Connexin 43 mimetic peptide Gap26 confers protection to intact heart against myocardial ischemia injury

Unapposed connexin 43 hemichannels (Cx43Hc) are present on sarcolemma of cardiomyocytes. Whereas Cx43Hc remain closed during physiological conditions, their opening under ischemic stress contributes to irreversible tissue injury and cell death. To date, conventional blockers of connexin channels act unselectively on both gap junction channels and unapposed hemichannels. Here, we test the hypothesis that Gap26, a synthetic structural mimetic peptide deriving from the first extracellular loop of Cx43 and a presumed selective blocker of Cx43Hc, confers resistance to intact rat heart against ischemia injury. Langendorff-perfused intact rat hearts were utilized. Regional ischemia was induced by 40-min occlusion of the left anterior descendent coronary and followed by 180 min of reperfusion. Gap26 was applied either 10 min before or 30 min after the initiation of ischemia. Interestingly, myocardial infarct size was reduced by 48% and 55% in hearts treated with Gap26 before or during ischemia, respectively, compared to untreated hearts. Additionally, myocardial perfusate flow was increased in both groups during reperfusion by 37% and 32%, respectively. Application of Gap26 increased survival of isolated cardiomyocytes after simulated ischemia–reperfusion by nearly twofold compared to untreated cells. On the other hand, superfusion of tsA201 cells transiently expressing Cx43 with Gap26 caused 61% inhibition of Cx43Hc-mediated currents recorded using the patch clamp technique. In summary, we demonstrate for the first time that Cx43 mimetic peptide Gap26 confers protection to intact heart against ischemia–reperfusion injury whether administered before or after the occurrence of ischemia. In addition, we provide unequivocal evidence for the inhibitory effect of Gap26 on genuine Cx43Hc.     

Ceramide is involved in triggering of cardiomyocyte apoptosis induced by ischemia and reperfusion.

Involvement of ceramide signaling in the initiation of apoptosis induction in myocardial cells by in vitro and in vivo ischemia and reperfusion was analyzed. Synthetic cell permeable C2-ceramide induced apoptotic death of rat neonatal cardiomyocytes in vitro. In vitro ischemia (oxygen/serum/glucose deprivation) led to a progressive accumulation of ceramide in cardiomyocytes. After 16 hours of simulated in vitro reperfusion (readdition of oxygen, serum and glucose), the level of ceramide in surviving cells was found to have returned to baseline, whereas, levels in nonadherent dead cells remained high. In the rat heart left coronary artery occlusion model, ischemia with the subsequent reperfusion, but not ischemia alone, induced apoptosis in myocardial cells as demonstrated by DNA electrophoresis and measurement of soluble chromatin degradation products. The content of ceramide in ischemic area was elevated to 155% baseline levels at 30 minutes, and to 330% after 210 minutes of ischemia. Ischemia (30 minutes) followed by reperfusion (180 minutes) increased the ceramide level to 250% in the ischemic area. The combination of results obtained in both in vitro and animal models demonstrate for the first time that ceramide signaling can be involved in ischemia/reperfusion death of myocardial cells.     

Netrin-1 overexpression in kidney proximal tubular epithelium ameliorates cisplatin nephrotoxicity

Netrin-1, a multifunctional laminin-related protein is widely expressed in various tissues, including kidney. The pathophysiological roles of netrin-1 in toxic acute kidney injury are unknown. To determine the role of netrin-1 in cisplatin-induced nephrotoxicity, we used netrin-1 transgenic mice that overexpress netrin-1 in the proximal tubular epithelium using the fatty acid binding protein promoter. Administration of cisplatin caused severe renal injury in WT mice but not in netrin-1 transgenic mice. Functional improvement was associated with better preservation of morphology, reduced cytokine expression and oxidative stress in the kidney, and reduced serum and urine cytokine and chemokine levels of transgenic mice as compared with WT mice. Cisplatin induced an increase in neutrophil infiltration into the kidney of WT mice, which was not significantly reduced in netrin-1 transgenic mice. Interestingly, ischemia reperfusion induced a large increase in apoptosis in WT mice but not in netrin-1 transgenic mice (215 ± 40 vs 94 ± 20 cells/5 HPF ( × 400), P < 0.0001), which was associated with reduced caspase-3 and p53 activation in the transgenic kidney. These results suggest that netrin-1 protects renal tubular epithelial cells against cisplatin-induced kidney injury by suppressing apoptosis and inflammation.     

Non-hypoxic stabilization of HIF-Iα during coordinated interaction between Akt and angiopoietin-1 enhances endothelial commitment of bone marrow stem cells

We previously reported that mesenchymal stem cells (MSC) co-expressing Akt and angiopoietin-1 (Ang-1) preserved infarcted heart function via angiomyogenesis. The present study determined the mechanism of co-overexpression of Akt and Ang-1 in promoting endothelial commitment of MSC. The cells were transduced with vectors encoding for Akt ((Akt)MSC), Ang-1 ((Ang-1)MSC), and both Akt and Ang-1 ((AA)MSC) using Empty vector transduced MSC ((Emp)MSC) as control. Molecular studies indicated a coordinated interaction between Akt and Ang-1 in (AA)MSC and led to non-hypoxic stabilization of hypoxia inducible factor-1α (HIF-Iα) which accentuated under 4-h anoxia. We also observed HIF-Iα dependent induction of hemeoxygenase-1, endothelial specific markers and VEGF in (AA)MSC. Vascular commitment of (AA)MSC was confirmed by immunostaining, Western blotting and flow cytometry for endothelial specific early and late markers including Flt1, Flk1, Tie2, VCAM-1, and von Willebrand Factor-VIII (vWF-VIII) in HIF-Iα dependent fashion besides exhibiting higher emigrational activity and angiogenesis in vitro. (AA)MSC transplanted into rat model of myocardial infarction showed higher Flk1 and Flt1 positivity and also promoted intrinsic Flk1(+) and Flt1(+) cell mobilization into the infarcted heart. Given the ease of availability of MSC and simplicity of approach to co-overexpress Ang-1 and Akt to enhance their endothelial commitment, the strategy will be significant for cellular angiogenesis to treat ischemic heart.     

Nanoparticle based delivery of hypoxia-regulated VEGF transgene system combined with myoblast engraftment for myocardial repair

A regulated promoter system to control gene expression is desirable for safe and efficacious over-expression of therapeutic transgene. Combined with skeletal myoblast (SkMs), we report the efficacy of hypoxia-regulated VEGF gene delivery for myocardial repair during acute myocardial infarction (AMI). A?hypoxia-regulated VEGF plasmid (pHRE-VEGF) was developed. After optimization, ～30% SkMs were transfected using polyethyleneimine (PEI) nanoparticles. The peak VEGF expression was higher in pHRE-VEGF transfected SkMs ((VEGF)SkMs) under hypoxia (151.34?±?8.59?ng/ml) than that with normoxia (16.92?±?2.74?ng/ml). The efficacy of hypoxia-regulated gene expression system was assessed in a rabbit model of AMI. The animals were grouped to receive basal M199 without cells (group-1) or containing non-transfected SkMs (group-2) or (VEGF)SkMs (group-3). In group-4, (VEGF)SkMs were injected into normal heart to serve as normoxia control. Improved SkM survival was observed in group-3 and -4 (p?相似文献   

Anti-inflammatory Effect of Protocatechuic Aldehyde on Myocardial Ischemia/Reperfusion Injury In Vivo and In Vitro

Myocardial ischemia/reperfusion (MI/R) injury, in which inflammatory response plays a vital role, is frequently encountered in clinical practice. The present study was aimed to investigate the anti-inflammatory effect and the possible mechanism of protocatechuic aldehyde (PAl) on MI/R injury both in vivo and in vitro. The rat model of MI/R injury was induced by ligation of the left anterior descending coronary artery for 30 min, followed by 3-h reperfusion, and pretreatment with PAl could protect the heart from MI/R injury by reducing myocardial infarct size and the activities of creatine kinase-MB and cardiac troponin I (cTn-I) in serum. Also, PAl administration markedly reduced cellular injury induced by simulated ischemia/reperfusion (SI/R) in cultured neonatal rat cardiomyocytes, which was evidenced by increased cell viability, reduced lactate dehydrogenase and cTn-I activities in the culture medium, and greatly decreased percentage of cell apoptosis. Moreover, the levels of tumor necrosis factor-α, interleukin-6, intracellular adhesion molecule-1, phosphorylated IκB-α, and the nuclear translocation of nuclear factor-kappa B (NF-κB) were all evidently decreased by PAl both in vivo and in vitro. Taken together, these observations suggested that PAl could exert great protective effects against MI/R injury in rats and SI/R injury in cultured neonatal rat cardiomyocytes, and the cardioprotective mechanism might be involved in the suppression of inflammatory response via inhibiting the NF-κB signaling pathway.     

Absence of PKR attenuates the anti-HSV-1 activity of an adenoviral vector expressing murine IFN-beta.

A study was undertaken to evaluate the efficacy of an adenoviral vector containing the murine interferon-beta (IFN-beta) transgene (Ad:IFN-beta) against herpes simplex virus type 1 (HSV-1) infection in two transduced cell lines. The transduction of the adenoviral vector efficiency, ranging from 2% to 100%, was dependent on the multiplicity of infection (moi) (0.4-50 plaque-forming units [pfu]/cell). Supernatants from cells transduced with the Ad:IFN-beta but not the adenoviral null vector (Ad:Null) contained biologically active IFN-beta (6.6-106 U/ml depending on the moi). Cells transduced with the Ad:IFN-beta displayed up to 25-fold reduction in viral titers compared with cells transduced with the Ad:Null or nontransduced cell controls. The suppression in viral titer correlated with a reduction in viral gene (alpha, beta, and gamma) and protein expression. The expression of IFN beta-responsive genes, including protein kinase R (PKR) and 2',5'-oligoadenylate synthetase (OAS), were significantly elevated in the Ad:IFN-beta-transduced cells by 12-fold and 25-fold, respectively. However, after infection with HSV-1, a transient but significant drop in PKR but not OAS gene expression was observed 10 h postinfection. The absence of PKR but not RNase L significantly attenuated the antiviral efficacy of the transgene. Collectively, these results illustrate the feasibility of employing a viral vector to deliver a potent antiviral gene to targeted cells without any obvious detriment to the vector itself and support an important role for PKR as a mediator of the anti-HSV-1 activity of type I IFN.     

Transduction of Wnt11 promotes mesenchymal stem cell transdifferentiation into cardiac phenotypes

Transplantation of mesenchymal stem cells (MSCs) has emerged as a potential treatment for ischemic heart repair. Previous studies have suggested that Wnt11 plays a critical role in cardiac specification and morphogenesis. In this study, we examined whether transduction of Wnt11 directly increases MSC differentiation into cardiac phenotypes. MSCs harvested from rat bone marrow were transduced with both Wnt11 and green fluorescent protein (GFP) (MSC(Wnt11)) using the murine stem cell virus (pMSCV) retroviral expression system; control cells were only GFP-transfected (MSC(Null)). Compared with control cells, MSC(Wnt11) was shown to have higher expression of Wnt11 by immunofluorescence, real-time polymerase chain reaction, and western blotting. MSC(Wnt11) shows a higher expression of cardiac-specific genes, including GATA-4, brain natriuretic peptide (BNP), islet-1, and α-actinin, after being cultured with cardiomyocytes (CMs) isolated from ventricles of neonatal (1-3 day) SD rats. Some MSC(Wnt11) were positive for α-actinin when MSCs were cocultured with native CMs for 7 days. Electron microscopy further confirmed the appearance of sarcomeres in MSC(Wnt11). Connexin 43 was found between GFP-positive MSCs and neonatal rat CMs labeled with red fluorescent probe PKH26. The transdifferentiation rate was significantly higher in MSC(Wnt11) than in MSC(Null), as assessed by flow cytometry. Functional studies indicated that the differentiation of MSC(Wnt11) was diminished by knockdown of GATA-4 with GATA-4-siRNA. Transduction of Wnt11 into MSCs increases their differentiation into CMs by upregulating GATA-4.     

Activation of mu-opioid receptors and cardiomyocyte resistance to free radical damage

It has been found that after intravenous administration of selective agonist of mu-opioid receptors DAGO (0.1 mg/kg 15 min before heart excision) isolated rat heart becomes resistant to ischemia (45 min) and reperfusion (60 min) ex vivo. The in vivo pretreatment with DAGO prevented reperfusion injury of cardiac cells and decreased myocardial content of conjugated dienes during ischemia and reperfusion of the heart in vitro. In addition, similar mu-opioid receptor stimulation promotes a postischemic recovery of myocardial contractility in the postischemic period. However, this receptor activation does not affect heart tolerance to free radical damage during perfusion of isolated heart by a solution containing Fe(2+)-ascorbic acid.     

Cardioprotective effect of κ<Subscript>1</Subscript>-opioid receptor activation and role of cAMP in its realization

The cardioprotective and antiarrhythmic effects of a selective κ₁-opioid receptor agonist U-50,488 were studied during experimental 45-min total ischemia and 30-min reperfusion of isolated rat heart. The opioid had no effect on the incidence and type of reperfusion arrhythmias. U-50,488 in a concentration of 0.1 μM inhibited reperfusion-induced release of creatine phosphokinase and decreased cAMP concentration in the myocardium by 2 times. These parameters remained unchanged after treatment with U-50,488 in a concentration of 1 μM. The cardioprotective effect of U-50,488 was probably associated with a decrease in cAMP concentration in heart cells. U-50,488 in a concentration of 1 μM produced no cardioprotective effect, which can be explained by its interaction with an unknown non-opioid receptor in cardiomyocytes. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 1, pp. 28–31, January, 2007     

大鼠在体心肌缺血再灌注损伤细胞膜钙转运通道蛋白mRNA的表达变化

目的 探讨大鼠心肌在体缺血再灌注(IR)损伤后细胞膜钙转运通道蛋白的mRNA变化对钙超载的作用。方法 12只SD大鼠按随机数字法分为IR组和对照组。IR组通过结扎（缺血20 min）后松解(再灌注60 min)前降支造成心肌IR,对照组则免除结扎松解前降支。应用生理记录仪连续监测两组大鼠缺血开始前及再灌注60 min后心率、平均动脉压等血流动力学指标。全自动生化仪检测缺血前及再灌注60 min后两组大鼠血钙及肌钙蛋白T(cTnT)的水平。荧光定量PCR检测再灌注60 min后两组大鼠左心室缺血区和右心室心肌细胞膜钙转运通道蛋白即心肌细胞膜钠钙交换器1(NCX1),L型钙通道(LVDCC)α-1C和胞膜钙转运ATP酶1(PMCA1 )mRNA的表达。结果两组大鼠缺血前的心率、平均动脉压均高于再灌注60 min后,而两组间缺血前和再灌注60 min后的心率、平均动脉压差异则无统计学意义。两组血浆Ca2+浓度在缺血前与再灌注60 min后差异无统计学意义,同时间点两组之间的差异也没有统计学意义。缺血前IR组与对照组血浆cTnT浓度水平相近,缺血60 min后IR组血浆cTnT浓度较对照组升高[(4.29±2.22) μg/L比(1.62±0.60)μg/L,P=0.031];两组血浆cTnT浓度在缺血前与再灌注60 min后差异也有统计学意义（均P＜0.05）。NCX1,LVDCCα-1C和PMCA1的mRNA表达在再灌注60 min后同心室两组间和同组内左右心室之间的差异均无统计学意义（NCX1：对照组左心室为50±4,右心室为47±9;IR组左心室为55±6,右心室为53±11;LVDCCα-1C:对照组左心室为33±7,右心室为30±7;IR组左心室为28±3,右心室为37±5;PMCA1,对照组左心室为70±10,右心室为53±11;IR组左心室为66±12,右心室为78±8;均P＞0.05）。结论 大鼠在体心肌缺血20 min再灌注60 min后,NCX1、LVDCCα-1C和PMCA1的mRNA表达水平均无显著改变,提示钙超载并非由细胞膜钙转运通道蛋白数量改变引起。     

降纤酶对离体大鼠工作心脏缺血再灌注损伤的作用

目的 :探讨降纤酶 ( Df)对心脏缺血再灌注损伤的作用。方法 :采用离体大鼠工作心脏缺血再灌注损伤模型 ,通过检测缺血再灌注损伤前后对照组、Df低剂量组及 Df高剂量组的心功能参数 ,观察Df的作用。结果 :Df可降低实验动物的室颤发生率 ,增强心肌收缩力 ( P<0 .0 5 ) ,防止冠脉流量下降 ( P<0 .0 5 )及左室内压下降 ( P<0 .0 1) ,促进压力最大上升速度的恢复 ( P<0 .0 5 )。结论 :Df对心脏缺血再灌注损伤具有显著的保护作用。     

Exercise-induced physiological hypertrophy initiates activation of cardiac progenitor cells

Objective: Physiological hypertrophy is featured by the hypertrophy of pre-existing cardiomyocytes and the formation of new cardiomyocytes. C-kit positive cardiac progenitor cells increased their numbers in exercise-induced physiological hypertrophy. However, the participation of Sca-1 positive cells in the physiological adaptation of the heart to exercise training is unclear. Methods: Physiological hypertrophy was induced by swimming and the mRNA levels of GATA binding protein 4 (GATA4), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), endogenous hepatocyte growth factor (HGF), and insulin like growth factor-1 (IGF-1) from the whole heart were determined by real-time polymerase chain reactions (RT-PCRs) analysis. Immunofluorescent staining was used to compare the number of C-kit and Sca-1 positive cardiac progenitor cells. In addition, mRNA levels of C-kit and Sca-1 in left ventricle (LV), right ventricle (RV), and outflow tract (OFT) were determined in mice swimming for 7, 14, and 21 days by RT-PCRs. Results: The ratio of heart weight (HW) to body weight and HW to tibia length and the mRNA level of GATA4 were increased while mRNA levels of ANP and BNP remained unchanged. C-kit and Sca-1 positive cardiac progenitor cells were activated by swimming training. An increased endogenous production of HGF and IGF was observed at least at the mRNA level. Swimming induced a significant up-regulation of C-kit in LV of mice swimming for 1, 2 and 3 weeks and in RV of mice swimming for 3 weeks. Sca-1 positive cardiac progenitor cells were increased in LV and OFT in mice swimming for 3 weeks. Conclusion: This study presents that swimming-induced physiological hypertrophy initiates activation of cardiac progenitor cells.     

MC-3 receptor and the inflammatory mechanisms activated in acute myocardial infarct

Investigation of the mechanisms activated by endogenous inhibitory pathways can lead to identification of novel targets for cardiovascular inflammatory pathologies. Here we exploited the potential protective role that melanocortin receptor type 3 (MC3-R) activation might play in a myocardial ischemia-reperfusion injury model. In resting conditions, mouse and rat heart extracts expressed MC3-R mRNA and protein, without changes following ischemia-reperfusion. At the cellular level heart macrophages, but not fibroblasts or cardiomyocytes, expressed this receptor, as demonstrated by immunogold labeling. In vivo, administration of the melanocortin agonist MTII (10 microg per mouse equivalent to 9.3 nmol) 30 min prior to ischemia (25 min) attenuated mouse heart 2 h reperfusion injury by approximately 40%, an effect prevented by the mixed MC3/4-R antagonist SHU9119 but not by the selective MC4-R antagonist HS204. Similar results were obtained when the compound was given at the beginning of the reperfusion period. Importantly, delayed myocardial damage as measured 24 h post-reperfusion was equally protected by administration of 10 microg MTII. The focus on MC3-R was also substantiated by analysis of the recessive yellow (e/e) mouse, bearing a mutated (inactive) MC1-R, in which MTII was fully protective. Myocardial protection was associated with reduced markers of systemic and local inflammation, including cytokine contents (interleukin-1 and KC) and myeloperoxidase activity. In conclusion, this study has highlighted a previously unrecognized protective role for MC3-R activation on acute and delayed heart reperfusion injury. These data may open new avenues for therapeutic intervention against heart and possibly other organ ischemia-reperfusion injury.     

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。     

Roles and relationship of macrophages and monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 in the ischemic and reperfused rat heart

Reperfusion injury is a troublesome and unresolved problem in acute myocardial infarction and is believed to be associated with inflammatory reactions in which various types of cells and cytokines participate, in particular, macrophages and monocyte chemoattractant protein-1 (MCP-1). We designed this study to clarify the role and relationship of macrophages and MCP-1 in ischemic and reperfused heart. The number and distribution of macrophages and MCP-1 messenger RNA (mRNA) in the ischemic and reperfused rat heart were examined with in situ hybridization and immunohistochemistry. Myocardial samples were obtained at several times. In situ hybridization was performed with digoxigenin-labeled antisense RNA probe for rat MCP-1 mRNA, and immunohistochemistry was performed with antimacrophage antibody. Double staining with in situ hybridization and immunohistochemistry was also performed. The number of MCP-1 mRNA-positive cells increased after reperfusion and peaked at 3 hours after reperfusion. Early infiltration of ischemic tissues by macrophages was also observed at the time of the absence of an increase of MCP-1 mRNA-positive cells, and this infiltration was not significantly accelerated by reperfusion, but by ischemia itself. The numbers of both MCP-1 mRNA-positive cells and macrophages increased in the ischemic marginal region over time. From the result of double staining, and based on the cellular morphology and the distribution, the majority of MCP-1 mRNA-positive cells appeared to be activated macrophages. This suggests that macrophages may not be attracted to cardiac tissue only by MCP-1 and that MCP-1 may have some roles other than attracting macrophages into ischemic heart. It also suggests that macrophages and MCP-1 may play an important role in reperfusion injury and that MCP-1 may be one of the key molecules of reperfusion injury. These observations may contribute to the development of a new therapeutic approach to the prevention of reperfusion injury.     

Immunohistochemical study of netrin-1 in the spinal cord with rat experimental autoimmune encephalomyelitis

To investigate whether netrin-1 is involved in autoimmune injury of the central nervous system, the expression of netrin-1 protein was analyzed in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis revealed significantly increased content of netrin-1 in the spinal cords of rats at the peak stage of EAE, as compared with the levels in normal control animals (p < 0.01). Immunohistochemistry detected the netrin-1 protein in neurons, oligodendrocytes, astrocytes and vascular endothelial cells in the spinal cords of normal controls. In EAE-affected spinal cords, netrin-1 immunoreactivity was detected in infiltrating inflammatory cells at the peak stage as well as in neurons, oligodendrocytes and astrocytes. These results suggest that netrin-1 is transiently increased in rat EAE lesions, where it contributes to the modulation of rat acute EAE.     

Immunohistochemical Study of Netrin-1 in the Spinal Cord with Rat Experimental Autoimmune Encephalomyelitis

To investigate whether netrin-1 is involved in autoimmune injury of the central nervous system, the expression of netrin-1 protein was analyzed in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis revealed significantly increased content of netrin-1 in the spinal cords of rats at the peak stage of EAE, as compared with the levels in normal control animals (p < 0.01). Immunohistochemistry detected the netrin-1 protein in neurons, oligodendrocytes, astrocytes and vascular endothelial cells in the spinal cords of normal controls. In EAE-affected spinal cords, netrin-1 immunoreactivity was detected in infiltrating inflammatory cells at the peak stage as well as in neurons, oligodendrocytes and astrocytes. These results suggest that netrin-1 is transiently increased in rat EAE lesions, where it contributes to the modulation of rat acute EAE.     

11,12-EET和缺血预处置对在体大鼠正常及再灌注心肌磷酸化ERK和p38MAPK表达的影响

目的:观察11,12-环氧二十碳三烯酸(11,12-EET)和缺血预处置对大鼠再灌注心肌组织磷酸化ERK1/ERK2和p38 MAPK表达的影响,了解大鼠心肌磷酸化ERK1/ERK2和p38 MAPK表达与预处置有否关系。方法: 使用雄性Wistar大鼠,通过结扎(60 min)和松开(30 min)冠状动脉左前降支,复制缺血/再灌注模型;采用缺血5 min,再灌注5 min两次造成缺血预处置。大鼠经手术并静脉给予6.24×10^-8mol/L 11,12-EET,稳定20 min,结扎冠脉复制缺血/再灌注模型。实验分5组:①正常组(norm);②假手术组(sham);③缺血再灌注组(I/R);④短阵缺血预处置组(SI+I/R);⑤11,12-EET预处置缺血/再灌注组(EET+I/R)。采用Western blot法测定心肌细胞外调节的蛋白激酶(ERK1/2)和p38 MAPK的表达程度,并观察再灌注过程中心功能的变化。结果: 再灌注30 min时,I/R组+dp/dt_max%、-dp/dt_max%和LVDP均显著低于sham组、SI+I/R组和EET+I/R组(P＜0.05);而I/R组大鼠心肌ERK1/2磷酸化表达明显高于sham组(P＜0.05),明显低于SI+I/R组和EET+I/R组(P＜0.05);I/R组大鼠心肌p38 MAPK磷酸化表达I/R组显著高于sham组、norm组、SI+I/R及EET+I/R组(P＜0.05)。结论:6.24×10^-8 11,12-EET mol/L具有保护心功能的作用,这种保护作用可能与大量激活磷酸化ERK1/2和抑制p38 MAPK有关。