神经干细胞（NSCs）,探讨PI3-K/AKT信号转导通路在NSCs中的作用。方法 体外分离、培养E15～16 胎鼠NSCs,应用不同浓度（0~40 μmol/L）的LY294002孵育NSCs后进行细胞存活(WST-8检测)、增殖(BrdU免疫组织化学鉴定)、分化(β Tubulin-Ⅲ免疫组织化学鉴定)和AKT磷酸化蛋白水平(Western Blotting)的检测。结果 LY294002对NSCs的存活、增殖和分化的影响呈剂量依赖性。在LY294002较高浓度（25、30、35、40 μmol/L）时,NSCs的存活率明显下降（P<0.05）;LY294002为30、35、40 μmol/L时,BrdU阳性细胞数明显少于正常对照组（LY294002 0 μmol/L）（P<0.05）;LY294002为35、40 μmol/L时,β Tubulin-Ⅲ的阳性细胞数明显少于正常对照组（P<0.05）。LY294002 阻断AKT表达的作用呈剂量依赖性。结论 PI3-K/AKT信号转导通路对大鼠NSCs的存活、增殖、分化起重要作用。     

大鼠神经干细胞中PI3-K/AKT信号转导通路的研究

目的 应用PI3-K特异性阻滞剂LY294002孵育大鼠神经干细胞(NSCs),探讨PI3-K/AKT信号转导通路在NSCs中的作用.方法 体外分离、培养E15～16 胎鼠NSCs,应用不同浓度(0~40 μmol/L)的LY294002孵育NSCs后进行细胞存活(WST-8检测)、增殖(BrdU免疫组织化学鉴定)、分化(β Tubulin-Ⅲ免疫组织化学鉴定)和AKT磷酸化蛋白水平(Western Blotting)的检测.结果 LY294002对NSCs的存活、增殖和分化的影响呈剂量依赖性.在LY294002较高浓度(25、30、35、40 μmol/L)时,NSCs的存活率明显下降(P<0.05);LY294002为30、35、40 μmol/L时,BrdU阳性细胞数明显少于正常对照组(LY294002 0 μmol/L)(P<0.05);LY294002为35、40 μmol/L时,β Tubulin-Ⅲ的阳性细胞数明显少于正常对照组(P<0.05).LY294002 阻断AKT表达的作用呈剂量依赖性.结论 PI3-K/AKT信号转导通路对大鼠NSCs的存活、增殖、分化起重要作用.     

p38抑制剂对缺血再灌注大鼠心肌细胞丝裂原活化蛋白激酶通路的影响

目的探讨p38抑制剂对心肌缺血再灌注大鼠心肌细胞丝裂原活化蛋白激酶(MAPK)通路的影响。方法将44只大鼠随机分为4组:对照组(9只)、缺血组(15只)、缺血再灌注组(10只)和干预组(10只)。对照组大鼠只暴露心脏,缺血组大鼠制备心肌缺血模型,缺血再灌注组大鼠制备心肌缺血再灌注模型,干预组大鼠在左冠状动脉前降支结扎前30 min注射p38抑制剂SB20358(100μg·kg-1),其余操作均与缺血再灌注组一致。观察各组大鼠心肌组织形态学变化及心肌组织中p38、c-Jun氨基端激酶(JNK)和细胞外调节蛋白激酶(ERK1/2)水平的变化。结果对照组大鼠心肌组织中未检测到p38、ERK1/2和JNK蛋白。缺血组缺血60、90 min时大鼠心肌组织中p38蛋白水平显著高于缺血30 min时,差异均有统计学意义(P<0.05);各个时间点,干预组大鼠心肌组织中p38蛋白水平显著低于缺血组和缺血再灌注组,差异均有统计学意义(P<0.05)。随着再灌注时间的延长,缺血再灌注组和干预组大鼠心肌组织中p38蛋白水平逐渐降低,但差异均无统计学意义(P>0.05)。随着缺血和再灌注时间延长,缺血组大鼠心肌组织中ERK1/2和JNK水平略有升高,缺血再灌注组和干预组大鼠心肌组织中ERK1/2和JNK水平略有降低,但差异均无统计学意义(P>0.05)。结论 MAPK中p38参与心肌缺血再灌注损伤,但JNK和ERK1/2的作用不显著。     

无患子皂苷对压力超负荷模型大鼠血流动力学及左心室肥厚的影响

目的:观察无患子皂苷对腹主动脉缩窄致压力超负荷模型大鼠抗心肌肥厚的作用及对血流动力学的影响。方法:采用腹主动脉缩窄法制备压力超负荷心肌肥厚大鼠模型,观察无患子皂苷对模型大鼠左心室重量指数及血流动力学的影响。结果:①无患子皂苷能够显著降低模型大鼠左心室重量指数;②无患子皂苷能够显著降低模型大鼠收缩压、左室压和左室舒张末期压。结论:无患子皂苷能够显著抑制压力超负荷模型大鼠的左心室肥厚,改善血流动力学改变,具有显著的逆转心肌肥厚的作用。     

Protective effect and mechanism of sodium tanshinone II A sulfonate on microcirculatory disturbance of small intestine in rats with sepsis

To explore the protective effect of sodium tanshinone ⅡA sulfonate(STS) on microcirculatory disturbance of small intestine in rats with sepsis,and the possible mechanism,a rat model of sepsis was induced by cecal ligation and puncture(CLP).Rats were randomly divided into 3 groups:sham operated group(S),sepsis group(CLP) and STS treatment group(STS).STS(1 mg/kg) was slowly injected through the right external jugular vein after CLP.The histopathologic changes in the intestinal tissue and changes of mesenteric microcirculation were observed.The levels of tumor necrosis factor-α(TNF-α) in the intestinal tissue were determined by using enzyme-linked immunoabsorbent assay(ELISA).The expression of intercellular adhesion molecule-1(ICAM-1) in the intestinal tissue was detected by using immunohistochemisty and Western blot,that of nuclear factor κB(NF-κB) and tissue factor(TF) by using Western blot,and the levels of NF-κB mRNA expression by using RT-PCR respectively.The microcirculatory disturbance of the intestine was aggravated after CLP.The injury of the intestinal tissues was obviously aggravated in CLP group as compared with S group.The expression levels of NF-κB p65,ICAM-1,TF and TNF-α were upregulaed after CLP(P<0.01).STS post-treatment could ameliorate the microcirculatory disturbance,attenuate the injury of the intestinal tissues induced by CLP,and decrease the levels of NF-κB,ICAM-1,TF and TNF-α(P<0.01).It is suggested that STS can ameliorate the microcirculatory disturbance of the small intestine in rats with sepsis,and the mechanism may be associated with the inhibition of inflammatory responses and amelioration of coagulation abnormality.     

三七总皂苷抗大鼠心肌肥大作用

目的研究三七总皂苷(PNS)对体外培养大鼠心肌细胞肥大及在体大鼠心肌肥大的影响。方法体外培养大鼠心肌细胞,用血管紧张素(Ang)刺激制备心肌细胞肥大模型,分别采用Lowry法、3H-亮氨酸掺入法和激光共聚焦显微镜测定细胞内蛋白质的量、蛋白质合成速率和游离钙荧光强度。ip去甲肾上腺素法诱导大鼠心肌肥大,计算心脏指数和左心室指数。结果与模型组相比,0.05、0.10、0.15g/LPNS可显著降低细胞内蛋白质的量和3H-亮氨酸掺入量,减弱细胞内Ca2 荧光信号。25、50、75mg/kgPNS可显著降低心肌肥大大鼠左心室指数。结论PNS对Ang诱导的大鼠心肌细胞肥大和去甲肾上腺素诱导的在体大鼠心肌肥大均有明显的抑制作用。     

MAPK反义寡核苷酸对K562细胞系的增殖抑制作用

目的 :探讨MAPK在慢性髓细胞白血病 (CML)信号转导中的作用及其作用机制 .方法 :用脂质体转染法将MAPKASO导入K5 6 2细胞中 ,通过集落形成、DNA合成、蛋白质含量和MAPK活性的变化检测MAPKASO对K5 6 2细胞的作用 .结果 :MAPKASO可明显抑制细胞集落形成、DNA合成、蛋白质含量和MAPK活性 ,其抑制率分别为 4 6 .1% ,5 7.1% ,4 3.2 %和 6 2 .0 % ,与对照组比较有显著差异 (P <0 .0 5 ) .结论 :MAPK在CML信号转导中起重要作用 ,极有可能成为CML治疗的新靶点