Protection of sivelestat sodium in ischemia reperfusion brain injury in rats

Objective： To explore the protective effect and its mechanism of sivelestat sodium （SS） on cerebral ischemia reperfusion in rats. Methods： The neurons of neonate rat were cultured in vitro. The protective effects of sivelestat sodium on ischemic injury were observed by treating cells in glucose - free and oxygen -free medium. Neuron survival was measured by MTT reduction assay and the release of LDH; neutrophile elastase contents were determined by ELISA assay; intracellular calcium levers were measured by fluo -3 and confocal laser microscopy; malondialdehyde （MDA） contents, superoxide dismutase （SOD） activity and glutamic acid contents in neuron were measured by colorimetric method. The expression of Bcl - 2 and Bax in rat neurons were detected by immunohistochemistry ; the mrna sequences of Bcl - 2 and Bax were detected by RT - PCR; the expression of Bcl - 2, Bax and p - p38 MAPK proteins in rat neurons was detectd by Western blotting. Results： Sivelestat sodium protected the integrity and survival of cells and reduced LDH leakage, MDA contents, ncutrophile elastase contents, glutamic acid contents; increased SOD contents; decreased the mrna expression of bax and increased the mrna expression of Bcl - 2 ; decreased the protein expression of p - p38MAPK . The ratio of Bcl - 2/Bax in hippocampus neurons of models was lower than that of Sivelestat sodium groups. Conclusions： SS may play neuroprotectvie role by increasing neuron survival, reducing oxygen free radicals, calcium release, glutamate release and the expressions of bax and p -p38MAPK, and down - regulating the expressions of bcl -2.     

Effects of phytoestrogen genistein on myocardial ischemia／reperfusion injury and apoptosis in rabbits

INTRODUCTION Ischemia/reperfusion (I/R) injury is often seen inclinics which pathogenesis has not been elucidatedclearly. It is generally believed that its mechanism isrelated to lipid peroxide induced by oxygen free radi-cals and to irreversible damage caused by intracellularcalcium overload. The Ca2 overload is induced mainlyby the action of sarcolemmal Na /H and Na /Ca2 ex-changers[1,2], which then activates several intracellularprocess, resulting in myocyte death[3-5]. Genistein…     

Isoflurane preconditioning protects against ischemia-reperfusion injury partly by attenuating cytochrome c release from subsarcolemmal mitochondria in isolated rat hearts

AIM: To examine if isoflurane preconditioning can attenuate ischemia/reperfusion injury by reducing cytochrome c release from inner mitochondrial membrane. METHODS: Isolated hearts of Sprague-Dawley rats were perfused on Langendorff apparatus. Hearts were randomly assigned to a non-treated group (CON group, n=12) or three isoflurane preconditioning groups (0.5% ISC group, 1.0% ISC group, and 2.0% ISC group; n=12). In the latter three groups, isoflurane was given at concentrations of 0.5%, 1.0%, and 2.0% for 15 min with 15-min washout before 30-min ischemia. Subsarcolemmal mitochondria of the myocardium were isolated after 60-min reperfusion. Hemodynamics of the each heart was recorded, infarct size of the hearts and contents of cytosolic cytochrome or mitochondrial cytochrome c were measured at the end of reperfusion. Morphology of isolated mitochondria in the four groups was evaluated, respectively. RESULTS: Compared with the CON group, cytosolic cytochrome c in 0.5% ISC group, 1.0% ISC group, and 2.0% ISC group were significantly decreased along with a significant increase of mitochondrial cytochrome c. Infarct size of the hearts in the four groups were 56%+/-12%, 41%+/-12%, 32%+/-7% and 33%+/-11%, respectively. The values of the three isoflurane preconditioning groups were significantly lower than that of the CON group (P<0.05). Isoflurane exposure before ischemia can attenuate the change of morphology of mitochondria after reperfusion. The effects of 2.0% isoflurane on reducing cytochrome c release were more remarkable than 0.5% and 1.0% concentrations of isoflurane. CONCLUSION: Myocardioprotective effects of isoflurane preconditioning were associated with attenuation of cytochrome c loss from the inner membrane of subsarcolemmal mitochondria.     

Taurine inhibits ischemia／reperfusion-induced compartment syndrome in rabbits

Aim: To investigate effects of taurine on ischemia/reperfusion (I/R)-induced compartment syndrome in rabbit hind limbs. Methods: Rabbits underwent femoral artery occlusion after ligation of branches from terminal aorta to femoral artery. After a 7-h ischemia, reperfusion was established with the use of heparinized polyethylene shunts. Rabbits received taurine (1 g/kg) or normal saline (control) by iv infusion 10 min before shunt placement. During reperfusion, anterior compartment pressure (ACP) was monitored continuously in the left lower extremity. Gastrocnemius muscle triphenyltetrazolium chloride (TTC) level, taurine content and myeloperoxidase activity were assayed. Oxidative stress was induced in the in vitro gastrocnemius muscle slices by free radical generating systems (FRGS), and the malondialdehyde content was measured in presence or absence of taurine. Results: After 7 h of ischemia, none of the parameters that we measured were different from those before ischemia, except that TTC reduction decreased by 80%. In the control group, after 2 h of reperfusion, ACP increased 4.5-fold, and gastrocnemius muscle taurine content was reduced by 33%. In taurine-treated animals, at 2 h reperfusion, the mean arterial blood pressure and heart rate were increased, by 6% and 10%. ACP decreased by 39%, muscle edema decreased by 16%, TTC reduction increased by 150%, and lactate dehydrogenase decreased by 36% compared to control group. Plasma and muscle taurine content increased by 70% and 88%, respectively. In the taurine-treated group, at 2 h reperfusion, plasma malondialdehyde and conjugated diene content were decreased by 38% and 23%, respectively, and muscle malondialdehyde and conjugated diene content decreased by 22% and 30%, respectively compared to the control group. At 2 h reperfusion, myeloperoxidase activity was increased 3.5-fold in control animals. In the in vitro study, taurine decreased malondialdehyde content in muscle slices incubate dwith hypochlorous acid in a dose-dependent manner, but there was no change when incubated with hydrogen peroxide and xanthine oxidase. Conclusion: Treatment with taurine inhibited I/R-induced compartment syndrome by at least in part attenuating oxidative stress injury induced by I/R, suggesting clinical application of taurine might be a new strategy for the prevention and treatment of compartment syndrome.     

Chronic intermittent hypobaric hypoxia protects the heart against ischemia/reperfusion injury through upregulation of antioxidant enzymes in adult guinea pigs

Aim： To investigate the protection and the anti-oxidative mechanism afforded by chronic intermittent hypobaric hypoxia （ClHH） against ischemia/reperfusion （I/R） injury in guinea pig hearts.
Methods： Adult male guinea pigs were exposed to ClHH by mimicking a 5000 m high altitude （pB=404 mmHg, po2=84 mmHg） in a hypobaric chamber for 6 h/day for 28 days. Langendorff-perfused isolated guinea pig hearts were used to measure variables of left ventricular function during baseline perfusion, ischemia and the reperfusion period. The activity and protein expression of antioxidant enzymes in the left myocardium were evaluated using biochemical methods and Western blotting, respectively. Intracellular reactive oxygen species （ROS） were assessed using ROS-sensitive fluorescence.
Results： After 30 min of global no-flow ischemia followed by 60 min of reperfusion, myocardial function had better recovery rates in ClHH guinea pig hearts than in control hearts. The activity and protein expression of superoxide dismutase （SOD） and catalase （CAT） were significantly increased in the myocardium of ClHH guinea pigs. Pretreatment of control hearts with an antioxidant mixture containing SOD and CAT exerted cardioprotective effects similar to ClHH. The irreversible CAT inhibitor aminotriazole （ATZ） abolished the cardioprotection of ClHH. Cardiac contractile dysfunction and oxidative stress induced by exogenous hydrogen peroxide （H2O2） were attenuated by CIHH and CAT.
Conclusions： These data suggest that CIHH protects the heart against I/R injury through upregulation of antioxidant enzymes in guinea pig.     

谷氨酰胺预处理对大鼠肠缺血再灌注损伤的保护作用及其对eNOS-NO通路的影响

目的探讨谷氨酰胺(Gln)预处理对大鼠肠缺血再灌注损伤(IR)后的保护作用及其对内皮型一氧化氮合酶(e NOS)/一氧化氮(NO)信号通路的影响。方法将30只健康雄性Wistar大鼠随机分为假手术(Sham)组、IR组、Gln组,每组10只,Gln组给予谷氨酰胺1 g/(kg·d),连续灌胃7 d。Sham组和IR组以同等剂量生理盐水灌胃7 d。Sham组仅分离肠系膜上动脉(SMA)而根部不夹闭。IR组和Gln组均用无损伤血管夹夹闭SMA根部,30 min后放松血管夹形成再灌注损伤模型。各组大鼠均于制模后24 h采集腹主动脉血和回肠标本。应用HE染色法观察肠黏膜组织形态学改变,ELISA试剂盒双抗体夹心法测定D-乳酸、内毒素含量,硝酸还原酶法检测血清NO的含量,比色法检测血清e NOS、诱导型一氧化氮合酶(i NOS)的含量,荧光定量PCR(RT-PCR)检测大鼠肠组织e NOS、i NOS m RNA表达水平。结果再灌注后,与Sham组相比,IR组肠黏膜绒毛上皮脱落,固有层崩解,部分绒毛顶端出血,腺体明显受损;Gln组表现为肠黏膜绒毛上皮下间隙扩大,但不明显,绒毛轻度水肿,腺体大致正常,固有层轻度水肿。IR组血清D-乳酸、内毒素、i NOS水平及肠组织i NOS m RNA表达水平均高于Sham组和Gln组(P<0.05)。IR组血清NO、e NOS含量及组织中e NOS的m RNA表达量均低于Sham组和Gln组(P<0.05)。结论谷氨酰胺预处理可以减轻肠缺血再灌注后的组织形态学改变及损伤,其机制可能与抑制i NOS表达,增加e NOS表达,从而增加NO活性有关。     

慢性间歇性低压低氧对成年和幼年大鼠缺血/再灌注心脏保护作用的比较

目的观察慢性间歇性低压低氧(CIHH)对成年和幼年大鼠心脏缺血/再灌注损伤的保护作用的异同点。方法♂成年和新生Sprague-Dawlay(SD)大鼠随机分为4组:对照28d组(CON28)、对照42d组(CON42)、CIHH处理28d组(CIHH28)和CIHH处理42d组(CIHH42)。间歇性低氧处理组动物于低压氧舱分别接受28d、42d模拟3000米海拔高度(PB=525mmHg,PO2=108.8mmHg)的低压低氧处理,每天5h。对照组动物除了不接受低氧处理外,其它处理均与间歇性低氧组动物相同。应用Langendorff离体心脏灌流技术,给予心脏缺血(停灌30min)/再灌注(复灌60min)处理,记录离体大鼠心脏在不同时期的心功能变化。心功能参数包括左室发展压(left ventricular developing pressure,LVDP)、左室压力最大变化速率(maximum changerate of LVDP,±LVdp/dtmax)、左室舒张末压(left ventricularend di-astolic pressure,LVEDP)、冠脉流量(coronary flow,CF)和心率(heartrate,HR)。结果①对成年大鼠,基础状态和缺血/再灌注状态下CIHH28d组各心功能参数与CON28d组相比差异均无统计学意义。CIHH42d组各心功能参数均好于CON42d组,表现为LVDP、LVEDP、±LVdp/dtmax和CF恢复均增加(P<0.05)。②对幼年大鼠,基础状态下,CIHH大鼠CF较对照大鼠明显增多,其余心功能参数与对照大鼠无差异。CIHH大鼠缺血/再灌注后心脏功能的恢复明显好于对照动物,表现为LVDP、LVEDP、±LVdp/dtmax和CF恢复均增加(P<0.05),且CIHH42d组比CIHH28d组心功能改善更明显。结论CIHH可增强成年和幼年大鼠抗心肌缺血/再灌注损伤的能力,具有明显的心脏保护作用,CIHH42d组保护作用更为明显;CIHH的心脏保护作用有明显的年龄差异,在幼年大鼠更易产生保护作用。     

Inducible nitric oxide synthase and inflammation

Nitric oxide (NO), derived from L-arginine (L-Arg) by the enzyme nitric oxide synthase (NOS), is involved in acute and chronic inflammatory events. In view of the complexity associated with the inflammatory response, the dissection of possible mechanisms by which NO modulates this response will be profitable in designing novel and more efficacious NOS inhibitors. In this review we describe the consequences associated with the induction of inducible nitric oxide synthase (iNOS) and its therapeutic implications.     

Inducible nitric oxide synthase inhibition by mycophenolic acid

The focus of this review is the influence of an immunosuppressive xenobiotic drug mycophenolic acid on the induction of nitric oxide production in various cell types. The potential therapeutic significance of the cell-specific fine-tuning of nitric oxide release by mycophenolic acid, as well as the mechanisms behind the drug action are discussed.     

The effect of inducible nitric oxide synthase inhibitor on microvascular permeability after cerebral ischemia/reperfusion

Our objective was to study the effect of inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (AG) on microvascular permeability after cerebral ischemia/reperfusion (I/R) injury. Cerebral I/R injury was produced by occlusion of both the carotid arteries for 60 min with restitution of blood flow for 60 min. AG (200 mg/kg) was intraperitoneally administrated 5 min before the onset of ischemia and again 5 min before reperfusion. Microvascular permeability was evaluated by 0.75% sodium fluorescein (FINa) extravasation during early 300 s. Cerebral I/R injury increased the permeability of microvessel to fluorescein and the concentration of fluorescein outside of microvessels was significantly higher than that in microvessels after 110 s. However, after AG administration, FINa extravasation appears much faster. From 80 s on, the fluorescence intensity outside is higher. I/R increased microvascular permeability. Nitric oxide derived from iNOS may maintain microvascular permeability at the early stage after I/R.     

Aliskiren protects against myocardial ischaemia‐reperfusion injury via an endothelial nitric oxide synthase dependent manner

Aliskiren, a direct renin inhibitor, has shown potent ability to attenuate hypertension. Our previous research has found that aliskiren protected against myocardial ischaemia‐reperfusion (I/R) injury and enhanced phosphorylation of endothelial nitric oxide synthase (eNOS) in spontaneously hypertensive rats. However, whether the cardioprotective effect of aliskiren against myocardial I/R injury was eNOS‐dependent is unknown. In the present study, 12‐week‐old male eNOS knockout (eNOS^?/?) and wild‐type C57BL/6J mice (WT) were orally administrated with the dose of 50 mg/kg per day of aliskiren. After a 4‐week treatment, aliskiren decreased blood pressure in eNOS^?/? mice, and reduced renin‐angiotension II levels in both eNOS^?/? and WT mice. Aliskiren also improved left ventricular ejection fraction (EF) and fractional shortening (FS), decreased myocardial infarct size, reduced creatine kinase (CK) and lactate dehydrogenase (LDH) activity in plasma, attenuated dihydroethidium (DHE) fluorescence and levels of malondialdehyde (MDA), enhanced superoxide dismutase (SOD) activity and total antioxidant capacity (T‐AOC) in myocardium, increased SOD and thioredoxin (Trx) proteins expression in WT mice subjected to 30 minutes of ischaemia followed by reperfusion for 24 hours. However, aliskiren failed to restore all of the above indices in eNOS^?/? mice subjected to the same I/R injury. Our study indicated that aliskiren protected against myocardial I/R injury via an eNOS dependent manner.     

Glutamine contributes to ameliorate inflammation after renal ischemia/reperfusion injury in rats

The aim of this study was to investigate the effects of glutamine in an in vivo rat model of renal ischemia/reperfusion (I/R) injury. Male Wistar rats underwent bilateral renal pedicle clamping for 45 min followed by reperfusion for 6 h. Glutamine (1.5 mg/kg) was administered intraperitoneally (i.p.) 15 min prior to reperfusion. Plasma concentrations of urea, creatinine, γ-glutamyl transferase (γ-GT), and aspartate aminotransferase (AST) were measured for the assessment of renal function and reperfusion injury. Markers of oxidative stress, expression of the pro-inflammatory mediators inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), AT-1 expression, and changes in the oxidative stress-sensitive nuclear factor kappa B (NF-κB) signaling pathway were measured to investigate whether glutamine can reduce the renal dysfunction. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of polymorphonuclear (PMN) cell infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of nitrotyrosine and poly(ADP-ribose) synthetase (PARS). In vivo, glutamine significantly reduced the increase in urea, creatinine, γ-GT, AST, produced by renal ischemia/reperfusion (I/R), suggesting an improvement in both renal function and injury. Glutamine significantly reduced iNOS and NF-κB, kidney MPO activity and MDA levels, indicating a reduction in PMN infiltration and lipid peroxidation, respectively. Glutamine reduced the histological evidence of renal damage associated with I/R and caused a substantial reduction in the staining for nitrotyrosine and PARS, suggesting reduced nitrosative and oxidative stress. Moreover, glutamine attenuated the reduction of COX-2 expression and prevented the increased AT-1 expression after I/R. Our results suggest that glutamine reduces the renal dysfunction and injury associated with I/R of the kidney.     

Role of polyamines in myocardial ischemia/reperfusion injury and their interactions with nitric oxide

Polyamines (putrescine, spermidine, and spermine) are present in all higher eukaryotic cells and are essential for cell growth, differentiation and apoptosis. Sharing common precursor with polyamines, nitric oxide (NO) is associated with myocardial ischemia/reperfusion injury by the generation of peroxynitrite. Although polyamines have been implicated in tissue ischemia injury, their metabolism and interactions with NO in myocardial ischemia/reperfusion injury have not been fully understood. In our experiment, when Langendorff perfused rat hearts were subjected to 40 min ischemia without reperfusion, both ornithine decarboxylase (ODC) and Spermidine/spermine N(1)-acetyltransferase (SSAT) activities were up-regulated and putrescine accumulated. While after reperfusion, ODC activity decreased and SSAT activity increased, resulting in putrescine accumulation and decreased spermidine and spermine. Meanwhile NO content was increased. In addition, sodium nitroprusside (SNP, a NO donor) decreased ODC activity in cardiac tissue homogenate but increased SSAT activity in a dose-dependent manner. Pre-treatment of isolated heart with N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME, an inhibitor of NO synthase) increased ODC activity. Exogenous spermine (1 mM) administration prior to ischemia prevented spermine decrease, reduced cardiac myocyte necrosis and apoptosis, and promoted the recovery of cardiac function after ischemia/reperfusion. These results suggest that acute heart ischemia activates myocardial polyamine stress response characterized by increased ODC and SSAT activities and accumulation of putrescine. Ischemia/reperfusion disturbs polyamine metabolism, and the loss of spermine might be associated with NO increase and thereby influences myocardial cell viability. Exogenous spermine may protect the hearts from myocardial ischemia/reperfusion injury.     

丹参对兔缺血再灌注肾组织一氧化氮及其合成酶的影响

目的：探讨丹参保护兔缺血再灌注肾损伤的内皮机制。方法：以兔肾缺血再灌注为模型，采用自动生化分析技术，检测不同实验条件下肾组织一氧化氮合成酶（NOS）总活性及一氧化氮（NO），并用Elasa法测定尿视黄醇结合蛋白（RBP）及尿微量白蛋白（Alb) ,试图分析NOS总活性，NO与肾功能之间的相互关系。结果：（1）正常肾髓质较皮质能产生更多的NO。（2）缺血肾组织NOS总活性显著下降，NO显著增高。（3）再灌注后，肾组织NOS总活性显著升高，而NO显著下降，肾功能明显受损。（4）肾缺血注射丹参后再灌注，肾组织NOS总活性进一步升高，而NO变化不明显，尿RBP及Abl下降至正常水平。结论：（1）生理情况下，肾髓质较皮质能产生更多的NO，这对肾髓质血流分布及其功能的调节可能具有重要作用。（2）缺血再灌注后，肾组织NOS活性升高与肾功能受损密切相关。（3）丹参对缺血再灌注所致的肾小管及肾小球损伤均有明显的保护作用。（4）丹参对缺血再灌注肾组织中的NO具有抑制作用。     

大鼠缺血再灌注肾组织一氧化氮合成酶mRNA表达的研究

目的 探讨大鼠缺血再灌注肾组织一氧化氮合成酶（NOS）mRNA表达的特点,分析缺血再灌注肾损伤的分子机制。方法 建立大鼠肾缺血再灌注模型。采用组织细胞原位杂交及图像分析技术对不同实验条件下各型NOS（eNOS,nNOS及iNOS)mRNA表达的定位及含量进行检测。并对肾组织NOS总活性及血肌酐（Cr)值进行生化测定。结果（1）正常情况下,eNOS,nNOS及iNOS在正常肾组织中均有表达,cNOS/iNOS比值为2.29;eNOS和nNOS主分布于肾小球及血管内皮;iNOS仅分布于皮质远,近曲小管上皮。（2）缺血时,肾组织NOS总活性显著下降,三种NOSmRNA在皮,髓质及小球中的表达均下调,以eNSO最明显,cNSO/iNOS比值降为2.01。（3）再灌注后,三种NOSmRNA的表达明显上调,以iNOSmRNA最明显,cNOS/iNOS比值降为1.77,eNOS及nNOS上调部位仅限于肾皮,髓质血管,而小球及小管中则表现为下调,尤以nNOSmRNA在小球中的下调最明显。结论 （1）缺血再灌注后,皮质肾小管上皮中iNOSmRNA的高表达是导致肾缺血再灌注损伤的重要分子机制。（2）cNOS/iN-OS比值与缺血再灌注过程中肾功能的变化密切相关,该比值的恒定对肾血流量和肾小球滤过率（GFR）的调节可能具有重要意义。