NF-kB在大鼠肝缺血再灌注损伤中的活化及意义

目的探讨NFkB在肝缺血再灌注损伤过程中的作用。方法选择健康雌性Wistar大鼠24只,随机分为手术对照组,肝缺血90min组,肝缺血90min/再灌注120min组,每组8只。常规方法观察肝脏组织学改变,检测血清酶学水平和肝组织中髓过氧化物酶(MPO)含量,采用sABC免疫组织化学方法测定肝组织中NFkB的活化程度。结果手术对照组肝组织形态正常,无NFkB活化,肝功能酶学和MPO正常水平;缺血组动物肝细胞索排列紊乱,肝小叶变形,肝细胞和内皮细胞普遍水肿变性,NFkB呈中重度活化,血清酶学和MPO水平升高(P<0.01);肝缺血/再灌注组肝组织在缺血组改变基础上合并中央区局灶性肝细胞坏死,血窦内微血栓形成,汇管区中性粒细胞浸润,NFkB活化最为明显,血清酶学和MPO升高最为显著(P<0.01)。结论肝缺血再灌注时,NFkB被活化,使中性粒细胞组织浸润,对肝脏缺血再灌注损伤病理过程起到重要的作用。     

Sexual dimorphism in reduced-size liver ischemia and reperfusion injury in mice: role of endothelial cell nitric oxide synthase

We have recently reported that female mice are protected to a much greater extent from the injurious effects of reduced-size liver ischemia and reperfusion (RSL+I/R) than are males by an estrogen-dependent mechanism. The objective of this study was to examine the possibility that the protective effect observed in female mice depends on the up-regulation and/or activation of endothelial cell NO synthase (eNOS). Anesthetized female and male wild-type or eNOS-deficient C57BL/6 mice were subjected to 70% liver ischemia for 45 min followed by resection of the remaining 30% nonischemic lobes and reperfusion of ischemic tissue. Survival was monitored daily, whereas liver injury was quantified by using serum alanine aminotransferase determinations and histopathology. Hepatic eNOS mRNA, protein, and enzymatic activity were determined in male and female mice subjected to RSL+I/R. We found that liver injury was reduced and survival increased in female mice compared with males. This protective effect correlated with significant increases in hepatic eNOS message levels and enzyme activity but not protein expression compared with males subjected to the surgery. Furthermore, N(omega)-nitro-L-arginine methyl ester-treated or eNOS-deficient female mice responded to RSL+I/R with dramatic increases in liver injury and 100% mortality within 2 days of surgery. Finally, we found that pravastatin pretreatment significantly attenuated hepatocellular injury and increased survival of male mice, which was associated with enhanced expression of eNOS message. We conclude that the protective effect afforded female mice is due to the activation of hepatic eNOS activity and enhanced NO production.     

Preconditioning by inhaled nitric oxide prevents hyperoxic and ischemia/reperfusion injury in rat lungs

Since the generation of nitric oxide (NO) is an essential step in the trigger phase of ischemic preconditioning, short-term inhalation of NO before ischemia should ameliorate ischemia/reperfusion (I/R) injury of the lung. We tested this hypothesis in high oxygen (>99%) ventilated rats in order to additionally evaluate compatibility of NO and exposure to hyperoxia. Male adult Sprague-Dawley rats inhaled NO (15 ppm, 10 min) before the left lung hilum was clamped for 1 h, and the reperfusion phase was observed for 4 h (NO group). Animals in the I/R group underwent the same treatment, but without NO inhalation. A third group without I/R served as time-matched controls. Animals in the I/R group showed severe I/R injury in terms of arterial pO2 (apO2), which was reduced to 22% of surgical controls (SCs) at time point 30 min reperfusion, and increased endothelial permeability (Evans blue procedure). The pretreatment with NO attenuated these effects. The pO2 after 4 h reperfusion was still 3.0-fold higher in the NO group compared to I/R. In contrast, the I/R- and hyperoxia-induced invasion of leukocytes, as determined by measuring myeloperoxidase (MPO) activity, was not affected by NO. These data were correlated with the activity of major cellular signaling pathways by measuring the phosphorylation at activating and inhibitory sites of extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, protein kinase B (AKT), and glycogen synthase kinase 3beta (GSK-3beta), and by determination of cGMP in plasma and lung tissue. Inhalation of NO partly prevented the loss of activation by I/R and hyperoxic ventilation of ERK, JNK, and AKT, and it reduced the I/R-induced activation of GSK-3beta. The level of cGMP in plasma and lung tissue was increased in the NO group after 4 h reperfusion. In conclusion, application of inhaled NO in the preconditioning mode prevented I/R injury in the rat lung without interfering effects of hyperoxic ventilation. The effects of NO on cellular signaling pathways resemble mechanisms of ischemic preconditioning, but further studies have to evaluate the physiological relevance of these results.     

Role of interleukin 18 in acute lung inflammation induced by gut ischemia reperfusion

AIM: To study the changes of endogenous interleukin 18 (IL-18) levels and evaluate the role of IL-18 on lung injury following gut ischemia/reperfusion. METHODS: A superior mesenteric artery occlusion model was selected for this research. The mice were randomly divided into four groups: Sham operation (sham), ischemia (0.5 h) followed by different times of reperfusion (I/R), and I/R pretreated with exogenous IL-18 (I/R+IL-18) or IL-18 neutralizing antibody (I/R+IL-18Ab) 15 min before ischemia. Serum IL-18 levels were detected by Western blot and ELISA, and the levels of IL-18 in lung tissue were evaluated by immunohistochemical staining. For the study of pulmonary inflammation, the lung myeloperoxidase (MPO) contents and morphological changes were evaluated. RESULTS: Gut ischemia/reperfusion induced rapid increase of serum IL-18 levels, peaked at 1 h after reperfusion and then declined. The levels of IL-18 in lung tissue were gradually enhanced as the progress of reperfusion. Compared with I/R group, exogenous administration of IL-18 (I/R+IL-18) further remarkably enhanced the pulmonary MPO activity and inflammatory cell infiltration, and in I/R+IL-18Ab group, the content of MPO were significantly reduced and lung inflammation was also decreased. CONCLUSION: Gut ischemia/reperfusion induces the increase of IL-18 expression, which may make IL-18 act as an important proinflammatory cytokine and contribute to gut ischemia/reperfusion-induced lung inflammation.     

Role of interleukin 18 in acute lung inflammation induced by gut ischemia repeifusion

AIM: To study the changes of endogenous interleukin 18 (IL-18) levels and evaluate the role of IL-18 on lung injury following gut ischemia/reperfusion. METHODS: A superior mesenteric artery occlusion model was selected for this research. The mice were randomly divided into four groups: Sham operation (sham), ischemia (0.5 h) followed by different times of reperfusion (I/R), and I/R pretreated with exogenous IL-18 (I/R+IL-18) or IL-18 neutralizing antibody (I/R+IL-18Ab) 15 min before ischemia. Serum IL-18 levels were detected by Western blot and ELISA, and the levels of IL-18 in lung tissue were evaluated by immunohistochemical staining. For the study of pulmonary inflammation, the lung myeloperoxidase (MPO) contents and morphological changes were evaluated. RESULTS: Gut ischemia/reperfusion induced rapid increase of serum IL-18 levels, peaked at 1 h after reperfusion and then declined. The levels of IL-18 in lung tissue were gradually enhanced as the progress of reperfusion. Compared with I/R group, exogenous administration of IL-18 (I/R+IL-18) further remarkably enhanced the pulmonary MPO activity and inflammatory cell infiltration, and in I/R+IL-18Ab group, the content of MPO were significantly reduced and lung inflammation was also decreased. CONCLUSION: Gut ischemia/reperfusion induces the increase of IL-18 expression, which may make IL-18 act as an important proinfiammatory cytokine and contribute to gut ischemia/reperfusion-induced lung inflammation.     

Ginkgo biloba extract （EGb 761） attenuates lung injury induced by intestinal ischemia/reperfusion in rats： Roles of oxidative stress and nitric oxide

AIM： To investigate the effect of ginkgo biloba extract （EGb 761） on lung injury induced by intestinal ischemia/ reperfusion （ Ⅱ/R）. METHODS： The rat model of Ⅱ/R injury was produced by damping the superior mesenteric artery for 60 min followed by reperfusion for 180 min. The rats were randomly allocated into sham, Ⅱ/R, and EGb ＋Ⅱ/R groups. In EGb ＋Ⅱ/R group, EGb 761 （100 mg/kg per day） was given via a gastric tube for 7 consecutive days prior to surgery. Rats in Ⅱ/R and sham groups were treated with equal volumes of the vehicle of EGb 761. Lung injury was assessed by light microscopy, wet-todry lung weight ratio （W/D） and pulmonary permeability index （PPT）. The levels of malondialdehyde （MDA） and nitrite/nitrate （NO2/NO3）, as well as the activities of superoxide dismutase （SOD） and myeloperoxidase （MPO） were examined. Western blot was used to determine the expression of inducible nitric oxide synthase （iNOS）. RESULTS： EGb 761 markedly improved mean arterial pressure and attenuated lung injury, manifested by the improvement of histological changes and significant decreases of pulmonary W/D and PPT （P 〈 0.05 or 0.01）.Moreover, EGb 761 markedly increased SOD activity, reduced MDA levels and MPO activity, and suppressed NO generation accompanied by down-regulation of iNOS expression （P 〈 0.05 or 0.01）. CONCLUSION： The results indicate that EGb 761 has a protective effect on lung injury induced by Ⅱ /R, which may be related to its antioxidant property and suppressions of neutrophil accumulation and iNOS- induced NO generation. EGb 761 seems to be an effective therapeutic agent for critically ill patients with respiratory failure related to Ⅱ/R.     

葛根素对大鼠心肌缺血再灌注后热休克蛋白70表达的影响

目的探讨葛根素对大鼠心肌缺血再灌注后热休克蛋白70(HSP70)表达的影响。方法结扎大鼠左冠状动脉前降支建立心肌缺血/再灌注模型,运用免疫组化法观察心肌细胞HSP70表达情况及血清髓过氧化物酶(MPO)活性。结果与对照组相比,葛根素组于心肌缺血/再灌注后0.5、4、8h时间点显著上调HSP70表达(P<0.01),抑制血清MPO活性(P<0.01)。结论葛根素可明显减轻心肌缺血再灌注损伤,其心肌保护机制可能是通过上调HSP70的表达、降低血清MPO的活性来实现的。     

The influence of iloprost on acute lung injury induced by hind limb ischemia-reperfusion in rats

The local ischemia-reperfusion (I/R) process gains a systemic nature and affects distal organs. The remote effects of I/R are most frequently observed in the lungs and pulmonary damage may vary from acute lung injury with mild dysfunction to severe respiratory failure or the acute respiratory distress syndrome. In this hind limb I/R induced experimental lung injury model two groups of rats as IR and ILO were determined. Both groups underwent 60 min of ischemia and 120 min of reperfusion. While ILO group received iloprost in saline, IR group received only saline before reperfusion period intravenously. Serum myeloperoxidase (MPO) activity, malondialdehyde (MDA) levels and total antioxidant capacity (TAC) and lung tissue MPO activity, MDA levels and Na+-K+ ATPase activity were measured and light microscopic analyses of lung specimens were performed. The MPO activities in serum and lung homogenates were found to be significantly decreased in ILO group (P < or = 0.01). The MDA levels in lung homogenates were found to be significantly decreased in ILO group (P < or = 0.01), but the decreases were not significant in serum MDA levels (P=0.052). Serum TAC and lung tissue Na+-K+ ATPase activity levels were found to be increased in ILO group compared to IR group (P < or = 0.01). Lung histology showed marked improvement by iloprost compared to the IR group in this study. Iloprost has been found to be effective in attenuating ischemia reperfusion-induced remote organ damage, in this case, lung injury, in rats.     

Protection of Veratrum nigrum L. var. ussuriense Nakai alkaloids against ischemia-reperfusion injury of the rat liver

AIM： TO investigate the protective effects and possible mechanisms of Veratrum nigrum L. var. ussuriense Nakai alkaloids （VnA） on hepatic ischemia/reperfusion （I/R） injury in rats.
METHODS： Forty male Wistar rats were randomly divided into four experimental groups （n = 10 in each）： （A） Control group （the sham operation group）; （8） I/R group （pretreated with normal saline）; （C） Small-dose （10 μg/kg） VnA pretreatment group; （D） Large-dose （20 μg/kg） VnA pretreatment group. Hepatic ischemia/ reperfusion （Hepatic I/R） was induced by occlusion of the portal vein and the hepatic artery for 90 min, followed by reperfusion for 240 min. The pretreatment groups were administered with VnA intraperitoneally, 30 min before surgery, while the control group and I/R group were given equal volumes of normal saline. Superoxide dismutase （SOD） activity, myeloperoxidase （MPO） activity and nitric oxide （NO） content in the liver tissue at the end of reperfusion were determined and liver function was measured. The expression of intercellular adhesion molecule-1 （ICAM-1） and E-selectin （ES） were detected by immunohistochemical examinations and Western blot analyses.
RESULTS： The results showed that hepatic I/R elicited a significant increase in the plasma levels of alanine aminotransferase （ALT： 74.53 ± 2.58 IU/L vs 1512.54 ± 200.76 IU/L, P 〈 0.01） and lactic dehydrogenase （LDH： 473.48 ± 52.17 IU/L vs 5821.53 ± 163.69 IU/L, P 〈 0.01）, as well as the levels of MPO （1.97 ± 0.11 U/g vs 2.57 ± 0.13 U/g, P 〈 0.01） and NO （69.37 ± 1.52 μmol/g protein vs 78.39 ± 2.28 μmol/g protein, P 〈 0.01） in the liver tissue, all of which were reduced by pretreatment with VnA, respectively （ALT： 1512.54 ± 200.76 IU/L vs 977.93 ± 89.62 IU/L, 909.81 ± 132.76 IU/L, P 〈 0.01, P 〈 0.01; LDH： 5821.53 ± 163.69 IU/L vs 3015.44 ± 253.01 IU/L, 2448.75 ± 169.4 IU/L, P 〈 0.01, P 〈 0.01; MPO： 2.57 ± 0.13 U/g vs 2.13 ± 0.13 U/g,     

Curcumin protects against hypertension aggravated retinal ischemia/reperfusion in a rat stroke model

The pathogenesis of visual dysfunction in stroke remains unclear. The objective of this study was to explore retinal damage in stroke spontaneously hypertensive rats (SHR) and evaluate the role of curcumin in the retinal injury after stroke. Mature male SHR were used as the animal model for hypertension and age-matched male Wistar-Kyoto (WKY) rats as the normotensive controls. The rat model of stroke was made by bilateral vertebral artery electrocoagulation combined with transient bilateral common carotid artery ligation. The animals were randomly divided into sham group, ischemia/reperfusion group, solvent control group, and curcumin treatment group. Each group was subdivided into 2 h, 6 h, 24 h, 72 h, and 7 day after reperfusion. Blood pressure was measured in SHR and WKY rats. Eye fundus was examined in living animals, and then, tissue specimens were collected for histologic examination, terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nick end labeling, and immunohistochemistry. Retinopathy, induced by I/R, was more serious in rats with hypertension than that in normotensive rats (retinal thickness index, p = 0.004). The number of apoptosis in retinal capillary cells and neurons reduced significantly in the curcumin-treated groups. Curcumin treatment inhibited phosphorylated c-Jun N-terminal kinase (JNK) expression in SHR after retinal I/R injury. Thus, hypertension aggravated retinal I/R injury after stroke. Curcumin, a specific inhibitor of JNK, can prevent the development of hypertensive retinopathy after I/R injury by inhibiting apoptosis in retinal capillary cells and neurons.     

U50,488H抑制中性粒细胞在大鼠缺血/再灌注心肌中的聚集和TNF-α生成

目的:观察К-阿片受体激活对大鼠心肌缺血/再灌注(MI/R)局部心肌中性粒细胞聚集的影响及其相关机制。方法:将60只成年健康SD大鼠随机分为假手术组、缺血/再灌注(I/R)生理盐水组、К-阿片受体激动剂(U50,488H)组、U50,488H+Nor-BNI组、U50,488H+Wortmannin组及U50,488H+L-精氨酸甲酯(L-NAME)组,每组9~10只大鼠。使心肌局部缺血30 min再灌注3 h后处死动物,检测大鼠心梗面积并按相应试剂盒提供方法检测血清肌酸激酶(CK)、心肌中髓过氧化物酶(MPO)活性、一氧化氮(NO)及心肌和血清中肿瘤坏死因子-α(TNF-α)的水平。结果:与I/R生理盐水组相比较,U50,488H组的心梗面积(P0.05)、血清CK活性(P0.05)、心肌MPO(P0.05)及心肌和血清TNF-α(P0.05)的水平明显下降,同时心肌中NO(P0.05)的水平上升;而К-阿片受体的选择性阻断剂(Nor-BNI)、PI3K的选择性抑制剂(Wortmannin)及NO合成酶(NOS)抑制剂(L-NAME)均可消除U50,488H的上述作用(P0.05)。结论:U50,488H可通过激活К-阿片受体,发挥对心脏的保护作用。U50,488H的保护作用可能与其激活PI3-激酶,增加心肌中NO的合成,减少中性粒细胞向I/R损伤心肌的聚集和MI/R诱导的TNF-α生成有关。     

Prophylaxis with carnosol attenuates liver injury induced by intestinal ischemia/reperfusion

AIM： To investigate the possible protective effects of carnosol on liver injury induced by intestinal ischemia reperfusion （I/R）.
METHODS： Rats were divided randomly into three experimental groups： sham, intestinal I/R and carnosol treatment （n = 18 each）. The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h. In the carnosol treatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg carnosol 1 h before the operation. At 2, 4 and 6 h after reperfusion, rats were killed and blood, intestine and liver tissue samples were obtained. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase （AST）, alanine aminotransferase （ALT） and interleukin （IL）-6 were measured. Liver tissue superoxide dismutase （SOD） and myeloperoxidase （IvIPO） activity were assayed. The liver intercellular adhesion molecule-1 （ICAM-1） and nuclear factor κB （NF-κB） were determined by immunohistochemical analysis and western blot analysis.
RESULTS： Intestinal I/R induced intestine and liver injury, characterized by histological changes, as well as a significant increase in serum AST and ALT levels. The activity of SOD in the liver tissue decreased after I/R, which was enhanced by carnosol pretreatment. In addition, compared with the control group, carnosol markedly reduced liver tissue MPO activity and serum IL-6 level, which was in parallel with the decreased level of liver ICAI-1 and NF-κB expression.
CONCLUSION： Our results indicate that carnosol pretreatment attenuates liver injury induced by intestinal I/R, attributable to the antioxidant effect and inhibition of the NF-κB pathway.     

Amelioration of hepatic ischemia/reperfusion injury in the remnant liver after partial hepatectomy in rats

BACKGROUND AND AIMS: Reactive oxygen species have been implicated in the development of hepatic ischemia/reperfusion (I/R) injury. I/R injury remains an important problem in massive hepatectomy and organ transplantation. The aim of this study was to examine the effect of edaravone, a newly synthesized free radical scavenger, on I/R injury in the remnant liver after partial hepatectomy in rats. METHODS: Partial (70%) hepatic ischemia was induced in rats by occluding the hepatic artery, portal vein, and bile duct to left and median lobes of liver. Total hepatic ischemia (Pringle maneuver) was induced by occluding the hepatoduodenal ligament. Edaravone was intravenously administered to rats just before reperfusion and partial (70%) hepatectomy was performed just after reperfusion. RESULTS: Edaravone significantly reduced the increases in the levels of serum alanine aminotransferase and aspartate aminotransferase in rats with liver injury induced by 90-min of partial ischemia followed by 120-min of reperfusion. Histopathological analysis showed that edaravone prevented inflammatory changes in the livers with I/R injury. Edaravone also decreased the levels of myeloperoxidase activity, which is an index of neutrophil infiltration, and interleukin-6 mRNA, which is a proinflammatory cytokine. Additionally, edaravone improved the survival rate in partial hepatectomy rats with I/R injury induced by the Pringle maneuver. CONCLUSIONS: Edaravone administration prior to reperfusion protected the liver against I/R injury. Edaravone also improved the function of the remnant liver with I/R injury after partial hepatectomy. Therefore, edaravone may have applicability for major hepatectomy and liver transplantation in the clinical setting.     

Japanese herbal medicine, Saiko-keishi-to, prevents gutischemia／reperfusion-induced liver injurv in rats via nitric oxide

AIM: To determine whether Saiko-keishi-to (TJ-10), a Japanese herbal medicine, could protect liver injury induced by gut ischemia/reperfusion (I/R), and to investigate the role of NO. METHODS: Male Wistar rats were exposed to 30-min gut ischemia followed by 60 min of reperfusion. Intravital microscopy was used to monitor leukocyte recruitment. Plasma tumor necrosis factor (TNF) levels and alanine aminotransferase (ALT) activities were measured. TJ-10 1 g/(kg.d) was intragastrically administered to rats for 7 d. A NO synthase inhibitor was administered. RESULTS: In control rats, gut I/R elicited increases in the number of stationary leukocytes, and plasma TNF levels and ALT activities were mitigated by pretreatment with TJ-10. Pretreatment with the NO synthase inhibitor diminished the protective effects of TJ-10 on leukostasis in the liver, and the increase of plasma TNF levels and ALT activities. Pretreatment with TJ-10 increased plasma nitrite/nitrate levels. CONCLUSION: TJ-10 attenuates the gut I/R-induced hepatic microvascular dysfunction and sequential hepatocellular injury via enhancement of NO production.     

姜黄素后处理通过JAK2/STAT3信号通路拮抗心肌缺血/再灌注损伤

目的:观察姜黄素（curcumin,Cur）后处理对离体心肌缺血/再灌注损伤(ischemia and reperfusion injury,I/RI)的拮抗作用及其可能的机制。方法: 40只SD大鼠随机分为5组(n=8),即空白对照(Ctl组)、缺血/再灌注组(I/R组)、I/R+姜黄素后处理组(I/R+Cur组)、I/R+姜黄素后处理+AG490组(I/R+Cur+AG组)及AG490组（AG组）,AG490为JAK2/STAT3信号通路特异性阻断剂。采用离体大鼠心脏Langendoff逆行灌注模型,缺血60 min,再灌注60 min。分别于缺血前及再灌注后15 min、30 min、45 min和60 min,测定血流动力学各项指标,包括:心率（HR）、左心室发展压（LVDP）、左心室内压力上升的最大速率(+dp/dtmax)、冠脉流量（CF）、计算出心率压力指数（DP,LVDP×HR/1000）。用氯化三苯基四氮唑(TTC)染色法测定各组心肌梗死(MI)的面积,用Western blot检测各组JAK2、磷酸化JAK2(p-JAK2)、STAT3以及磷酸化STAT3(p-STAT3)的表达。结果: 与Ctl组比较,I/R组各时间点的收缩及舒张功能均显著降低(P<0.01),MI面积显著增加(P<0.01),磷酸化JAK2和STAT3的表达明显升高(P<0.01)。与I/R组比较,I/R+Cur组各时间点的心肌收缩及舒张功能下降程度明显减轻(P<0.01),MI的面积明显减少(P<0.01),p-JAK2和STAT3的表达更高(P<0.01)。与I/R+Cur组相比,I/R+Cur+AG组各时间点的收缩及舒张功能显著降低(P<0.01),MI的面积显著增加(P<0.01),p-JAK2和STAT3的表达显著下降(P<0.01);和Ctl组相比,AG组血流动力学和MI的面积无统计学差异。结论: Cur后处理对I/R大鼠心肌具有保护作用,其作用机制与JAK2/STAT3信号通路的活化有关。     

硝酸甘油耐受加重缺血再灌注所引起的主动脉损伤及其机制的实验研究

目的研究硝酸甘油耐受对缺血再灌注动脉损伤的影响及其机制。方法雄性SD大鼠尾静脉给予硝酸甘油(60μg·kg-1·h-1)或生理盐水12 h,测量平均动脉压和离体血管张力以确定硝酸甘油耐受是否产生。耐受大鼠的离体动脉随机接受以下处理:(1)模拟缺血再灌注,(2)模拟缺血再灌注+还原型谷胱甘肽(GSH,终浓度0.1 mmol/L),(3)对照处理(n=16-18)。未产生耐受的大鼠随机接受模拟缺血再灌注或对照处理(n=16-18)。为了确定硝酸甘油耐受对缺血再灌注血管损伤和内皮功能的影响,检测各组再灌注液中一氧化氮含量、肌酸激酶(CK)和乳酸脱氢酶(LDH)活性,并观测血管收缩功能和对乙酰胆碱的反应。免疫组化标记血管中的硝基化酪氨酸(过氧亚硝基阴离子标志物),灰度扫描分析标记强度。结果与其他各组相比,接受模拟缺血再灌注处理的硝酸甘油耐受血管内皮功能(乙酰胆碱舒张反应和合成一氧化氮能力)显著下降,组织损伤明显增加(CK、LDH活性增加,血管收缩功能降低),血管内硝基化酪氨酸含量显著升高。GSH显著抑制了硝酸甘油耐受的血管损伤作用。结论实验结果显示硝酸甘油耐受显著增加了缺血再灌注诱导的血管损伤,这种作用可能是过氧亚硝基阴离子介导的。此外,我们的结果还表明GSH不仅降低了硝酸甘油耐受的血管损伤作用,而且抑制了血管内过氧亚硝基阴离子的形成。     

Modulation of liver oxidant-antioxidant system by ischemic preconditioning during ischemia／reperfusion injury in rats

AIM: To investigate effects of ischemic pre-conditioning on the liver endogenous oxidant-antioxidant system during ischemia/reperfusion injury. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into sham-operated (Sham), ischemia/ reperfusion (I/R), ischemic pre-conditioning plus ischemia/ reperfusion (IPC) groups. Serum ALT, AST and hyaluronic acid levels were assayed and pathologic alterations observed. Liver malondialdehyde (MDA) contents, endogenous antioxidant enzymes, superoxidase dismutase (SOD), catalase (CAT), gultathionine peroxidase (GSH-Px) activities, neutrophils accumulation marker, myeloperoxidase (MPO) activities were measured respectively. RESULTS: Compared with I/R group, sinusoidal endothelial cells as well as hepatocytes damages, as assessed biochemically and histochemically, were improved significantly in IPC group; neutrophils infiltration was also markedly reduced. In IPC group, liver peroxidation, as measured by MDA contents, was significantly decreased when compared with I/R group; endogenous antioxidant enzymes, SOD, CAT and GSH-Px activities were markedly higher than that in I/R group. CONCLUSION: Ischemic pre-conditioning exerts protective effects on both hepatic sinusoidal endothelial cells and hepatocytes during liver I/R injury. Its mechanisms may involve dimunition of neutrophils infiltration and modulation of the imbalance of endogenous oxidant-antioxidant system in the organism.     

姜黄素对大鼠肝缺血再灌注早期肝组织一氧化氮表达的影响

目的:探讨姜黄素对大鼠肝脏缺血再灌注早期损伤微循环的影响.方法:将大鼠随机分为假手术组、对照组和实验组(姜黄素40 mg/kg,2次给药).通过检测再灌注早期1、3 h血清转氨酶水平、肝组织中一氧化氮(nitricoxide,NO)、一氧化氮合酶(nitricoxide synthase,NOS),诱导型一氧化氮合酶(inducible nitricoxide synthase,iNOS)mRNA及内皮型一氧化氮合酶(endothelium nitricoxide synthase,eNOS)mRNA水平,以及肝组织病理学检查来评价姜黄素对大鼠肝脏缺血再灌注早期损伤微循环的影响.结果:相对于对照组,姜黄素可降低大鼠肝脏缺血再灌注早期损伤1、3 h血清谷丙转氨酶(ALT)的水平(603.8 U/L±64.5 U/L vs 758.1 U/L±114.7U/L,837.1 U/L±33.3 U/L vs 1012.7 U/L±119.8 U/L,均P<0.01)和谷草转氨酶(AST)的水平(605.7 U/L±65.7 U/L vs 779.5 U/L±124.3 U/L,849.6 U/L±36.0 U/L vs 1027.8 U/L±139.8 U/L,均P<0.01);改善肝组织病理学损害;减少肝脏缺血再灌注早期损伤1、3 h肝组织由iNOS产生的NO蛋白水平(0.455±0.056 vs 0.594±0.087.0.492±0.040 vs 0.671±0.079,均P<0.01);降低肝脏缺血再灌注早期损伤1、3 h肝组织iNOS mRNA的表达强度(0.426±0.075 vs 0.569±0.073,0.527±0.066vs 0.702±0.089,均P<0.01).结论:姜黄素可通过减轻肝组织中由iNOS产生的NO生成,来改善肝缺血再灌注早期损伤中微循环的紊乱,从而减少对肝缺血再灌注肝实质细胞的损伤.     

Low-dose ethanol attenuates gut ischemia/reperfusion-induced liver injury in rats via nitric oxide production

BACKGROUND AND AIMS: The acute administration of low-dose ethanol was demonstrated to attenuate liver injury elicited by gut ischemia/reperfusion (I/R). Nitric oxide (NO) has been found to be a modulator of adhesive interactions between leukocytes, platelets, and endothelial cells, but there has been much controversy about the effects of ethanol on NO regulation. The objective of this study was to investigate the role of NO in ethanol-reduced hepatic microvascular dysfunction elicited by gut I/R. METHODS: Male Wistar rats were exposed to 30 min of gut ischemia followed by 60 min of reperfusion. Intravital microscopy was used to monitor leukocyte recruitment and non-perfused sinusoids (NPS). Plasma alanine aminotransferase (ALT) activities were measured 6 h after the onset of reperfusion. In another set of experiments, ethanol (10%, 1 g/kg) was administered before ischemia. RESULTS: Gut I/R elicited increases in the number of stationary leukocytes, NPS, and plasma ALT activities; all of which were attenuated by pretreatment with ethanol or an NO donor. Gut I/R caused the apoptosis of hepatocytes, which was prevented by pretreatment with ethanol. Pretreatment with an NO synthase inhibitor diminished the protective effects of ethanol. The administration of ethanol increased plasma nitrite/nitrate levels. CONCLUSION: These results suggest that low-dose ethanol attenuates the gut I/R-induced hepatic microvascular dysfunction and sequential liver injury by increasing sinusoidal NO levels.