Mechanisms of hepatic ischemia-reperfusion injury and protective effects of nitric oxide

Hepatic ischemia-reperfusion injury(IRI) is a patho-physiological event post liver surgery or transplantation and significantly influences the prognosis of liver func-tion. The mechanisms of IRI remain unclear, and effec-tive methods are lacking for the prevention and therapy of IRI. Several factors/pathways have been implicated in the hepatic IRI process, including anaerobic metabo-lism, mitochondria, oxidative stress, intracellular cal-cium overload, liver Kupffer cells and neutrophils, and cytokines and chemokines. The role of nitric oxide(NO)in protecting against liver IRI has recently been report-ed. NO has been found to attenuate liver IRI through various mechanisms including reducing hepatocellular apoptosis, decreasing oxidative stress and leukocyte adhesion, increasing microcirculatory flow, and enhanc-ing mitochondrial function. The purpose of this review is to provide insights into the mechanisms of liver IRI, indicating the potential protective factors/pathways that may help to improve therapeutic regimens for control-ling hepatic IRI during liver surgery, and the potential therapeutic role of NO in liver IRI.     

Combined exogenous surfactant and inhaled nitric oxide therapy for lung ischemia-reperfusion injury in minipigs

BACKGROUND: The combined application of exogenous surfactant and inhaled nitric oxide was evaluated for prevention of ischemia-reperfusion injury of the lung. METHODS: Left lungs were selectively perfused in 18 minipigs in situ with cold preservation solution. After 90 min of warm ischemia, the lungs were reperfused and the right pulmonary artery and bronchus were ligated (control group, n=6). Exogenous surfactant was instilled via bronchoscopy during ischemia (surfactant group, n=6). In a third group, surfactant was applied, followed by administration of inhaled nitric oxide (surfactant+NO group, n=6). Hemodynamic and respiratory parameters were recorded for 7 hr, and bronchoalveolar lavage fluid (BALF) was obtained before and after reperfusion for measurement of surface tension, small aggregate/large aggregate ratio, protein and phospholipid contents, and a differential cell count. RESULTS: Control group animals survived for 3.7+/-1.4 hr. In both surfactant-treated groups, five out of six animals survived the observation period (P<0.001). Dynamic compliance of the lung was decreased in control animals (P<0.001). In the surfactant+NO group, arterial PO2 was higher than in both other groups (P<0.001). BALF cell count and histology showed reduced neutrophil infiltration in surfactant+NO-treated lungs. Surface tension assessed in BALF with a pulsating bubble surfactometer was severely impaired in control animals (gammamin, 14.82+/-9.95 mN/m), but maintained in surfactant-treated (gammamin, 1.11+/-0.56 mN/m) and surfactant+NO-treated animals (gammamin, 3.90+/-2.35 mN/m, P=0.02). CONCLUSIONS: Administration of exogenous surfactant in lung reperfusion injury results in improved lung compliance. The addition of inhaled NO improves arterial oxygenation and reduces neutrophil extravasation compared with surfactant treatment alone.     

一氧化氮在大鼠小肠移植缺血再灌注损伤和急性排斥反应中的作用

目的 探讨一氧化氮(NO)在大鼠小肠移植缺血再灌注损伤(IRI)和急性排斥反应(AR)中作用.方法 建立同种大鼠原位小肠移植模型,采用随机数字表法将受鼠分为4组.移植对照组、左旋精氨酸(L-Arg)组、左旋硝基精氨酸甲酯(L-NAME)Ⅰ组(Ⅰ组)和L-NAMEⅡ组(Ⅱ组)受鼠于手术当天开始分别每天给予生理盐水、L-Arg 150 mg·kg-1 ·d-1、L-NAME 4和8 mg·kg-1·d-1.术后观察各组受鼠的存活时间,行HE染色观察移植小肠的组织病理学改变,采用免疫组织化学法观察移植小肠一氧化氮合酶(NOS)的活性,以及检测血糖吸收功能和血清NO浓度.结果 移植对照组、L-Arg组、Ⅰ组及Ⅱ组受鼠的存活时间分别为(11.7±1.2)d、(10.2±1.0)d、(12.3±1.5)d和(17.3±1.9)d,Ⅱ组受鼠的存活时间明显延长(P＜0.01).与移植对照组相比,L-Arg组和Ⅰ组IRI的Park评分下降,IRI减轻;Ⅱ组Park评分显著升高(P＜0.01),IRI加重,但AR明显减轻.与移植对照组相比,IRI期间,Ⅰ组iNOS染色减弱,Ⅱ组iNOS和nNOS染色均减弱;AR期间,Ⅱ组iNOS染色明显减弱.各组血清NO浓度于再灌注后30min逐渐升高.与移植对照组相比,Ⅱ组血 NO浓度的升高延缓.与移植对照组相比,L-Arg组血糖吸收值于再灌注30 min至术后3d明显增高(P＜0.01);Ⅰ组和Ⅱ组血糖吸收值术后处于较低水平.结论 NO在大鼠小肠移植IRI中起到了细胞毒和细胞保护的双重作用;在AR中加重了组织损伤.术后早期补充L-Arg可促进移植肠管对糖类的吸收.     

Differential nitric oxide synthase expression during hepatic ischemia-reperfusion

BACKGROUND: In recent years the important role of nitric oxide in hepatic ischemia-reperfusion injury has been increasingly recognised. The prevailing consensus is that reperfusion injury may be partly the result of decreased production of nitric oxide from endothelial nitric oxide synthase and excessive production of nitric oxide from the inducible isoform. We therefore undertook this study to characterize the expression of different nitric oxide synthase isoforms during hepatic reperfusion. METHODS: Male Wistar rats (n = 6) were subjected to 45 minutes of partial hepatic ischemia (left lateral and median lobes) followed by 6 hours of reperfusion. Control animals (n = 6) were subjected to sham laparotomy. The expression of endothelial and inducible nitric oxide synthase was examined using immunohistochemistry and Western blotting. Liver sections were also stained with nitrotyrosine antibody, a specific marker of protein damage induced by peroxynitrite (a highly reactive free radical formed from nitric oxide). RESULTS: Liver sections from all the control animals showed normal expression of the endothelial isoform and no expression of inducible nitric oxide synthase. Livers from all the animals subjected to hepatic ischemia showed decreased expression of endothelial nitric oxide synthase, and all but one animal from this group showed expression of the inducible isoform both in inflammatory cells and in hepatocytes. Western blotting confirmed these findings. Staining with the antinitrotyrosine antibody was also confined to five liver sections from animals subjected to hepatic ischemia. CONCLUSIONS: During the reperfusion period after hepatic ischemia, endothelial nitric oxide synthase is downregulated while inducible nitric oxide synthase is expressed in both hepatocytes and inflammatory cells. The presence of nitrotyrosine in livers subjected to hepatic ischemia-reperfusion suggests that the expression of inducible nitric oxide synthase plays an important role in mediating reperfusion injury in this model.     

The effect of nitric oxide in testicular ischemia-reperfusion injury

This experiment was carried out to investigate the effect of endogenous nitric oxide (NO) on the ischemia-reperfusion injury of testis.Testicular ischemia was achieved by twisting the right testis and spermatic cord 1080 counter-clockwise for 30 minutes and reperfusion was allowed for 30 minutes after detorsion of 33 rats. Animals were treated with normal saline in controls just before detorsion,NG-nitro-L-arginine methyl ester (L-NAME), and L-arginine (L-arg) in others.The tissue damage was evaluated with light microscopy, malondialdehyde (MDA) level in tissue, and the blood flow measurement using ¹³³xenon (Xe) clearence technique.MDA indicator of reperfusion injury increased 25% after detorsion when only normal saline was given, L-NAME further increased MDA, L-arginin decreased MDA to control level.Conclusion: L-arginin infusion during the detorsion reduced the reperfusion injury of testis and improved the testicular blood flow after the detorsion.     

The role of nitric oxide in testicular ischemia-reperfusion injury

PURPOSE: This study was designed to determine the role of nitric oxide (NO) in the ischemia-reperfusion (I/R) injury process in testes. METHODS: Fifty prepubertal male rats were divided into 5 groups each containing 10 rats. After 4-hour torsion and 4-hour detorsion, bilateral orchiectomies were performed for measurement of tissue malondialdehyde (MDA) level and histopathologic examination. The results were compared statistically. The groups were labeled as group 1, basal values of biochemical parameters in testes; group 2 (control group), torsion plus detorsion; group 3, torsion plus N-monomethyl-L-arginine (L-NMMA) plus detorsion; group 4, torsion plus L-arginine plus detorsion; group 5, sham operation. RESULTS: The highest MDA values were determined in the L-arginin group in ipsilateral testes. Group 3 and group 4 were statistically different from control group. Histological examination showed that specimens from group 4 had a significantly (P < .05) greater histological injury than group 3, and contralateral testes showed normal testicular architecture in all groups. CONCLUSIONS: These results suggest that NO plays an important role in damaging the testis with I/R. Although inhibition of NO synthesis with L-NMMA significantly improves I/R injury in testes, enhancing NO production by providing excess of L-arginine increases such damage. In the early periods of detorsion, there is no damage to contralateral testes after unilateral testicular torsion.     

依布西林对大鼠无心跳供体肺的保护作用

目的 观察依布西林在大鼠无心跳供体(NHBD)肺保护中的作用.方法 将60只SD大白鼠随机分为A组:有心跳供体(HBD)组;B组:NHBD组;C组:NHBD+依布硒林(Ebselen)组.B组、C组供体处死后维持辅助呼吸,放置室温中30 min,再灌注低钾右旋糖苷(LPD)液.受体鼠行"原位左肺移植术".C组受体在肺移植前1 h给予Ebselen.结果 C组移植后肺顺应性为0.1740±0.0100,结扎右肺门后15、30 min动脉血氧分压分别为(93.97±5.94)、(92.30±6.57)mm-Hg,肺组织丙二醛(MDA)含量为(0.63±0.23)nmol/mg蛋白,肺组织能量代谢物总量为(821.51±29.70)mol/g,与B组比较差异均有统计学意义(P＜0.05).结论 给予受体一定浓度的Ebselen可改善NHBD肺保护作用.

Abstract：

Objective To evaluate the protective effect of ebselen on the rat lungs from non-heartbeating donors (NHBD). Methods Sixty Sprague-Dawley rats were randomly divided into 3 groups:group A, heart-beating donor; group B, NHBD with 30 min of warm ischemia time (WIT); group C, NHBD with 30 min of WIT and administration of ebselen. The donor lungs in groups B and C maintained ventilation at room temperature for 30 min after asystolia and then were flushed with LPD solution. The recipient rats underwent left lung transplantation. The recipients in group C were administered with ebselen 1 h before transplantation. Results All the recipients survived during the observation period. The pulmonary compliance of group C was 0. 1740 ±0. 0100. The PaO2 at 15 min and 30 min after the ligation of the right pulmonary hila was (93.97 ±5.94), (92. 30 ±6. 57) mmHg, respectively. Malondialdehyde (MDA) of the pulmonary tissue was (0. 63 ±0. 23) nmol/mg pro and the energy metabolism was (821.51 ±29.70)mol/g. The difference between group B and group C was significant (P ＜ 0. 05 ). Conclusion The administration of ebselen is a safe and effective treatment in the preservation of the rat lungs from NHBD.     

Milrinone-induced postconditioning reduces hepatic ischemia-reperfusion injury in rats: the roles of phosphatidylinositol 3-kinase and nitric oxide

Background

Ischemic postconditioning (PostC) protects the liver against ischemia-reperfusion (IR) injury. Milrinone, a phosphodiesterase 3 inhibitor, has been reported to exhibit preconditioning properties against hepatic IR injury; however, its PostC properties remain unknown. This study investigated whether milrinone has PostC properties against hepatic IR injury and the roles of phosphatidylinositol 3-kinase (PI3K) and nitric oxide synthase (NOS).

Materials and methods

Male Wistar rats were separated into six groups: (1) group S: animals that underwent sham operation without ischemia, (2) group C: ischemia followed by reperfusion with no other intervention, (3) group M: milrinone administered immediately after reperfusion, (4) group MW: wortmannin, a PI3K inhibitor, injected before milrinone administration, (5) group MN: l-NAME, a NOS inhibitor, injected before milrinone administration, and (6) group MD, milrinone administered 30 min after reperfusion. Except for group S, all groups underwent 1 h of warm ischemia of median and left lateral lobes, followed by 5 h of reperfusion. Biochemical liver function analysis and histologic examination were performed.

Results

Serum aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels, histologic damage scores, and apoptotic rate in group M were significantly lower than those in group C. The inhibition of PI3K or NOS prevented this protective effect. Milrinone administered 30 min after reperfusion did not show obvious protective effects.

Conclusions

Milrinone-induced PostC protects against hepatic IR injury when it is administered immediately after reperfusion, and PI3K and NOS may play an important role in this protective effect.     

一氧化氮对大鼠肝缺血-再灌注损伤中肝细胞凋亡的影响

目的探讨一氧化氮对肝缺血-再灌注损伤及肝细胞凋亡的影响。方法在一氧化氮合酶（NOS）增强剂和抑制剂条件下,观察大鼠肝缺血-再灌注后1、3、6、24h肝的血清门冬氨酸氨基转移酶（AST）和丙氨酸氨基转移酶（ALT）变化,同时进行肝细胞胞浆诱导型一氧化氮合酶（iNOS）表达及肝细胞凋亡的免疫组织化学分析。结果在再灌注6h点AST、ALT、肝细胞凋亡水平上,L-精氨酸（L-arg）组值分别为（341.88±111.24）IU/L、（311.75±139.41）IU/L和2.80±1.79,显著低于L-硝基精氨酸甲酯（L-NAME）组（2080.88±241.98）IU/L、（1153.00±110.92）IU/L和5.50±0.71（P值均＜0.05）。而L-arg组的肝细胞内iNOS表达灰度值152.07±3.46显著高于L-NAME组灰度值180.45±4.46（P＜0.05）。结论一氧化氮可减少肝缺血-再灌注损伤后肝酶的释放,抑制肝细胞的凋亡,改善肝缺血-再灌注损伤。     

Real-time monitoring of nitric oxide in ischemia-reperfusion rat kidney

In this study we attempted to clarify the release of nitric oxide (NO) and its role in the ischemia-reperfusion rat kidney. After right nephrectomy, male Wistar rats were divided into four groups: one sham operated and three groups who underwent ischemia (30 min) and reperfusion of the left renal artery. Thirty minutes prior to ischemia-reperfusion, two groups were injected intraperitoneally with 10 and 30 mg/kg of NG-nitro-L-arginine methylester (L-NAME). Real-time monitoring of blood flow and NO release in the rat kidney was measured with a laser Doppler flowmeter and an NO-selective electrode, respectively. Serum creatinine and blood urea nitrogen (BUN) levels were measured 1 and 7 days after the induction of ischemia-reperfusion. Clamping of the renal artery decreased blood flow to 1-5% of the basal level measured before clamping. After removal of the clip, the blood flow of the 30 mg/kg L-NAME rats was significantly lower than that of the controls. Immediately following the clipping of the renal artery, NO release rapidly increased. After removing the clip, NO release immediately returned to three-quarters of the basal level. Serum creatinine and BUN levels of the ischemia-reperfusion rats were slightly but not significantly higher and those of 30 mg L-NAME rats were significantly higher than those of the control or ischemia-reperfusion rats 1 day and 7 days after ischemia-reperfusion. Our data suggest that NO acts as a cytoprotective agent in ischemia-reperfusion injury of the rat kidney.     

Inhaled nitric oxide attenuates apoptosis in ischemia-reperfusion injury of the rabbit lung

Background

Lung ischemia-reperfusion injury occurs after lung transplantation and various clinical procedures. Recently, apoptosis was reported to be induced after ischemia-reperfusion. We investigated the effects of inhaled nitric oxide (NO) on lung ischemia-reperfusion and apoptosis after ischemia-reperfusion.

Methods

As a control group, the left pulmonary hilum of Japanese white rabbits (n = 10) was occluded for 120 minutes and reperfused for 120 minutes. In the inhaled NO group (n = 10), 20 parts per million nitric oxide was inhaled during reperfusion. The sham-operated group was ligated at the right hilum and perfused by the left lung only for 120 minutes. The mean pulmonary arterial pressures and Pao₂ were measured during reperfusion. The wet-to-dry weight ratio of the left lower lobe of the lung was calculated. The number of apoptotic cells was estimated using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) technique. The TUNEL staining for a time course study was done using 15 control animals that were killed by exsanguination at 15, 30, and 60 minutes after reperfusion.

Results

After 120 minutes of reperfusion, the mean pulmonary arterial pressures in the control group and in the inhaled NO group were 23.0 ± 3.2 mm Hg and 13.6 ± 2.4 mm Hg, respectively (p < 0.01). At the same time point, the Pao₂ in the control group and in the inhaled NO group were 46.1 ± 15.9 mm Hg and 88.1 ± 14.7 mm Hg, respectively (p < 0.01). The wet-to-dry weight ratios in the control group and in the inhaled NO group were 0.856 ± 0.024 and 0.808 ± 0.006, respectively (p < 0.01). Apoptotic cells appeared in the early phase of reperfusion (after 15 minutes' reperfusion). The number of apoptotic cells was significantly lower in the inhaled group than in the control group after 120 minutes' reperfusion (1.76% versus 2.87%, p < 0.01).

Conclusions

Our results suggest that the inhaled NO prevents lung ischemia-reperfusion injury and attenuates apoptosis after reperfusion in the rabbit lung.     

The role of nitric oxide pathway in the renal ischemia-reperfusion injury in rats

INTRODUCTION: Nitric oxide (NO), synthesized from L-arginine by the enzyme nitric oxide synthase (NOS), seems to play an ambiguous role during tissue ischemia-reperfusion injury. Our objective was to evaluate the effects of L-arginine, a NO donor, and N(G)-nitro-L-arginine-methylester (L-NAME), a NOS inhibitor, on oxidative stress, renal dysfunction, histologic alterations and surgical mortality rate induced by renal ischemia-reperfusion (RIR) in uninephrectomized rats. MATERIALS AND METHODS: One-hundred and ninety-seven Wistar rats were randomized into five experimental groups. Group 1: sham operation; group 2: right uninephrectomy (UNI); group 3: UNI + RIR in the contralateral kidney; group 4: UNI + L-NAME (20 mg/kg; intraperitoneally) + RIR; and group 5: UNI + L-arginine + RIR. The effect of the drugs was evaluated by lipid peroxidation measured by the renal malondialdehyde (MD) content and chemiluminescence (CL) levels, serum creatinine (Cr) levels, urinary volume, tubular necrosis and athrophy, inflammatory infiltrate, interstitial fibrosis as histologic evaluation and surgical mortality rate after the procedures. A P value less than 0.05 was considered significant. RESULTS: Right uninephrectomy did not alter the renal parameters. RIR increased Cr levels (at 24 and 96 h of reperfusion), index of lipid peroxidation (both MD and QL levels), and worsened the histologic aspects. Pretreatment with L-arginine reduced the kidney levels of QL when compared with the non-treated group (5574 +/- 909 vs. 13 660 +/- 1104 cps/mg of protein; P < 0.05) but increased the MD levels (0.97 +/- 0.24 vs. 0.79 +/- 0.06 nmol/mg of protein; P < 0.05). Moreover, L-arginine attenuated the increment of Cr levels, inflammatory infiltrate and tubular athrophy in rats subjected to RIR (P < 0.05). On the other hand, pretreatment with L-NAME increased both CL (17 482 +/- 4397 vs. 13 660 +/- 1104 cps/mg of protein; P < 0.05) and MD levels (1.16 +/- 0.11 vs. 0.79 +/- 0.06 nmol/mg of protein; P < 0.05). Furthermore, L-NAME worsened the renal dysfunction (P < 0.05) at 192 h after the RIR, and surgical mortality rates were similar (P > 0.05). CONCLUSION: L-arginine has a tendency to exert a beneficial effect on renal damage during RIR in rats. Moreover, L-NAME seems to worsen the renal damage by increasing the kidney-levels of CL and impairment of renal function probably due to reduction of NO production.     

Preventive influence of inhaled nitric oxide on lung ischemia-reperfusion injury

n = 9). The control group was not administered NO (group II, n = 8). Severe ischemia-reperfusion injury occurred as evidenced by hypoxia and lung edema. PaO₂ at 120 min after reperfusion was 325 ± 41 mmHg in group I and 40 ± 6 mmHg in group II. The pulmonary blood flow of the left lung at 120 min after reperfusion was 51% ± 3% in group I and 20% ± 5% in group II. The wet-to-dry weight ratio was 5.5 ± 0.2 for the right lungs, 5.8 ± 0.8 for the left lung in group I, and 6.1 ± 0.4 for the left lung in group II. Histopathologically, marked hemorrhage, hyaline membrane formation, and leukocyte infiltration were observed in group II but not in group I. These data suggested that inhaled NO reduced warm ischemia-reperfusion injury in the lung, and also contributed to a better preserved lung function. (Received for publication on July, 13, 1998; accepted on Mar. 11, 1999)     

短时长吸入一氧化氮预处理对大鼠肺缺血再灌注损伤的保护作用

目的 探讨低浓度一氧化氮(NO)预处理及其吸入时长的不同对肺缺血再灌注损伤(IRI)的影响和机制.方法 成年雄性SD大鼠77只,分为空白组(Sham组)、缺血再灌注组(I/R组)、NO预处理10 min组(NO-10 min组)、NO预处理1 min组(NO-1 min组),NO预处理60 min组(NO-60 min组),分别于不同的再灌注时间点采集标本,检测动脉血氧分压(PaO2),肺组织干/湿重比(W/D),丙二醛(MDA)浓度,髓过氧化酶(MPO)活性,肺损伤组织学评价等指标,比较前3组再灌注后2、6、24 h结果,取IRI最严重时间点,比较各组肺功能指标以及血清和左肺组织NO浓度.结果 再灌注6h肺损伤最严重;与I/R组比较,NO-10 min组各时间点指标明显改善(P＜0.05),NO吸入具有肺保护作用;NO-1 min组肺损伤无改善(P＞0.05);NO-10 min组与NO-60 min组对肺损伤保护作用相似(P＞0.05).结论 短时长吸入低浓度NO预处理可以改善IRI,但是其保护作用不与NO预处理时长成正比.     

诱导性一氧化氮合酶在大鼠肝缺血再灌注损伤中的作用

目的 探讨诱导性一氧化氮合酶（iNOS）在大鼠部分肝缺血再灌注损伤中的作用.方法 选取雄性Sprague-Dawley大鼠30只,体重225～250 g,随机分成氨基胍（AG）组、脂多糖（LPS）组、对照组.每组均按相同方法建立70%的肝缺血再灌注损伤模型（缺血1 h,再灌注6 h）,取再灌注大鼠肝组织及血清样本.氨基胍（AG）组（n=10）：术前30 min尾静脉注射AG 100 mg/kg（质量浓度10 kg/L）;脂多糖（LPS）组（n=10）：术前30 min尾静脉注射LPS 10 mg/kg（质量浓度1 kg/L）;对照组（n=10）：术前30 min尾静脉注射生理盐水（10 μL/kg）.检测血清谷氨酸转氨酶（ALT）水平,实时荧光定量PCR测定肝组织iNOS mRNA的表达,Western blot测定肝组织iNOS蛋白的表达,考马斯法测定肝组织匀浆丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性,HE染色光镜下组织学观察等.结果 AG组与对照组相比,肝组织中iNOS mRNA表达量明显下降（P〈0.05）,iNOS蛋白表达量明显下降（P〈0.05）;ALT和MDA明显下降（P〈0.05）;SOD明显升高（P〈0.05）;肝细胞水肿较轻,排列相对整齐.LPS组与对照组相比,肝组织中iNOS mRNA表达量明显升高（P〈0.05）,iNOS蛋白表达量明显升高（P〈0.01）;ALT和MDA明显升高（P〈0.05）;SOD则明显降低（P〈0.05）;肝细胞水肿,排列紊乱,并且出现水样变性.结论 iNOS 升高会加重缺血再灌注损伤,这一过程可能通过改变氧化还原状态实现.