硝酸异山梨酯后处理对大鼠在体心肌缺血/再灌注损伤保护作用的研究

目的观察硝酸异山梨酯后处理对大鼠在体心肌缺血/再灌注损伤是否具有保护作用，并探讨其作用机制，为实现硝酸酯后处理心肌保护新模式的临床应用与推广提供理论依据。 方法清洁级雄性Wistar大鼠，32只，10-12周龄。建立大鼠在体心肌缺血-再灌注模型；随机分为以下四组：假手术组（sham组）；缺血/再灌注组（the ischemia/reperfusion group, I/R组）；缺血后处理组（ischemic postconditioning group ,IPostC组）；硝酸异山梨酯（异舒吉 UCB Pharma GmbH.德国）后处理组（ isosorbide dinitrate postconditioning group, PPostC组）。实验结束后，测定各组大鼠心肌梗死面积，并用Elisa法测定大鼠血清肌钙蛋白I(cTnI)、丙二醛（MDA）、超氧化物歧化酶（SOD）水平。 结果（1）各组大鼠左室质量和缺血面积无统计学差异，IPostC组及PPostC组与I/R组相比，心肌梗死面积减小有统计学意义（P<0.05），分别为（41.015±1.037）%、（38.129±0.726）、（52.190±1.216）%；而IPostC组与PPostC组之间无统计学差异（P=0.057）。（2）与sham组相比，I/R组、 IPostC组和PPostC组血清cTnI的水平显著升高（p<0.05）；与I/R组相比，IPostC组和PPostC组的大鼠血清cTnI水平明显降低（p<0.05），IPostC组和PPostC组相比无统计学差异（p>0.05）。（3）与sham组相比，I/R组、IPostC组及PPostC组血清MDA水平显著升高，SOD水平显著降低（p<0.05）；与I/R组相比，IPostC及PPostC能有效降低血清MDA水并显著升高血清SOD水平(P均<0.05)。 结论（1）缺血后处理能够减轻再灌注损伤；（2）硝酸异山梨酯后处理可以产生与缺血后处理相似的心肌保护效果；（3）硝酸异山梨酯的心肌保护作用机制可能与减少活性氧的产生以及提高心肌的抗氧化（应激）能力有关。     

CB2 cannabinoid receptor activation is cardioprotective in a mouse model of ischemia/reperfusion

Preventive treatment with cannabinoid agonists has been reported to reduce the infarct size in a mouse model of myocardial ischemia/reperfusion. Here we investigated the possible cardioprotective effect of selective CB₂ cannabinoid receptor activation during ischemia. We performed left coronary artery ligature in C57Bl/6 mice for 30 min, followed by 24 h of reperfusion. Five minutes before reperfusion, mice received intraperitoneal injection of the CB₂ selective agonist JWH-133 (20 mg/kg) or vehicle. Infarct size was assessed histologically and by cardiac troponin I (cTnI) ELISA. Immunohistochemical analysis of leukocyte infiltration, oxidative stress in situ quantification, real-time RT-PCR analysis of inflammatory mediators as well as western blots for kinase phosphorylation was also performed. In addition, we studied chemotaxis and integrin expression of human neutrophils in vitro. JWH-133 significantly reduced the infarct size (I/area at risk: 19.27% ± 1.91) as compared to vehicle-treated mice (31.77% ± 2.7). This was associated with a reduction of oxidative stress and neutrophil infiltration in the infarcted myocardium, whereas activation of ERK 1/2 and STAT-3 was increased. Preinjection of PI3K inhibitor LY294002, MEK 1/2 inhibitor U0126 and JAK-2 inhibitor AG-490 partially abrogated the JWH-133 mediated infarct size reduction. No changes in cardiac CXCL1, CXCL2, CCL3, TNF-α, and ICAM-1 expression levels were found. Furthermore, JWH-133 inhibited the TNF-α induced chemotaxis and integrin CD18/CD11b (Mac-1) upregulation on human neutrophils. Our data suggest that JWH-133 administration during ischemia reduces the infarct size in a mouse model of myocardial ischemia/reperfusion through a direct cardioprotective activity on cardiomyocytes and neutrophils.     

A role for heterotrimeric GTP-binding proteins and ERK1/2 in insulin-mediated, nitric-oxide-dependent, cyclic GMP production in human umbilical vein endothelial cells

Aims/hypothesis Insulin is known to stimulate endothelial nitric oxide synthesis, although much remains unknown about the intracellular mechanisms involved. This study aims to examine, in human endothelial cells, the specific contribution of heterotrimeric G_i proteins and extracellular signal-regulated protein kinases 1/2 (ERK1/2) in insulin signalling upstream of nitric-oxide-dependent cyclic GMP production.Methods Human umbilical vein endothelial cells were treated with 1 nmol/l insulin in the presence or absence of inhibitors of tyrosine kinases (erbstatin), G_i proteins (pertussis toxin) or ERK1/2 (PD098059 or U0126), and nitric oxide production was examined by quantification of intracellular cyclic GMP. Activation/phosphorylation of ERK1/2 by insulin was examined by immunoblotting with specific antibodies, and direct association of the insulin receptor with G_i proteins was examined by immunoprecipitation.Results Treatment of cells with a physiological concentration of insulin (1 nmol/l) for 5 min increased nitric-oxide-dependent cyclic GMP accumulation by 3.3-fold, and this was significantly inhibited by erbstatin. Insulin-stimulated cyclic GMP production was significantly reduced by pertussis toxin and by the inhibitors of ERK1/2, PD098059 and U0126. Immunoblotting indicated that insulin stimulated the phosphorylation of ERK1/2 after 5 min and 1 h, and that this was completely abolished by pertussis toxin, but insensitive to the nitric oxide synthase inhibitor l-NAME. No direct interaction of the insulin receptor with could be demonstrated by immunoprecipitation.Conclusions/interpretation This study demonstrates, for the first time, that nitric oxide production induced by physiologically relevant concentrations of insulin, is mediated by the post-receptor activation of a pertussis-sensitive GTP-binding protein and subsequent downstream activation of the ERK1/2 cascade.     

CircRNA 010567 plays a significant role in myocardial infarction via the regulation of the miRNA-141/DAPK1 axis

BackgroundMyocardial infarction (MI), caused by temporary or permanent coronary artery occlusion, poses a serious threat to patients’ lives. Circular RNAs (circRNAs), a new kind of endogenous noncoding RNAs, have been widely studied recently. This study was designed to illustrate and potential molecular mechanisms of circRNA 010567 in hypoxia-induced cardiomyocyte injury in vitro, so as to provide new strategies for the therapy of MI.MethodsH9c2 cells were cultured in anoxic conditions with 94% N₂, 5% CO₂, and 1% O₂ to establish the in vitro MI model. Cell viability and apoptosis were checked using MTT and flow cytometry assay, respectively, Moreover, the levels of circRNA 010567, miR-141, and DAPK1 was determined using qRT-PCR. The putative targets of circRNA 010567 and miR-141 were confirmed by dual-luciferase reporter system and the RNA immunoprecipitation (RIP) assay. The release of creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), and the viability of mitochondria were detected using assay kits.ResultsThe current study revealed that circRNA 010567 and DAPK1 were over-expressed, and miR-141 was low-expressed in hypoxia-induced MI. circRNA 010567 sponges miR-141 and DAPK1 was a direct target of miR-141. Mechanistic investigations revealed that circRNA 010567-siRNA impaired the release of CK-MB and cTnI, and promoted the viability of mitochondria in hypoxia-induced H9c2 cells, while these findings were reversed by the miR-141 inhibitor. In addition, the miR-141 mimic markedly reduced the release of CK-MB and cTnI, and promoted the viability of mitochondria, and these results were reversed by the DAPK1-plasmid. Subsequently, functional experiments revealed that hypoxia-stimulated decreases in H9c2 cell viability, as well as increases in apoptosis and caspase-3 activity, were induced by the miR-141 mimic and circRNA 010567-siRNA. However, these results were reversed by the miR-141 inhibitor and DAPK1-plasmid.ConclusionsOur results demonstrated that circRNA 010567-siRNA played a protective role in hypoxia-induced cardiomyocyte damage via regulating the miR-141/DAPK1 axis, indicating that circRNA 010567-siRNA may be a promising target for MI therapy.     

氧化槐定碱通过Nrf2/HO-1通路抑制大鼠急性心肌梗死后心肌细胞凋亡

目的观察氧化槐定碱(OSR)通过核因子E2相关因子2(Nrf2)/血红素氧化酶1(HO-1)通路抑制大鼠急性心肌梗死(AMI)后心肌细胞凋亡的作用。方法成年健康雄性SD大鼠分为假手术组、AMI组、OSR组、OSR+锌原卟啉9(ZPP-Ⅸ)组,后3组采用左冠状动脉前降支结扎的方式建立AMI模型,OSR组给予OSR腹腔注射,OSR+ZPP-Ⅸ组给予OSR及HO-1抑制剂ZPP-Ⅸ腹腔注射。检测血清心肌酶含量、心肌细胞凋亡率、心肌中凋亡基因及Nrf2、HO-1的表达量。结果 AMI组大鼠的血清乳酸脱氢酶(LDH)、磷酸肌酸激酶(CK)、磷酸肌酸激酶同工酶(CK-MB)含量、心肌细胞凋亡率以及心肌中Bcl-2相关X蛋白(Bax)、Cleaved-caspase-3、Nrf2、HO-1的表达量均明显高于假手术组,心肌中B淋巴细胞瘤2蛋白(Bcl-2)的表达量明显低于假手术组。OSR组大鼠的血清LDH、CK、CK-MB含量、心肌细胞凋亡率以及心肌中Bax、Cleaved-caspase-3的表达量均明显低于AMI组,心肌中Bcl-2、Nrf2、HO-1的表达量明显高于AMI组。OSR+ZPP-Ⅸ组大鼠的血清LDH、CK、CK-MB含量、心肌细胞凋亡率以及心肌中Bax、Cleaved-caspase-3的表达量均明显高于OSR组,心肌中Bcl-2的表达量明显低于OSR组。结论OSR通过激活Nrf2/HO-1通路抑制大鼠AMI后心肌细胞的凋亡。     

苦参碱对心肌梗死大鼠辅助性T细胞1/辅助性T细胞2平衡及钾通道Kv2.1和KIR2.1的影响

目的探讨苦参碱对心肌梗死大鼠辅助性T细胞1(Th1)/辅助性T细胞2(Th2)平衡及钾通道Kv2.1、KIR2.1的影响。方法制备心肌梗死大鼠模型60只,随机分为模型组、低剂量苦参碱组(50 mg/kg)、中剂量苦参碱组(100 mg/kg)、高剂量苦参碱组(200 mg/kg)、硫氮卓酮组(2.61 mg/kg),每组各12只,药物处理组大鼠于建模成功后每天灌胃治疗,持续7 d,另取12只大鼠作为假手术组。末次给药24 h后处死大鼠检测心肌梗死面积,采用酶联免疫吸附试验(ELISA)检测血清肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白I(cTnI)、干扰素γ(IFN-γ)、白细胞介素(IL)-2、IL-4、IL-10水平,流式细胞术检测外周血Th1/Th2比值,蛋白质印迹法(Western blot)检测心肌组织钾通道Kv2.1、KIR2.1蛋白水平。结果与假手术组比较,模型组大鼠心肌梗死面积比例、血清CK-MB、cTnI、IFN-γ、IL-2水平、外周血Th1/Th2比值升高,血清IL-4、IL-10、心肌组织钾通道Kv2.1及KIR2.1蛋白水平降低(P<0.05);与模型组比较,各药物处理组大鼠心肌梗死面积比例、血清CK-MB、cTnI、IFN-γ、IL-2水平、外周血Th1/Th2比值降低,血清IL-4、IL-10水平、心肌组织钾通道Kv2.1及KIR2.1蛋白水平升高且各苦参碱组呈剂量依赖性(P<0.05);高剂量苦参碱组和硫氮卓酮组大鼠上述指标比较差异均无统计学意义(P>0.05)。结论苦参碱可恢复心肌梗死大鼠Th1/Th2平衡,上调心肌组织钾通道Kv2.1和KIR2.1蛋白的表达,降低心肌梗死面积,起心肌保护作用。     

HB-EGF enhances restitution after intestinal ischemia/reperfusion via PI3K/Akt and MEK/ERK1/2 activation

BACKGROUND & AIMS: Early recovery of intestinal function after injury occurs by restitution, a complex process with a poorly understood molecular basis. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent chemotactic factor that is induced during ischemia/reperfusion in vivo and intestinal wounding in vitro. The role of HB-EGF in intestinal restitution and the underlying intracellular signaling pathways involved were investigated. METHODS: Adult rats were subjected to intestinal ischemia, with histologic and biochemical damage assessed during the first 3 hours of reperfusion. The effect of recombinant HB-EGF (rHB-EGF) on structural and functional recovery of the intestine by restitution was evaluated in vivo. Scrape wounding of intestinal epithelial cell monolayers was used to elucidate the mechanisms of intrinsic and rHB-EGF-induced restitution. RESULTS: Early structural recovery occurred within 3 hours of reperfusion and was attributed to restitution rather than proliferation. HB-EGF treatment significantly improved structural recovery and accelerated functional recovery of the gut barrier. In vivo restitution was preceded by activation of Akt and extracellular signal-regulated kinase (ERK) 1/2, which were accelerated and enhanced by HB-EGF treatment. Blocking of ErbB-1, phosphatidylinositol 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase/ERK kinase (MEK)/ERK activity resulted in significant reduction in intrinsic and HB-EGF-induced restitution in vitro. Endogenous HB-EGF was shown to play an essential role in wound-induced ErbB-1 and ERK1/2 activation and in intrinsic restitution. CONCLUSIONS: Endogenous HB-EGF, ErbB-1, PI3K/Akt, and MEK/ERK are involved in intrinsic restitution. rHB-EGF enhances restitution in vivo and in vitro in a PI3K/Akt- and MEK/ERK1/2-dependent fashion.