细胞自噬在大鼠缺血/再灌注肺损伤中的调控作用

目的: 探讨细胞自噬在大鼠缺血/再灌注肺损伤中的作用。方法: 随机将40只SD大鼠分为5组(n=8),分别为 ① 假手术组(Sham组):只开胸3.5 h;② 缺血/再灌注组(I/R组):开胸夹闭肺门缺血0.5 h后再灌注3 h;③ 溶剂组(DMSO组):术前1 h腹腔注射DMSO溶液;④自噬激动剂组(Rap组):术前腹腔注射雷帕霉素溶液;⑤自噬抑制剂组(3-MA组):术前1 h腹腔注射3-MA溶液;后三组的其余操作同I/R组。实验结束后处死大鼠,取肺组织,记录并计算肺组织湿/干重比(W/D)、总肺含水量变化(TLW) ,光镜和电镜观察肺组织及细胞形态,计算肺泡损伤率(IAR),Western blot检测自噬相关蛋白的表达情况。结果: 相对于sham组,其余四组肺W/D、TLW、IAR均明显升高,自噬相关蛋白表达明显上升,p-AMPK、Beclin 1、LC3 II 蛋白明显增多,p-mTOR、p62蛋白明显减少(P＜0.05或P＜0.01),光镜下其余各组肺组织有不同程度的水肿渗出,肺泡结构紊乱,电镜下细胞超微结构损伤加重,部分可见自噬小体;与DMSO组相比,3-MA组肺W/D、TLW、IAR明显下降(P＜0.05或P＜0.01),自噬相关蛋白表达明显下降,肺间质水肿较轻,细胞渗出较少,细胞超微结构损伤减轻,未见自噬小体。而I/R、DMSO、Rap组的各项指标变化无统计学差异(P＞0.05)。结论: 肺缺血/再灌注可诱发细胞自噬增强,从而引起大鼠肺损伤。     

脑缺血和缺血再灌注大鼠皮层神经元自噬的动态变化

目的：探讨脑缺血和缺血/再灌注不同时间大鼠大脑皮层神经元自噬的变化。方法：健康雄性SD大鼠60只,随机分为：假手术（Sham）组（n=10）,脑缺血和缺血/再灌注模型组（n=50）.模型组分别在缺血30min、2h,缺血2h再灌注1h、6h、24h五个时间点,随机抽取10只大鼠,测定脑梗死体积和脑含水量,同时采用Western印迹法测定各组大鼠大脑皮层中微管相关蛋白轻链3-Ⅱ（LC3-Ⅱ）的水平,透射电镜检测大脑皮层神经细胞自噬情况。结果：脑缺血30min时LC3-Ⅱ/Ⅰ比值未见明显上升,缺血2h时LC3-Ⅱ/Ⅰ比值开始升高,明显高于Sham组（P<0.01）;缺血/再灌注1h、6h时LC3-Ⅱ/Ⅰ比值虽较缺血2h组有所下降,但仍明显高于Sham组（P<0.05）;缺血/再灌注24h时LC3Ⅱ/Ⅰ比值达高峰,明显高于Sham组（P<0.01）。透射电镜观察进一步证实该现象。缺血/再灌注6h和24h时大鼠脑梗死体积明显增加,与Sham组比较有统计学差异（P<0.01）。缺血/再灌注24h大鼠脑组织含水量明显增加,明显高于Sham组（P<0.05）。HE染色显示：仅在缺血/再灌注24h组大鼠皮层见组织水肿、疏松,部分细胞变性、凋亡,海马区见大量神经元细胞核皱缩、深染呈变性凋亡状。结论：局灶性脑缺血和缺血/再灌注模型中大脑皮层缺血2 h神经元自噬即明显激活,缺血/再灌注1 h、6 h自噬均持续增高,缺血/再灌注24 h自噬达高峰。     

The fibrin-derived peptide Bbeta15-42 protects the myocardium against ischemia-reperfusion injury

In the event of a myocardial infarction, current interventions aim to reopen the occluded vessel to reduce myocardial damage and injury. Although reperfusion is essential for tissue salvage, it can cause further damage and the onset of inflammation. We show a novel anti-inflammatory effect of a fibrin-derived peptide, Bbeta15-42. This peptide competes with the fibrin fragment N-terminal disulfide knot-II (an analog of the fibrin E1 fragment) for binding to vascular endothelial (VE)-cadherin, thereby preventing transmigration of leukocytes across endothelial cell monolayers. In acute or chronic rat models of myocardial ischemia-reperfusion injury, Bbeta15-42 substantially reduces leukocyte infiltration, infarct size and subsequent scar formation. The pathogenic role of fibrinogen products is further confirmed in fibrinogen knockout mice, in which infarct size was substantially smaller than in wild-type animals. Our findings conclude that the interplay of fibrin fragments, leukocytes and VE-cadherin contribute to the pathogenesis of myocardial damage and reperfusion injury. The naturally occurring peptide Bbeta15-42 represents a potential candidate for reperfusion therapy in humans.     

Cardioprotection by adaptation to ischaemia augments autophagy in association with BAG-1 protein

Autophagy is an intracellular process in which a cell digests its own constituents via lysosomal degradative pathway. Though autophagy has been shown in several cardiac diseases like heart failure, hypertrophy and ischaemic cardiomyopathy, the role and the regulation of autophagy is still largely unknown. Bcl-2-associated athanogene (BAG-1) is a multifunctional pro-survival molecule that binds with Hsp70/Hsc70. In this study, myocardial adaptation to ischaemia by repeated brief episodes of ischaemia and reperfusion (I/R) prior to lethal I/R enhanced the expression of autophagosomal membrane specific protein light chain 3 (LC3)-II, and Beclin-1, a molecule involved in autophagy and BAG-1. Autophagosomes structures were found in the adapted myocardium through electron microscopy. Co-immunoprecipitation and co-immunofluorescence analyses revealed that LC3-II was bound with BAG-1. Inhibition of autophagy by treating rats with Wortmannin (15 μg/kg; intraperitoneally) abolished the ischaemic adaptation-induced induction of LC3-II, Beclin-1, BAG-1 and cardioprotection. Intramyocardial injection of BAG-1 siRNA attenuated the induction of LC3-II, and abolished the cardioprotection achieved by adaptation. Furthermore, hypoxic adaptation in cardiac myoblast cells induced LC3-II and BAG-1. BAG-1 siRNA treatment attenuated hypoxic adaptation-induced LC3-II and BAG-1, and abolished improvement in cardiac cell survival and reduction of cell death. These results clearly indicate that myocardial protection elicited by adaptation is mediated at least in part via up-regulation of autophagy in association with BAG-1 protein.     

Protective effects of autophagy against blue light-induced retinal degeneration in aged mice

The aim of this study was to explore the role of autophagy in response to blue light damage in aged mice and in human retinal pigmented epithelium(hRPE) cells. Blue light damage to the retina was induced in 10-month-old(10 mo) C57 mice and hRPE cells. Flash electroretinography was used to assess retinal function. Retinal structure changes were observed by electron microscopy. Western blot was conducted to determine the expression levels of the following proteins: cleaved caspase-3, p38 mitogen-activated protein kinases, protein kinase R-like endoplasmic reticulum kinase(PERK), autophagy marker light chain 3(LC3), P62, and Beclin-1. On day 1 after light damage to the 10 mo mice, retinal function was changed. The latent periods of awave and b-wave were delayed, and amplitude was reduced. The electron microscopy results revealed mitochondria damage in the retinal pigmented epithelium and a disorganized photoreceptor outer segment(OS). PERK, LC3, and Beclin-1 were upregulated, whereas P62 was not. On day 5 after the blue light damage, restoration of electroretinography and OS was observed.PERK, LC3, and Beclin-1 were downregulated, whereas P62 was not. Protein changes in vitro were consistent with in vivo. The present study provided structural and functional evidence that autophagy plays an important role in the response to blue lightinduced retinal damage.     

Activation of poly(ADP-ribose) polymerase by myocardial ischemia and coronary reperfusion in human circulating leukocytes

Reactive free radical and oxidant production leads to DNA damage during myocardial ischemia/reperfusion. Consequent overactivation of poly(ADP-ribose) polymerase (PARP) promotes cellular energy deficit and necrosis. We hypothesized that PARP is activated in circulating leukocytes in patients with myocardial infarction and reperfusion during primary percutaneous coronary intervention (PCI). In 15 patients with ST segment elevation acute myocardial infarction, before and after primary PCI and 24 and 96 h later, we determined serum hydrogen peroxide concentrations, plasma levels of the oxidative DNA adduct 8-hydroxy-2'-deoxyguanosine (8OHdG), tyrosine nitration, PARP activation, and translocation of apoptosis-inducing factor (AIF) in circulating leukocytes. Plasma 8OHdG levels and leukocyte tyrosine nitration were rapidly increased by PCI. Similarly, poly(ADP-ribose) content of the leukocytes increased in cells isolated just after PCI, indicating immediate PARP activation triggered by reperfusion of the myocardium. In contrast, serum hydrogen peroxide concentrations and the translocation of AIF gradually increased over time and were most pronounced at 96 h. Reperfusion-related oxidative/nitrosative stress triggers DNA damage, which leads to PARP activation in circulating leukocytes. Translocation of AIF and lipid peroxidation occurs at a later stage. These results represent the first direct demonstration of PARP activation in human myocardial infarction. Future work is required to test whether pharmacological inhibition of PARP may offer myocardial protection during primary PCI.     

雷米普利对糖尿病大鼠心肌缺血／再灌注损伤保护作用的超微结构研究

目的：研究雷米普利对糖尿病大鼠心肌缺血／再灌注损伤的保护作用,并从超微结构的角度初步探讨其作用机制。方法：链脲佐菌素致糖尿病大鼠被随机分为3组（n=16）：缺血／再灌注（I／R）、缺血预适应（IPC）和雷米普利（RAM）组。RAM组每天用雷米普利（1mg／kg）灌胃,I／R和IPC组用等体积生理盐水灌胃。4周后各组动物均经历心肌缺血／再灌注损伤,IPC组于缺血前行心肌缺血预适应。连续监测心电图变化,测定心肌梗死面积,光、电镜下观察心肌形态学改变。结果：与I／R组比较,RAM及IPC组缺血期心脏ST-段抬高幅度降低,室早出现时间推迟,持续时间缩短,室速、室颤发生率降低,心肌梗死面积缩小,形态学观察心肌损伤减轻,心肌纤维及线粒体特征性结构保持清晰,血管通畅,内皮损伤减轻。结论：连续4周使用RAM对实验性糖尿病大鼠具有与IPC相似的心脏保护效应,机制可能与保护心肌细胞及线粒体、改善内皮功能等有关。     

Cytoprotection of rat hepatocytes by desipramine in a model of simulated ischemia/reperfusion

We investigated the cytoprotective effect of desipramine (DMI) during in vitro simulated ischemia/reperfusion (I/R) of rat hepatocytes. Primary hepatocytes isolated from male Sprague-Dawley rats were subjected to 4 h of anoxia at pH 6.2 followed by normoxia at pH 7.4 for 2 h to simulate ischemia and reperfusion, respectively. During simulated reperfusion, some hepatocytes were reoxygenated using media containing 5 μM DMI. Necrotic cell death and the onset of mitochondrial permeability transition (MPT) were assessed using fluorometry and confocal microscopy. Changes in autophagic flux and autophagy-related proteins (ATGs) were analyzed by immunoblotting. DMI was shown to substantially delay MPT onset and suppress I/R related cell damage. Mechanistically, DMI treatment during reperfusion increased the expression level of the microtubule-associated protein 1A/1B-light chain 3 (LC3) processing enzymes, ATG4B and ATG7. Genetic knockdown of ATG4B abolished the cytoprotective effect of DMI. Together, these results indicate that DMI is a unique agent which enhances LC3 processing in an ATG4B-dependent way.     

缺血性心肌病大鼠心肌细胞自噬在心肌重塑中的作用*

目的:研究缺血性心肌病大鼠心肌细胞自噬在心肌重塑中的作用。方法:36 只雄性SD大鼠分为正常对照组、假手术组、缺血性心肌病组( n=12),3组大鼠术前行心脏彩超检查,正常对照组大鼠不进行处理;假手术组大鼠开胸后不结扎冠状动脉,关闭胸腔;缺血性心肌病组大鼠开胸结扎冠状动脉20 min后,解开结扎线行再灌注后关闭胸腔,3组大鼠术后4周行心脏彩超检查后处死大鼠取心脏行HE染色、masson染色,观察心肌病理改变,用Western blot技术检测各组大鼠心肌细胞GRP78、LC3-I、LC3-II、Beclin-I表达及LC3-II/LC3-I比值的变化。结果:与正常组及假手术组比较,缺血性心肌病大鼠心室扩大,EF值降低;心肌排列紊乱,心肌纤维化增加;线粒体空泡化严重;内质网应激关键蛋白GRP78上调;自噬相关蛋白LC3-I、LC3-II、Beclin-I及LC3-II/LC3-I比值增加。结论:缺血性心肌病大鼠心肌细胞中内质网应激和自噬可能在心肌重塑中具有重要作用。     

Enhanced IPC by activation of pertussis toxin-sensitive and -insensitive G protein-coupled purinoceptors

Extracellular ATP plays an important role in ischemic preconditioning (IPC) through the activation of P(2y) purinoceptors. This study examined whether ATP-stimulated P(2y) purinoceptors are coupled to pertussis toxin (PTX)-insensitive G protein and whether activation of this pathway enhances myocardial protection afforded by IPC. The rat was treated with PTX for 48 h, and the heart was then isolated and buffer perfused. The heart underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. Isovolumic left ventricular function was measured, and functional recovery at 30 min after reperfusion was taken as an end point of myocardial protection. PTX pretreatment partially inhibited functional protection by IPC. Treatment with 100 microM 8-(p-sulfophenyl) theophylline (SPT) during IPC had no further effect on PTX-induced inhibition of functional protection by IPC, whereas suramin (300 microM) or reactive blue (RB) (10 microM) completely abolished myocardial protection in the preconditioned heart pretreated with PTX. Supplementation with adenosine (30 microM), ATP (30 microM), or UTP (50 microM) significantly enhanced IPC-induced functional protection, although preconditioning with these nucleotides without IPC had no protective effect. Adenosine-enhanced IPC was inhibited by pretreatment with PTX and SPT but not by suramin or RB, whereas ATP-enhanced IPC was inhibited by suramin or RB in combination with PTX pretreatment. On the other hand, UTP-enhanced IPC was not affected by PTX pretreatment but was inhibited by suramin or RB. Adenosine supplemented IPC without PTX pretreatment and ATP supplemented IPC with PTX pretreatment were not affected by nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (100 microM). Although the protein kinase C inhibitor Ro318425 (0.3 microM) or tyrosine kinase inhibitor genistein (50 microM) had no significant effect on the functional protection afforded by adenosine-supplemented IPC without PTX pretreatment and ATP-supplemented IPC with PTX pretreatment, the combination of Ro318425 and genistein attenuated functional protection afforded by both the purinoceptor agonist-supplemented IPC. These results suggest the crucial involvement of PTX-sensitive and -insensitive G protein coupled purinoceptors in enhanced IPC by supplementation with adenosine, ATP, and UTP.     

细胞自噬在大鼠肺缺血/再灌注引发肝脏损伤中的作用*

目的: 探讨肺缺血/再灌注(LI/R)时肝脏损伤的影响,并初步探索细胞自噬(Autophagy)在其中发挥的作用。方法: 构建大鼠缺血/再灌注肺损伤(LI/RI)模型,模型制备方法为大鼠麻醉后切开气管进行机械通气,使用动脉夹将肺门夹闭模拟缺血过程,30 min后松开动脉夹,恢复灌注3 h。24只大鼠随机分为伪手术组(Sham组)、缺血/再灌注组(I/R组)、溶剂组(DMSO组)和自噬抑制剂组(3-MA组),每组均6只,后2组大鼠术前分别腹腔注射DMSO和3-MA,造模结束后使用肺湿/干重比判断造模是否成功;抽取静脉血测定肝脏转氨酶指标ALT与AST;取肝脏组织,光镜下观察肝脏形态改变,以及电镜下观察肝细胞超微结构;使用RT-qPCR和Western blot实验分别检测肝脏组织细胞中自噬相关蛋白的基因mRNA表达水平和蛋白表达水平。结果: 与Sham组相比,其余各组肺湿/干重比均升高;血AST和ALT均有大幅升高且肝脏组织损伤明显,其中以I/R组升高最为明显,光镜下组织形态学及电镜下细胞微细结构均有不同程度的破坏;肝脏中自噬相关蛋白的基因表达水平与蛋白表达水平均有明显不同,表现为自噬上升 (P＜0.01或P＜0.05)。I/R组和DMSO组肝脏组织均有较重损伤,肝细胞结构破坏严重,自噬小体形成,而AST、ALT、自噬相关蛋白转录和表达水平等各项指标均无统计学差异(P＞0.05)。而相较于DMSO组,3-MA组肝脏组织损伤有所减轻,肝细胞微细结构损伤程度低,且无自噬小体形成,血中AST和ALT下降,肝脏组织内自噬水平均下降 (P＜0.05)。结论: 肺缺血/再灌注可引起大鼠肝损伤;细胞自噬可介导大鼠肺缺血/再灌注引起的肝损伤,抑制细胞自噬可以有效减轻大鼠LI/R引起的肝损伤。     

Selenium supplementation and ischemia-reperfusion injury in rats

Cardiac ischemia--reperfusion injury results in oxidative stress and poor physiological recovery. This study examined the amount of lipid and protein oxidation during ischemia-reperfusion to assess the degree of oxidative stress. Selenium supplementation was used to alter the antioxidant status of rats and the recovery of myocardial function post ischemia-reperfusion was investigated. Male Wistar rats were fed diets containing 0, 50, and 1000 microg/kg sodium selenite for 5 weeks, whilst controls received normal rat food containing 240 microg/kg selenium. Langendorff-perfused hearts were subjected to 22.5 min global ischemia and 45 min reperfusion, with functional recovery assessed. Heart tissues were assayed for the presence of lipid peroxides and protein carbonyls and correlated to cardiac recovery. Following ischemia and reperfusion there was a significant increase in both protein oxidation and lipid peroxidation. Hearts from selenium-deficient animals demonstrated higher levels of both protein carbonyls and lipid peroxides and were more susceptible to ischemia-reperfusion injury when compared to controls (38% versus 47% recovery of rate pressure product (RPP)). Selenium supplementation lowered the levels of protein carbonyls and lipid peroxides and resulted in improved recovery of cardiac function post ischemia-reperfusion (57% recovery of RPP). These data suggest that selenium supplementation may provide an effective method for reducing oxidative damage post cardiac ischemia-reperfusion.     

Matrix metalloproteinases and collagen ultrastructure in moderate myocardial ischemia and reperfusion in vivo

Severe ischemic injury or infarction of myocardium may cause activation of matrix metalloproteinases (MMPs) and damage the interstitial matrix. However, it is unknown whether MMP activation and matrix damage occur after moderate ischemia and reperfusion that result in myocardial stunning without infarction, and if so whether such changes contribute to postischemic myocardial expansion and contractile dysfunction. To address these questions, open-chest anesthetized pigs underwent 90 min of regional ischemia (subendocardial blood flow 0.4 +/- 0.1 ml. g(-1). min(-1)) and 90 min of reperfusion. After ischemia plus reperfusion, histological and ultrastructural examination revealed no myocardial infarction or inflammatory cell infiltration. Myocardial MMP-9 content increased threefold with a fourfold increase in the active form (P < 0.001). Myocardial collagenase content doubled (P < 0.01) but remained in latent form. MMP-2 and tissue inhibitors of metalloproteinases were unaffected. Despite increases in MMPs, collagen ultrastructure (assessed by cell maceration scanning electron microscopy) was unaltered. Intracoronary administration of the MMP inhibitor GM-2487 did not prevent or attenuate myocardial expansion (assessed by regional diastolic dimensions at near-zero left ventricular pressure) or contractile dysfunction. We conclude that although moderate ischemia and reperfusion alter myocardial MMP content and activity, these effects do not result in damage to interstitial collagen, nor do they contribute to myocardial expansion or contractile dysfunction.     

Valsartan preconditioning protects against myocardial ischemia–reperfusion injury through TLR4/NF-κB signaling pathway

Toll-like receptor 4 (TLR4) activation has been implicated in the pathogenesis of myocardial ischemia/reperfusion (I/R) injury. The activated TLR4 is capable of activating a variety of proinflammatory mediators, such as tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6). Valsartan as a kind of Angiotensin II type 1 receptor blockers is gradually used for the treatment of ischemic heart disease depending on its anti-inflammation function. Therefore, we hypothesized that valsartan protects against myocardial I/R injury by suppressing TLR4 activation. We constructed the rat model of myocardial I/R injury. The rats were pretreated with valsartan for 2 weeks, and then subjected to 30 min ischemia and 2 h reperfusion. TLR4 and Nuclear factor kappa-B (NF-κB) levels were detected by quantitative real-time PCR and western blot. In order to evaluate myocardial damage, the myocardial infarct size, histopathologic changes, and the release of myocardial enzymes, proinflammation cytokines and Angiotensin II were analyzed by triphenyl tetrazolium chloride (TTC) staining, light microscopy, and enzyme-linked immunosorbent assay (ELISA), respectively. Valsartan preconditioning inhibited TLR4 and NF-κB expressions concomitant with an improvement in myocardial injury, such as smaller infarct size, fewer release of myocardial enzymes, and proinflammation mediators. These findings suggest that valsartan plays a pivotal role in the protective effects on myocardial I/R injury. This protection mechanism is possibly due to its anti-inflammation function via TLR4/NF-κB signaling pathway.     

缺血后处理降低高胆固醇血症大鼠心肌对缺血/再灌注损伤的易感性及其机制

目的:探讨缺血后处理对高胆固醇血症基础上发生的心肌缺血/再灌注损伤的影响及其可能的机制。方法:建立食源性高胆固醇血症大鼠模型,运用TTC染色、酶活性检测等方法测定缺血/再灌注所致的心肌损伤,用实时定量RT-PCR方法检测心肌组织中低氧诱导因子-1α(HIF-1α)mRNA水平,用Western blot方法检测HIF-1α蛋白水平。结果:高胆固醇血症加重了缺血/再灌注造成的心肌损伤,而缺血后处理显著缩小了高胆固醇血症大鼠缺血/再灌注所致的心梗面积,降低了血清肌酸激酶(CK)的活性,减少了心肌细胞凋亡。同时,缺血后处理提高了高胆固醇血症大鼠缺血心肌组织中HIF-1α的蛋白水平。结论:缺血后处理可以降低高胆固醇血症大鼠心肌对缺血/再灌注损伤的敏感性,其效应与心肌组织中HIF-1α的蛋白水平存在着相关性。     

Adenosine A1/A2a receptor agonist AMP-579 induces acute and delayed preconditioning against in vivo myocardial stunning

The purpose of this study was to determine whether the adenosine A1/A2a receptor agonist AMP-579 induces acute and delayed preconditioning against in vivo myocardial stunning. Regional stunning was produced by 15 min of coronary artery occlusion and 3 h of reperfusion (RP) in anesthetized open-chest pigs. In acute protection studies, animals were pretreated with saline, low-dose AMP-579 (15 microg/kg iv bolus 10 min before ischemia), or high-dose AMP-579 (50 microg/kg iv at 14 microg/kg bolus + 1.2 microg.kg(-1).min(-1) for 30 min before coronary occlusion). The delayed preconditioning effects of AMP-579 were evaluated 24 h after administration of saline vehicle or high-dose AMP-579 (50 microg/kg iv). Load-insensitive contractility was assessed by measuring regional preload recruitable stroke work (PRSW) and PRSW area. Acute preconditioning with AMP-579 dose dependently improved regional PRSW: 129 +/- 5 and 100 +/- 2% in high- and low-dose AMP-579 groups, respectively, and 78 +/- 5% in the control group at 3 h of RP. Administration of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.7 mg/kg) blocked the acute protective effect of high-dose AMP-579, indicating that these effects are mediated through A1 receptor activation. Delayed preconditioning with AMP-579 significantly increased recovery of PRSW area: 64 +/- 5 vs. 33 +/- 5% in control at 3 h of RP. In isolated perfused rat heart studies, kinetics of the onset and washout of AMP-579 A1 and A2a receptor-mediated effects were distinct compared with those of other adenosine receptor agonists. The unique nature of the adenosine agonist AMP-579 may play a role in its ability to induce delayed preconditioning against in vivo myocardial stunning.     

Protective effects of leukopenia and tissue plasminogen activator in microvascular ischemia-reperfusion injury

Ischemia shifts the anticoaugulant/procoagulant balance of the endothelium in favor of activation of coagulation. We studied whether cheek pouch microcirculation of leukopenic hamsters was protected by tissue plasminogen activator (tPA) (50 microg/100 g body wt) against ischemia-reperfusion injury. Adherent leukocytes, total perfused capillary length (PCL), permeability increase, and arteriolar and venular red blood cell (RBC) velocity were investigated by fluorescence microscopy. Measurements were made at control, 30 or 60 min of ischemia, and at 30 or 60 min of reperfusion. Hamsters were made leukopenic by treatment with cyclophosphamide (20 mg/100 g body wt ip, 4 days before the experiment), which decreased circulating leukocyte count by 85-90%. Leukopenic hamsters undergoing 30 min of ischemia followed by 30 min of reperfusion showed no significant decrease in PCL or increased permeability. Leukopenic hamsters undergoing 60 min of ischemia followed by 60 min of reperfusion presented a significant decrease in microvascular perfusion where PCL was 28 +/- 7% of baseline, low-flow conditions, and increased permeability. In leukopenic hamsters treated with tPA there was complete protection of capillary perfusion with no significant changes in permeability or arteriolar and venular RBC velocity. In conclusion, thrombus formation may be an additional and independent factor that with leukocyte-mediated mechanisms determines ischemia-reperfusion injury.     

海马神经元缺血/再灌注后自噬的表达及其作用

目的：研究自噬在大鼠海马神经元缺血缺氧/再灌注过程中的表达及自噬在神经元缺血缺氧/再灌注损伤中的作用。方法：原代培养的大鼠海马神经元经2 h的氧糖剥夺和不同时段的再灌注处理,MTT法检测细胞活性,透射电镜下检测自噬的特异性结构,免疫荧光化学法检测自噬特异性蛋白微管相关蛋白1轻链3（LC3B）的表达。应用自噬抑制剂3-甲基腺嘌呤（3-MA）检测神经元的活性。结果：经氧糖剥夺/再灌注后,海马神经元的活性比未经氧糖剥夺/再灌注组显著地降低。透射电镜和免疫荧光检测,未经氧糖剥夺/再灌注的神经元自噬的发生率极低,氧糖剥夺后和再灌注的不同时间段,均有自噬的发生。应用自噬抑制剂3-MA阻断自噬后,神经元的存活率显著降低。结论：缺血缺氧/再灌注能激活海马神经元的自噬,并可能在缺血缺氧/再灌注过程中起对抗损伤的作用。     

Knockdown of lncRNA AK139328 alleviates myocardial ischaemia/reperfusion injury in diabetic mice via modulating miR‐204‐3p and inhibiting autophagy

This study was aimed at investigating the effects of lncRNA AK139328 on myocardial ischaemia/reperfusion injury (MIRI) in diabetic mice. Ischaemia/reperfusion (I/R) model was constructed in normal mice (NM) and diabetic mice (DM). Microarray analysis was utilized to identify lncRNA AK139328 overexpressed in DM after myocardial ischaemia/reperfusion (MI/R). RT‐qPCR assay was utilized to investigate the expressions of lncRNA AK139328 and miR‐204‐3p in cardiomyocyte and tissues. Left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular ejection fraction (LVEF) and fractioning shortening (FS) were obtained by transthoracic echocardiography. Haematoxylin‐eosin (HE) staining and Masson staining were utilized to detect the damage of myocardial tissues degradation of myocardial fibres and integrity of myocardial collagen fibres. Evans Blue/TTC staining was used to determine the myocardial infarct size. TUNEL staining was utilized to investigate cardiomyocyte apoptosis. The targeted relationship between lncRNA AK139328 and miR‐204‐3p was confirmed by dual‐luciferase reporter gene assay. MTT assay was used for analysis of cardiomyocyte proliferation. Western blot was utilized to investigate the expression of alpha smooth muscle actin (α‐SMA), Atg7, Atg5, LC3‐II/LC3‐I and p62 marking autophagy. Knockdown of lncRNA AK139328 relieved myocardial ischaemia/reperfusion injury in DM and inhibited cardiomyocyte autophagy as well as apoptosis of DM. LncRNA AK139328 modulated miR‐204‐3p directly. MiR‐204‐3p and knockdown of lncRNA AK139328 relieved hypoxia/reoxygenation injury via inhibiting cardiomyocyte autophagy. Silencing lncRNA AK139328 significantly increased miR‐204‐3p expression and inhibited cardiomyocyte autophagy, thereby attenuating MIRI in DM.