NCA对受磷蛋白敲除小鼠心肌兴奋-收缩偶联的作用

 目的: 硝酰基(HNO)在轻微增加细胞内钙的基础上可以显著增加心肌肌丝对钙离子的反应性。本研究中,我们应用崭新的HNO供体乙酸1-亚硝基环己酯(NCA)来观察HNO对受磷蛋白敲除(PLB-KO)小鼠心室梳状肌的作用。方法: 小鼠右心室的完整梳状肌被连接在张力换能器与刺激电极之间,肌小节长度设定在2.2~2.3μm之间,K-H液表面灌流后,Fura-2经玻璃微电极负载进行离子透入法检测[Ca²⁺]_i,同时测定心肌收缩张力的变化。结果: PLB-KO小鼠心室梳状肌比野生型(WT)小鼠具有更高的钙瞬变及收缩力,同时展示负性收缩力-收缩频率相关性(FFR)。NCA(2.5μmol/L)在不同浓度细胞外钙([Ca²⁺]_o)条件下增加PLB-KO及WT小鼠心肌收缩力,但并不影响PLB-KO小鼠的负性FFR。稳态条件下2组小鼠去肌膜梳状肌的收缩力-钙离子相关性无显著性差异,NCA则增加去肌膜梳状肌的钙离子的反应性。结论: NCA提供的HNO通过增加PLB-KO及WT小鼠心肌肌丝对钙离子的反应性而增强心肌收缩力;心肌细胞内钙瞬变的增加伴随收缩力的增强表明HNO可改善钙离子活性,进一步证实HNO作为正性肌力药物的作用效果。     

过度表达人β_1-AR通过非cAMP途径激活心肌收缩反应

目的本研究通过过度表达人β 1-AR观察其对心肌收缩反应、cAMP水平及L -型Ca 通道的影响。方法心肌细胞可通过酶催化的方法获得 ,并与人β1-AR腺病毒 (100MOI,1000MOI)共培养48h。受体配基结合测定受体数量 ,ELISA法测定cAMP水平等。结果与100MOI或1000MOIβ1-AR共培养48h后 ,受体数分别增加18倍和30倍。基础收缩 (在1mmolCa 浓度下 )由对照组(5.4±2.7) %缩短率 (平均值±标准差 ,n=265)分别增加到(7.1±3.7) % (n=151,P<0.001)和(9.1±3.8) % (n=201 ,P<0.001)。收缩增加的幅度通过β1-拮抗剂CGP20712A(作为反向激动剂 )可恢复到正常对照水平。cAMP水平没有增加 ,而且cAMP拮抗剂(RpcAMPS)或Carbachol(其抑制cAMP刺激的收缩反应 )对增大的基础收缩没有显著的影响。在100MOI时 ,L-型Ca 通道电流显著增强[对照组(4.5±0.6)pA/pF,β1 过度表达组(9.0±1.2)pA/pF ,n=4,P<0.01]。结论激活的心肌收缩反应是由过度表达β 1受体引起 ,β 1受体可能是通过非限定膜途径直接偶联到L -型Ca 通道上     

丹参酮ⅡA对压力负荷增加大鼠心肌纤维化的改善作用

目的:观察丹参酮ⅡA(TanⅡA)对压力负荷增加大鼠心肌纤维化的改善作用。方法:60只SD大鼠随机分为假手术组(Sham组,n=8)和手术组(n=52),手术组均行腹主动脉缩窄术制备压力负荷增加心肌纤维化模型,术后4周,存活的32只成模大鼠中8只留作模型组(Model组),余24只分为TanⅡA低剂量组(L-TanⅡA组,10mg/Kg/天)、TanⅡA高剂量组(H-TanⅡA组,20mg/Kg/天)及阳性对照药物卡托普利组(Captopril组,100mg/Kg/天),每组8例。给药4周后,检测5组大鼠心肌肥厚指数和心肌组织形态、心肌羟脯氨酸(HYP)含量、心肌组织Rho相关卷曲螺旋蛋白激酶1(ROCK1)、转化生长因子-β1(TGF-β1)和核转录因子-κBp65(NF-κBp65)蛋白含量。结果:与Sham组比较,Model组大鼠的心肌肥厚指数、心肌HYP含量及心肌组织中ROCK1、TGF-β1和NF-κBp65蛋白含量均显著升高(P均<0.01),组织病理学改变明显;与Model组比较,L-TanⅡA和H-TanⅡA组心肌肥厚指数、心肌HYP含量及心肌组织中ROCK1、TGF-β1和NF-κBp65蛋白含量均降低(P<0.05或P<0.01),组织病理学改变明显。结论:TanⅡA能改善压力负荷增加大鼠心肌纤维化,此作用可能与其抑制ROCK1表达,下调TGF-β1和NF-κBp65水平有关。     

神经节苷酯对急性脊髓损伤治疗作用的实验研究

目的研究神经节苷酯对大鼠急性脊髓损伤后心肌营养素-1表达的影响,探寻神经节苷酯治疗急性脊髓损伤的机制.方法 Wistar脊髓损伤大鼠模型随机分为神经节苷酯治疗组、损伤对照组、假手术组,用原住杂交化学和RT-PCR方法检测损伤脊髓心肌营养素-1 mRNA的表达.结果脊髓损伤后1、7d神经节苷酯组心肌营养素-1 mRNA阳性表达显著高于损伤对照组(P>0.01).结论神经节苷酯可上调心肌营养素-1 mRNA表达,从而促进脊髓损伤后神经再生.     

环氧化酶-2参与血红素氧化酶1对抗大鼠心肌缺氧-复氧的损伤

目的 研究环氧化酶-2是否参与血红素氧化酶1(HO-1)对抗大鼠心肌缺氧.复氧的损伤及其可能的机制.方法 采用离体大鼠心脏Langendorff灌流法观察左室舒张末压(LVEDP)、左室发展压(LVDP)和最大左室收缩、舒张速率(±dp/dtmax).用全自动生化分析仪分析冠脉流出液乳酸脱氢酶(LDH)释放量.应用双波长分光光度计法间接测定大鼠血中COHb含量.心脏冷冻切片法观察心肌梗死面积.用6-keto-PGF1αRIA kit测定样本中前列腺素I2(PGI2)的稳定产物6-keto-PGF1α的含量.结果 ①HO-1的诱导剂高铁血红素明显抑制缺氧-复氧心脏LVEDP增高,降低LVDP和±dp/dtmax;减少复氧期LDH释放,缩小心肌梗死面积(P＜0.01).②HO-1的抑制剂可加重缺氧-复氧心脏LVDP和±dp/dtmax下降,LDH释放和梗死面积明显高于单纯缺氧-复氧组(P＜0.05).③环氧化酶-2(COX-2)抑制剂塞来昔布有部分取消高铁血红素降低缺氧-复氧心脏LVEDP、增加LVDP和±dp/dtmax,的作用,使LDH的释放和梗死面积明显增加(P＜0.05).结论 诱导HO-1增加可保护缺氧-复氧心肌,其作用可能通过调节COX-2的活性来完成.     

血红素加氧酶-1过表达延长肝脏低温保存时间的研究

目的 研究血红素加氧酶-1(heme oxygenase-1,HO-1)过表达延长肝脏低温保存的时间及其机制.方法 利用大鼠肝脏离体再灌注模型,用钴-原卟啉(cobalt protoporphyrin,CoPP)和锌-原卟啉(zincprotoporphyrin,ZnPP)特异地诱导和抑制HO-1,观察肝脏保存0、6、24h,灌注2h后的胆汁生成量,灌流液AST、LDH、TNF-α和IL-6的活性,肝脏MDA的含量,肝组织HO-1蛋白表达的Western印迹,细胞凋亡情况等.结果 CoPP诱导了肝组织HO-1的表达,与未诱导24h保存组相比,CoPP诱导组的肝脏灌流液AST、LDH、TNF-α和IL-6的活性以及肝脏MDA含量显著降低,胆汁生成量明显增加,凋亡细胞数量减少(P<0.05),并与未诱导6h保存组接近.给予ZnPP后,这些保护作用消失.结论 HO-1过表达延长了肝脏低温保存时间,原因可能与抗氧化应激、抑制炎性因子的表达和细胞凋亡有关.     

缺氧诱导因子-1在缺氧预处理心肌保护中的作用及其机制研究

目的:研究缺氧诱导因子-1(HIF-1)在缺氧预处理(HPC)心肌细胞保护中的作用及其机制。方法:在培养的SD乳鼠心肌细胞缺氧/复氧(H/R)模型上,观察HPC对于24h后心肌细胞H/R损伤的影响,以MTT法测定心肌细胞存活率,试剂盒测定培养液中乳酸脱氢酶(LDH)活性。制备心肌细胞蛋白提取物,以磷酸化的细胞外信号调节激酶(ERK1/2)抗体测定HPC后不同时间ERK1/2活性,以聚丙烯酰胺电泳迁移实验观察HIF-1α磷酸化,并观察蛋白磷酸酶激动剂BDM和ERKs的上游激酶(MEK1/2)抑制剂PD98059对于HPC诱导的HIF-1α磷酸化以及心肌细胞保护作用的影响。结果:HPC可以提高心肌细胞H/R后存活率、减少LDH漏出,并激活ERK1/2,使HIF-1α发生磷酸化;蛋白磷酸酶激动剂BDM和ERKs的上游激酶MEK抑制剂PD98059可以消除HPC诱导的HIF-1α磷酸化和心肌细胞保护作用。结论:HPC可以提高乳鼠心肌细胞对于H/R的耐受性,其机制涉及ERKs介导的HIF-1α磷酸化。     

清开灵对家兔内毒素性发热的作用及机制研究

目的:探讨清开灵(QKL)注射液对家兔内毒素性发热的解热作用和机制。方法:复制家兔内毒素(ET)性发热模型, 用数字温度计测量家兔的直肠温度, 用放免法测定下丘脑的IL-1β和cAMP、脑脊液中的cAMP、腹中隔区的AVP含量。结果:QKL+ET组的△T(0.24±0.10)℃、TRI₁(1.02±0.81)、下丘脑IL-1β(3.02±0.58)ng/g、下丘脑cAMP(1.37±0.23)nmol/g、CSF中cAMP(14.13±3.80)nmol/L、腹中隔区AVP(25.24±2.61)ng/g, 分别低于ET组的△T(0.40±0.11)℃、TRI₁(1.78±0.79)、下丘脑IL-1β(6.08±0.79)ng/g、下丘脑cAMP(2.90±0.40)nmol/g、CSF中cAMP(32.10±4.51)nmol/L、腹中隔区AVP(47.32±3.77)ng/g, 两者相比差异显著(P<0.01)。结论:QKL抑制下丘脑内生致热原和中枢发热介质的生成, 促进解热物质的释放, 可能是QKL对内毒素性发热的重要解热机制。     

不同剂量(+)-2-(1-羟基-4-环己酮)乙基咖啡酸酯对大鼠关节炎模型和全血炎症模型的药效学研究

目的:对(+)-2-(1-羟基-4-环己酮)乙基咖啡酸酯(HOEC)在大鼠关节炎模型上进行药效学评价,并利用大鼠全血花生四烯酸(AA)代谢模型,探究引起HOEC对关节炎的非剂量依赖性治疗作用的可能机理。方法:(1)采用大鼠胶原诱导性关节炎(CIA)模型,研究3个不同剂量的HOEC对CIA的治疗情况,并用免疫组化方法检测关节组织中胞质磷脂酶A_2(cPLA_2)、5-脂氧合酶(5-LOX)和环氧合酶2 (COX-2)的表达水平;(2) ELISA检测HOEC和其体内代谢物咖啡酸对大鼠全血AA代谢模型中代谢产物的作用。结果:(1) HOEC对大鼠CIA有治疗作用,但高剂量(10 mg/kg)组的治疗效果不如低(1 mg/kg)和中剂量(3 kg/kg)组;(2) HOEC对大鼠关节组织中cPLA2、5-LOX和COX-2的表达水平均有抑制作用;(3) HOEC对大鼠全血AA代谢模型中LOX和COX通路的代谢产物均有抑制作用,而咖啡酸对这些代谢产物的抑制作用弱于HOEC。结论:HOEC对大鼠CIA模型的抗炎作用可能与抑制关节组织中cPLA_2、5-LOX和COX-2的表达有关; HOEC对治疗大鼠CIA的非剂量依赖现象可能与其代谢产物咖啡酸对AA代谢产物生成的抑制作用弱于HOEC有关。     

Influence of maximal muscle strength and intrinsic muscle contractile properties on contractile rate of force development

‘Explosive’ muscle strength or contractile rate of force development (RFD) is a term to describe the ability to rapidly develop muscular force, and can be measured as the slope of the torque–time curve obtained during isometric conditions. Previously, conflicting results have been reported regarding the relationship between contractile RFD and various physiological parameters. One reason for this discrepancy may be that RFD in various time intervals from the onset of contraction is affected by different physiological parameters. The aim of the present study was to investigate the relationship between voluntary contractile RFD in time intervals of 0–10, 0–20,..., 0–250 ms from the onset of contraction and two main parameters: (1) voluntary maximal muscle strength and (2) electrically evoked muscle twitch contractile properties. The main finding was that voluntary RFD became increasingly more dependent on MVC and less dependent on muscle twitch contractile properties as time from the onset of contraction increased. At time intervals later than 90 ms from the onset of contraction maximal muscle strength could account for 52–81% of the variance in voluntary RFD. In the very early time interval (<40 ms from the onset of contraction) voluntary RFD was moderately correlated to the twitch contractile properties of the muscle and was to a less extent related to MVC. The present results suggest that explosive movements with different time spans are influenced by different physiological parameters. This may have important practical implications when designing resistance training programs for specific sports.     

The contractile function of cardiac muscle in experimental myocarditis

Summary The study is devoted to the examination of the contractile function of the cardiac muscle in myocarditis caused by adrenaline and theophylline. It was shown that in animals with acute and chronic myocarditis the contractile function of the left ventricle is not, on the whole, changed substantially, whereas the contractile capacity of the unit of the myocardial tissue is distinctly reduced. This defect in the contractile function of the cardiac muscle in myocarditis is not manifest in determination of the strength of ventricular contraction as a whole, since it is compensated for by an increase in the mass of the ventricle and can be detected only by determining the contractile capacity of the unit of the myocardial tissue mass.(Presented by Active Member AMN SSSR, V. V. Parin) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 60, No. 8, pp. 29–32, August, 1965     

Megestrol acetate increases short-term food intake in zinc-deficient rats

Rats offered a zinc-deficient (-Zn) diet voluntarily reduce their food intake within 3-4 days. Megestrol acetate (MA) is an appetite-stimulating drug used to treat cachexia of chronic diseases. In previous work, we found MA administration to male rats increased consumption of a -Zn diet. This approach would provide a useful tool for nutritional studies in which nutrient intake, except for zinc, would be maintained. The present study further examined the use of MA to increase consumption of a -Zn diet over a longer time period in both male and female rats. Rats were fed either a -Zn or a zinc-adequate (+Zn) diet. In Experiment 1, rats were treated orally with 0, 20, 50 or 100 mg MA/kg BW in corn oil for 21 days. MA stimulated intake of the -Zn diet in a linear manner. In Experiments 2 and 3, male and female rats, respectively, were fed the -Zn or +Zn diets and treated with 100 mg MA/kg BW for 21 days. In both experiments, MA administration increased intake of the -Zn diet to levels similar to the +Zn diet through Day 14. MA increased the hypothalamic neuropeptide Y (NPY) concentration in male rats, but did not affect serum IGF-I. MA administration improved growth of female but not male rats fed the -Zn diet. In females, serum IGF-I was not lower in zinc-deficient rats, which may have allowed the improved growth response with MA. Hence, MA administration may be a useful tool to increase consumption of a -Zn diet in short-term studies.     

Local tissue geometry determines contractile force generation of engineered muscle networks

The field of skeletal muscle tissue engineering is currently hampered by the lack of methods to form large muscle constructs composed of dense, aligned, and mature myofibers and limited understanding of structure-function relationships in developing muscle tissues. In our previous studies, engineered muscle sheets with elliptical pores ("muscle networks") were fabricated by casting cells and fibrin gel inside elastomeric tissue molds with staggered hexagonal posts. In these networks, alignment of cells around the elliptical pores followed the local distribution of tissue strains that were generated by cell-mediated compaction of fibrin gel against the hexagonal posts. The goal of this study was to assess how systematic variations in pore elongation affect the morphology and contractile function of muscle networks. We found that in muscle networks with more elongated pores the force production of individual myofibers was not altered, but the myofiber alignment and efficiency of myofiber formation were significantly increased yielding an increase in the total contractile force despite a decrease in the total tissue volume. Beyond a certain pore length, increase in generated contractile force was mainly contributed by more efficient myofiber formation rather than enhanced myofiber alignment. Collectively, these studies show that changes in local tissue geometry can exert both direct structural and indirect myogenic effects on the functional output of engineered muscle. Different hydrogel formulations and pore geometries will be explored in the future to further augment contractile function of engineered muscle networks and promote their use for basic structure-function studies in vitro and, eventually, for efficient muscle repair in vivo.     

Changes in contractile force by barium in the frog skeletal muscle

Effects of Ba2+ ions on the contractility were investigated in the frog skeletal muscle under the current clamp condition. The membrane potential was depolarized by 10 to 20 mV by perfusing 2 mM Ba2+ for 5 to 10 min. Membrane resistance was first increased and then decreased. The mechanical threshold examined in TTX-containing solution was shifted to more positive potential by applying Ba2+. The electrically induced contracture in TTX-solution as well as twitch in normal Ringer were enhanced by superfusing Ba2+. In both types of contraction an afterpotentiation was observed on washing out of Ba2+ ions, reflecting the existence of an inhibitory action during Ba2+ perfusion which could be masked by potentiation. Caffeine was able to induce contracture even in muscles soaked in "zero" Ca2+ solution for 24 to 48 h. In these muscles Ba2+ was more effective than Ca2+ to potentiate the contracture tension presumably by releasing Ca2+ ions from the sarcoplasmic reticulum whereas Mg2+ inhibited it.     

Calcium fraction correlating with contractile force of ventricular muscle of guinea-pig heart

Ca shifts in the isolated, perfused ventricular muscle of guinea-pig hearts were investigated with the aid of⁴⁵Ca under the conditions of complete equilibration of preparations with the isotope-containing solution. In some series of experiments total Ca content was also measured by means of atomic absorption spectrophotometry.The content of⁴⁵Ca at the end of 70 min equilibration period during which the ventricles were stimulated at a rate of 60/min was 1.66±0.09 mmol/kg w.w. This content dropped to 0.42±0.09 mmol/kg w.w. within 4 min of rest. Contractile force also decreased to 21% of control. Both the content of⁴⁵Ca and contractile force returned to the prerest values within the 4 min of post-rest stimulation. The difference between the total Ca content in the rested and stimulated muscle was comparable to the respective difference in⁴⁵Ca contents.A significant, linear correlation between this⁴⁵Ca fraction which was lost at rest and recovered during the postrest stimulation, and contractile force was found under the following experimental conditions: I. post-rest recovery, II. decay during rest, III. post-rest stimulation at various rates. These results are consistent with the hypothesis proposing that this Ca fraction is involved in the frequency-dependent control of contractile force.This work has been supported by the grant No. W. 10.8.4.14.01 of the National Institute of Cardiology, Warsaw.