Effect of nitric oxide on toll-like receptor 2 and 4 gene expression in rats with acute lung injury complicated by acute hemorrhage necrotizing pancreatitis

BACKGROUND: Toll-like receptor (TLR) 2/4 might play important roles in mediating proinflammatory cytokine synthesis and release. And nitric oxide (NO) has been used to treat acute respiratory distress syndrome (ARDS). This study aimed to investigate the changes in TLR2/4 gene expression in the lungs of rats with acute lung injury (ALI) complicated by acute hemorrhage necrotizing pancreatitis (AHNP) and the effect of NO on the TLR2/4 gene expression. METHODS: One hundred and ten SD male rats were randomly divided into sham-operated group ( n = 10) , AHNP group (n = 30) , chloroquine-treated group ( n = 30) , and L-Arg-treated group (n =40). The lungs were dissected for lung histological scoring, and bronchoalveolar lavages were harvested for lung injury indexing. TLR2/4 mRNA expression in the lungs was measured by RT-PCR. RESULTS: TLR2/4mRNA was detected in the lungs with low values in the sham-operated group (0.016±0. 210E-2, 0.112 ±0.750E-2) , but it was markedly increased at 3 hours in the AHNP group (0.787±0.751E-2, 1.512 ±1.794E-2) , peaking at 12 hours (1.113 ±6.141E-2, 2.957±2.620E-2; P <0.05 or P <0.01). When lung injuries were aggravated, TNF-α concentrations in the lungs were increased, but NO concentrations were decreased ( P < 0.05 or P < 0.01 ) . When TLR2/4mRNA was inhibited by CQ (3h: 0.313 ± 5.491E-2, 0.005 ±1.419E-3 ; 6h: 0.488 ±7.442E-2, 0.010 ± 1.518E-3; 12h: 0.883 ± 8.911E-2, 0.024 ± 2.760E-3; P< 0.05 or P <0.01) , lung injuries were relieved. NO concentrations in the lungs were increased but TNF-α concentrations were decreased (P <0. 05 or P <0.01). When the rats with AHNP were treated with L-Arg, TLR2/4mRNA expression in the lungs could be effectively inhibited (50mg-T: 0.656 ±3. 977E-2, 1. 501 ±6.111E-2; 100mg-T: 0.260± 0.891E-2, 0.732 ±5.135E-2; 200mg-T: 0.126 ±0.914E-2, 0.414 ± 1.678E-2; 400mg-T: 0.091 ±0.399E-2, 0.287 ± 0.176E-2; P <0.05 or P <0. 01) and lung injuries were relieved. At the same time, NO concentrations in the lungs were markedly increased, but TNF-α concentrations were decreased (P <0.05 or P <0.01). CONCLUSIONS: The expression of TLR2/4mRNA is increased in the lungs in rats with AHNP and lung injuries are aggravated. TLR2/4mRNA gene expression of the lungs of rats with AHNP could be markedly inhibited by NO, leading to the relief of lung injuries.

Research Progress on the Pathogenesis of Acute Lung Injury(ALI)

In this review,the databases searched were PubMed and Web of Science.It is believed that the main causes of acute lung injury(ALI)and acute respiratory distress syndrome(ARDS)are inflammatory response disorders,excessive oxidative stress,cell death,endoplasmic reticulum stress,coagulation dysfunction,and weakened aquaporin function.

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.

Effects of glucocorticoid dexamethasone on serum nitric oxide synthase activity and nitric oxide levels in a rat model of lung disease-induced brain injury

In this study, we investigated the effects of dexamethasone, pertussis toxin （a Gi protein inhibitor） and actinomycin （a transcription inhibitor） on serum nitric oxide synthase activity and nitric oxide content in a rat model of lung disease-induced brain injury. High-dose dexamethasone （13 mg/kg） and dexamethasone ＋ actinomycin reduced lung water content, increased serum nitric oxide synthase activity and nitric oxide content, diminished inflammatory cell infiltration in pulmonary alveolar interstitium, attenuated meningeal vascular hyperemia, reduced glial cell infiltration, and decreased cerebral edema. These results demonstrate that high-dose glucocorticoid treatment can reduce the severity of lung disease-induced brain injury by increasing nitric oxide synthase activity and nitric oxide levels.

Effects of n-butanol extract of Rumex gmelini Turcz on the endotoxin-induced acute lung injury in mice

Objective: To study the effects of n-butanol extract of Rumex gmelini Turcz on the endotoxin-induced acute lung injury (ALI) in mice. Methods: Kunming mice were selected as experimental animals and randomly divided into normal control group (NC group), acute lung injury group (ALI group) and n-butanol extract of Rumex gmelini Turcz group (Rum group). ALI group and Rum group were established into ALI models by intraperitoneal injection of endotoxin, and Rum group were given intragastric administration of n-butanol extract of Rumex gmelini Turcz for intervention before model establishment. 12 h after endotoxin injection, the superior lobe of right lung was taken to determine the water content, and the inferior lobe of right lung was taken to determine the contents of AQPs molecules, inflammatory response molecules and oxidative stress molecules. Results: 12 h after endotoxin injection, the water content of lung tissue in Rum group was (6.82±0.97)%. After variance analysis, the water content of lung tissue in ALI group was significantly higher than that in NC group, AQP1 and AQP5 protein levels in lung tissue were significantly lower than those of NC group, AQP3 and AQP4 protein levels were not different from those of NC group, and MPO, NF-kB, TNF-α, HMGB1, IL-8, ROS, ATP, MDA, Bax and Caspase-3 protein levels were significantly higher than those of NC group;the water content of lung tissue in Rum group was significantly lower than that in ALI group, AQP1 and AQP5 protein levels in lung tissue were significantly higher than those of ALI group, AQP3 and AQP4 protein levels were not different from those of ALI group, and MPO, NF-kB, TNF-α, HMGB1, IL-8, ROS, ATP, MDA, Bax and Caspase-3 protein levels were significantly lower than those of ALI group. Conclusion:The n-butanol extract of Rumex gmelini Turcz reduce the pulmonary edema, inflammatory response, oxidative stress and apoptosis during the endotoxin-induced ALI in mice.

α_1-ANTITRYPSIN ATTENUATES ENDOTOXIN-INDUCED ACUTE LUNG INJURY IN RABBITS

Objective To investigate whether pretreatment with α 1-AT can attenuate acute lung injury (ALI) in rabbits induced with endotoxin. Methods Thirty-two New Zealand rabbits were randomly assigned to four groups(n=8):1.Infusion of endotoxin(Lipopolysaccharide,LPS 500μg/kg)without α 1-AT (group LPS).2.Infusion α 1-AT 120mg/kg at 15min before challenge with LPS(group LAV).3.Infusion of α 1-AT 120mg/kg(group AAT).4 Infusion of saline 4ml/kg as control (group NS).Arterial blood gases,peripheral leukocyte counts and airway pressure were recorded every 1h.Physiologic intrapulmonary shunting (Qs/Qt) was measured every 4h.After 8h the bloods were collected for measurement of plasma concentration and activity of α 1-AT.Then bronchoalveolar lavage fluid (BALF)was collected for measurement of concentrations of total protein (TP),interleukin-8(IL-8),tumor necrosis factor(TNF-α),the activities of elastase-like and α 1-AT,total phospholipids(TPL) and disaturated phosphatidylcholine (DSPC).In addition,the wet-to-dry lung weight ratio(W/D) was measured. Results After infusion of endotoxin,it was observed that PaO 2,peripheral luekocyte counts,total respiratory compliance progressively decreased and P peak and Qs/Qt increased comparing with the baseline values.In contrast to group NS,the increased plasma concentration but reduced activity of α 1-AT was found in group LPS.In the BALF,the activity of α 1-AT,TPL,DSPC/TPL were lower,but the concentrations of albumin,IL-8,TNF-α,and the activity of NE were higher.The ratio of W/D also increased.The pretreatment of α 1-AT attenuated the deterioration of oxygenation,the reduction of compliance and the deterioration of other physiological,biochemical parameters mentioned above. Conclusion Pretreatment with α 1-AT could attenuate endotoxin-induced lung injury in rabbits.Those beneficial effects of α 1-AT might be due in part to the inhibitory effect on neutrophil elastase.

Acute respiratory distress syndrome and lung injury: Pathogenetic mechanism and therapeutic implication

To review possible mechanisms and therapeutics for acute lung injury(ALI) and acute respiratory distress syndrome(ARDS). ALI/ARDS causes high mortality. The risk factors include head injury, intracranial disorders, sepsis, infections and others. Investigations have indicated the detrimental role of nitric oxide(NO) through the inducible NO synthase(i NOS). The possible therapeutic regimen includes extracorporeal membrane oxygenation, prone position, fluid and hemodynamic management and permissive hypercapnic acidosis etc. Other pharmacological treatments are anti-inflammatory and/or antimicrobial agents, inhalation of NO, glucocorticoids, surfactant therapy and agents facilitating lung water resolution and ion transports. β-adrenergic agonists are able to accelerate lung fluid and ion removal and to stimulate surfactant secretion. In con-scious rats, regular exercise training alleviates the endotoxin-induced ALI. Propofol and N-acetylcysteine exert protective effect on the ALI induced by endotoxin. Insulin possesses anti-inflammatory effect. Pentobarbital is capable of reducing the endotoxin-induced ALI. In addition, nicotinamide or niacinamide abrogates the ALI caused by ischemia/reperfusion or endotoxemia. This review includes historical retrospective of ALI/ARDS, the neurogenic pulmonary edema due to head injury, the detrimental role of NO, the risk factors, and the possible pathogenetic mechanisms as well as therapeutic regimen for ALI/ARDS.

Acute lung injury and acute respiratory distress syndrome： experimental and clinical investigations

Acute lung injury （ALl） or acute respiratory distress syndrome （ARDS） can be associated with various disorders. Recent investigation has involved clinical studies in collaboration with clinical investigators and pathologists on the pathogenetic mechanisms of ALl or ARDS caused by various disorders. This literature review includes a brief historical retrospective of ALI/ARDS, the neurogenic pulmonary edema due to head injury, the long-term experimental studies and clinical investigations from our laboratory, the detrimental role of NO, the risk factors, and the possible pathogenetic mechanisms as well as therapeutic regimen for ALI/ARDS.

The Role of Neutrophil Collagenase in Endotoxic Acute Lung Injury

The aim of this study was to determine the role of neutrophil collagenase in the pathogenesis of acute lung injury induced by endotoxin. 28 Sprague-Dawley were randomized into control group and LPS-enduced groups. Samples of left lung were obtained in 2 h (group L 1), 6 h (group L 2), 12 h (group L 3) after intravenous LPS. Immunohistochemsitry was employed for detection of expression of neutrophil collagenase. Pathological scores, lung wet/dry weight ratio and the number of neutrophils were measured. The results showed that the concentration of neutrophil collagenase in LPS-enduced groups (group L 1, L 2, L 3) were significantly higher than that of control group (P<0.01). Pathological scores, lung wet/dry weight ratio and the number of neutrophils in LPS-enduced groups (group L 1, L 2, L 3) were also significantly higher than that of control group (P<0.01). Moreover, among group L 1, L 2 and L 3, there were significant correlations in concentration of neutrophil collagenase and pathological scores, lung wet/dry weight ratio, the number of neutrophils (P<0.05). The present study showed that neutrophil collagenase play an important role in the pathogenesis and progress of endotoxic acute lung injury.

Changes of pulmonary beta-adrenergic receptors and their relationship with membranous phospholipid metabolism in endotoxin-induced lung injury in rats

The changes of beta-adrenergic receptors (AARs) in lung tissue in endotoxin-induced acute lung injury was investigated with radioligand bindig assay in rats. The lipid fluidity and phospholipid content of the cellular membrane of lung tissue were measured with fluorescent polarization and high performance liquid chromatography respectively. The findings were as follows:1- Four hours after endotoxin injection, there was a 47% decrease of the maximal binding capacity of fyARsas compared with the control.2. Endotoxin was able to decrease the lipid fluidity and phospholipid content of the pulmonary cellular membrane markedly and at the same time. There was an elevated activity of phospholipase A2 in the pulmonary tissueThese findings suggest that the decrease of the binding capacity of &ARs results in a decrease of the PAR mediated functions, which plays a ro1e in the pathogensis of endotoxin-induced acute lung injury and the activation of phospholipase A2 which is an important factor to reduce the phospholipid content of cell membrane and subsequently to decrease its lipid fluidity, can result in a reduction of the lateral diffusion and rotatory movement of β-ARs and to decrease the chances of β-ARs to bind with the ligands.

Antithrombin-Ⅲ without concomitant heparin improves endotoxin-induced acute lung injury rats by inhibiting the activation of mitogen-activated protein kinase

Background Antithrombin-Ⅲ （AT-Ⅲ）, the major inhibitor of thrombin in plasma, also has anti-inflammation property and might have positive effect on sepsis. The present study aimed to investigate the effects of AT-Ⅲ on inflammatory reaction and pulmonary protection in endotoxin-induced acute lung injury （ALI） rat. Methods Sixty male Sprague-Dawley rats were randomly assigned equally to normal control group, ALl group, AT-Ⅲ treatment group, AT-Ⅲ＋heparin treatment group, and heparin treatment group. The pulmonary vascular permeability index （PVPI） was measured by single nuclide tracer technique. The activity of AT-Ⅲ in plasma was determined by the method of synthetic chromogenic substrata. Tumor necrosis factor-a （TNF-a） and interleukin-6 （IL-6） levels in serum were determined by enzyme-linked immunosorbent assay. The expressions of lung tissue mitogen-activated protein kinases （ERK1/2, P38 and JNK MAPK） were determined by Western blotting. Results Rats had significantly improved lung histopathology in the AT-Ⅲ treatment group and heparin treatment group compared with the ALl group, The PVPI of the ALl group was 0.38±0.04, significantly higher than that of the normal control group （0.20±0.02, P 〈0.01）, AT-Ⅲ treatment group （0.30±0.04, P 〈0.01） and heparin treatment group （0.28±0.04, P 〈0.01） respectively. There were no significant differences of PVPI in the ALl group and AT-Ⅲ＋heparin treatment group. The activity of AT-Ⅲ in plasma in the ALl group was （76±8）%, significantly lower than that of the normal control group （（96±11）%, P 〈0.05） and AT-Ⅲ treatment group （（105±17）%, P 〈0.05） respectively. The serum levels of TNF-α and I L-6 of the ALl group were （2.770±0.373） μg/L and （1.615±0.128） ng/ml respectively, significantly higher than those of the normal control group （0.506±0.093） μg/L and （0.233±0.047） ng/ml respectively, all P 〈0.01）, AT-Ⅲ treatment group （（1.774±0.218） pg/L and （1.140±0145） ng/ml respectively, all P 〈0.01） and heparin treatment group （（1.924±0.349） μg/L and （1.223±0.127） ng/ml respectively, all P 〈0.01）. The lung tissue levels of phospho-ERK1/2 and phospho-P38 MAPK expressions were markedly higher in the ALl group than in the normal control group, AT-Ⅲ treatment group and heparin treatment group respectively. Conclusions AT-Ⅲ without concomitant heparin inhibited the activation of ERK1/2 and P38 MAPK, down-regulated the levels of downstream cytokines TNF-a and IL-6, relieved endothelial permeability, and improved the ALl in endotoxin-induced rats. It might be helpful to administrate AT-Ⅲ alone, not with concomitant heparin, to those patients with ALl and sepsis.

Protective effects of α_1 -antitrypsin on acute lung injury in rabbits induced by endotoxin

Objective To investigate whether pretreatment with α1,-antitrypsin (AAT) can attenuate acute lung injury (ALI) in rabbits induced with endotoxin.Methods Thirty-two healthy adult New Zealand rabbits were anaesthetized, tracheotomized and mechanically ventilated. They were then randomly divided into four groups (n =8): (1) Infusion of Escherichia coli endotoxin [ Lipopolysaccharide (LPS) 500μg/kg ] without AAT (Group LPS). (2) Infusion of AAT 120 mg/kg at 15 minutes after LPS (Group LAV). (3) Infusion of AAT 120 mg/kg without endotoxin (Group AAT). (4) Infusion of saline 4 ml/kg as control (Group NS). Arterial blood gases, peripheral leukocyte counts and airway pressure were recorded every hour for eight hours. Physiologic intrapulmonary shunting (Qs/Qt) was measured every four hours. After eight hours, blood samples were collected for measurement of plasma concentration and activity of AAT. Then, the animals were sacrificed, and bronchoalveolar lavage fluid (BALF) was collected for measurement of concentrations of total protein (TP), interleukin-8 (IL-8), tumor necrosis factor (TNFa, the activities of NE and AAT, total phospholipids (TPL) and disaturated phosphatidylcholine (DSPC). In addition, the wet-to-dry lung weight ratio (W/D) was measured.Results The infusion of endotoxin induced decreases in arterial oxygen pressure (PaO2), peripheral leukocyte counts, total respiratory compliance (TLC) and the increases in peak pressure (Ppeak), Qs/ Qt compared with the baseline values ( P < 0. 05). The increased plasma concentration but reduced activity of AAT was also found in contrast to that in Group NS (P<0. 05). In the BALF, the activity of AAT, TPL, DSPC/TPL were lower than those in Group NS (P<0. 05), but the concentrations of albumin, IL-8, TNFα, the activity of NE and the ratio of W/D were higher than those in Group NS (P <0. 05). The pretreatment of AAT attenuated the deterioration of oxygenation, the reduction of compliance and the deterioration of other physiological and biochemical parameters mentioned above.Conclusion Pretreatment with AAT could attenuate endotoxin-induced lung injury in rabbits. Those beneficial effects of AAT might be due, in part, to reduction in the levels of mediators that could activate neutrophils, in addition to the direct inhibitory effect on neutrophil elastase.

15-deoxy-△12,14-prostaglandin J2 ameliorates endotoxin-induced acute lung injury in rats

Background A proinflammatory milieu emerging in the lung due to neutrophil accumulation and activation is a key in the pathogenesis of acute lung injury （ALI）.15-deoxy-△12,14-prostaglandin J2 （15d-PGJ2）,one of the terminal products of the cyclooxygenase-2 pathway,is known to be the endogenous ligand of peroxisome proliferator-activated receptor y （PPAR-y） with multiple physiological properties.Growing evidence indicates that 15d-PGJ2 has anti-inflammatory,antiproliferative,cytoprotective and pro-resolving effects.We investigated whether 15d-PGJ2 has a protective effect against endotoxin-induced acute lung injury in rats.Methods Twenty-four male Wistar rats were randomly assigned into four groups （n=6 per group）：sham＋vehicle group,sham＋15d-PGJ2 group,LPS＋vehicle group,and LPS＋15d-PGJ2 group.The rats were given either lipopolysaccharide （LPS,6 mg/kg intravenously） or saline,and pretreated with 15d-PGJ2 （0.3 mg/kg intravenously） or its vehicle （dimethyl sulphoxide） 30 minutes before LPS.Histological alterations,wet/dry weight （W/D） ratio and myeloperoxidase （MPO） activity as well as tumor necrosis factor （TNF）-α and cytokine-induced neutrophil chemoattractant-1 （CINC-1） levels were determined in lung tissues four hours after LPS injection.Immunohistochemical analysis for intercellular adhesion molecule-1 （ICAM-1） expression and Western blotting analysis for nuclear factor （NF）-κB p65 translocation and IκBα protein levels were also studied.Results 15d-PGJ2 pretreatment significantly attenuated LPS-induced lung injury,and reduced the increased W/D ratio,MPO activity,TNF-α,CINC-1 levels,and ICAM-1 expression in the lung.15d-PGJ2 also suppressed the nuclear NF-ΚB p65 translocation and increased cytosolic IKBα levels.Conclusions 15d-PGJ2 protects against endotoxin-induced acute lung injury,most likely through the reduction of proinflammatory protein levels during endotoxemia subsequent to the inhibition of NF-ΚB activation.

Osteopontin protects against hyperoxia-induced lung injury by inhibiting nitric oxide synthases

Background Exposure of adult mice to more than 95% O_2 produces a lethal injury by 72 hours. Nitric oxide synthase （NOS） is thought to contribute to the pathophysiology of murine hyperoxia-induced acute lung injury （ALI）. Osteopontin （OPN） is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase （iNOS）, which generates large amounts of nitric oxide production. However, the relationship between nitric oxide and endogenous OPN in lung tissue during hyperoxia-induced ALI has not yet been elucidated, thus we examined the role that OPN plays in the hyperoxia-induced lung injury and its relationships with NOS.Methods One hundred and forty-four osteopontin knock-out （KO） mice and their matched wild type background control （WT） were exposed in sealed cages 〉95% oxygen or room air for 24-72 hours, and the severity of lung injury was assessed; expression of OPN, endothelial nitric oxide synthase （eNOS） and iNOS mRNA in lung tissues at 24,48 and 72 hours of hyperoxia were studied by reverse transcription-polymerase chain reaction （RT-PCR）; immunohistochemistry （IHC） was performed for the detection of iNOS, eNOS, and OPN protein in lung tissues.Results OPN KO mice developed more severe acute lung injury at 72 hours of hyperoxia. The wet/dry weight ratio increased to 6.85±0.66 in the KO mice at 72 hours of hyperoxia as compared to 5.31±0.92 in the WT group （P〈0.05）. iNOS mRNA （48 hours： 1.04±0.08 vs. 0.63±0.09, P〈0.01; 72 hours： 0.89±0.08 vs. 0.72±0.09, P〈0.05） and eNOS mRNA （48 hours： 0.62±0.08 vs. 0.43±0.09, P〈0.05; 72 hours： 0.67±0.08 vs. 0.45±0.09, P〈0.05） expression was more significantly increased in OPN KO mice than their matched WT mice when exposed to hyperoxia. IHC study showed higher expression of iNOS （20.54±3.18 vs. 12.52±2.46, P 〈0.05） and eNOS （19.83±5.64 vs. 9.45±3.82, P 〈0.05） in lung tissues of OPN KO mice at 72 hours of hyperoxia. Conclusion OPN can protect against hyperoxia-induced lung injury by inhibiting NOS.

Effect of Radix Paeoniae Rubra on the expression of HO-1 and iNOS in rats with endotoxin-induced acute lung injury

Objective ： To investigate the effect of Radix Paeoniae Rubra （RPR） on the expression of heme oxygenase （ HO- 1 ） and induced nitric oxide synthase （ iNOS ） in endotoxin- induced acute lung injury in rats and its protective mechanism. Methods： Forty Wistar rats were divided randomly into 5 groups with 8 rats in each group： saline control group （NS group ）, lipopolysaccharide group （LPS group）, RPR-treatment group, RPR-prevention group and Hemin group. The effect of RPR on protein content, the ratio of neutrophiles in bronchoalveolar lavage fluid, malondialdehyde （MDA） content in the lung and the activity of serum NO were observed. Arterial blood was drawn for blood-gas analysis. The expression of HO-1 and iNOS in lung tissues was detected by immunohistochemitry and morphometry computer image analysis. The histological changes of the lung were observed under fight microscope. Results： Compared with that in NS group, the expression of HO-1 and iNOS was markedly increased in LPS group （P 〈0.01）. In RPR-treatment, RPR-prevention, and Hemin groups, the expression of iNOS was significantly lower, while the expression of HO-1 was higher than that in LPS group （P〈0.05）. The protein content, the ratio of neutrophiles in bronchouiveolar lavage fluid, the content of MDA and the activity of serum NO in LPS group were significantly higher than those in NS group （ P〈0.01 ）. There was a significant decrease in the level of arterial bicarbonate and partial pressure of oxygen in the LPS group （P 〈0.01）; these parameters of lung injury however, were significantly lower in RPR-treatment, RPR- prevention, and Heroin groups than LPS group （P〈0.05 or P〈0.01 ）. The pathologic changes of lung tissues were substantially attenuated in RPR-treatment, RPR- prevention, and Hemin groups than LPS group. Conclusions ： The high expression of HO-1 reflects an important protective function of the body during lipopolysaccharide-induced acute lung injury. The acute lung injury is related to the inhibition of iNOS expression and the induction of HO-1 expression.

茶多酚通过抑制NLRP3炎症小体改善脓毒症小鼠的急性肺损伤

目的探讨茶多酚(TP)对NLRP3炎症小体的调控作用机制以及其对脓毒症相关急性肺损伤的治疗效用。方法将60只C57BL/6小鼠随机平均分为假手术组(单纯开腹探查盲肠后即关腹)、盲肠结扎穿孔组(开腹暴露并游离盲肠后行盲肠结扎与穿孔操作后关腹)以及盲肠结扎穿孔+TP治疗组(盲肠结扎穿孔前1周予TP灌胃),每组20只。建模完成后绘制生存曲线评估各组小鼠对脓毒症打击的耐受情况。测定各组小鼠肺湿/干质量比值、肺组织髓过氧化物酶(MPO)水平,并对肺组织进行HE染色和急性肺损伤评分,评估肺损伤程度。ELISA法测定肺组织内IL-1β、TNF-α及IL-6表达水平;蛋白免疫印迹实验检测肺组织内NLRP3、caspase-1 p10及ASC蛋白表达水平;免疫组化染色测定肺组织内MPO、caspase-8及NLRP3蛋白表达,评估各组小鼠肺内炎症情况。检测肺组织内MDA及H2O2以评估氧化应激水平;免疫荧光共染NLRP3和NOX4蛋白以探讨二者表达及共定位。结果TP治疗组小鼠术后72 h死亡率、小鼠肺湿/干质量比值、肺组织MPO水平以及急性肺损伤评分均较盲肠结扎穿孔组小鼠显著下降(P<0.05);TP治疗组小鼠肺组织内IL-1β、TNF-α及IL-6、NLRP3、caspase-1 p10、ASC等蛋白表达水平亦较盲肠结扎穿孔组小鼠显著降低(P<0.05);氧化应激相关检测结果提示,TP治疗组小鼠肺组织内MDA及H2O2水平较盲肠结扎穿孔组小鼠显著减低(P<0.05);免疫荧光共染则提示TP治疗组小鼠NOX4蛋白与NLRP3蛋白在肺组织内的表达和共定位较盲肠结扎穿孔组小鼠减少。结论TP可通过抑制NLRP3炎症小体相关炎症改善盲肠结扎穿孔诱导的小鼠脓毒症肺损伤,且这一调控机制可能与其对肺组织内氧化应激相关蛋白NOX4的表达抑制相关。

白花败酱草总黄酮对实验性急性肺损伤模型大鼠的肺保护作用及其机制

目的:探讨白花败酱草总黄酮(total flavoniods from Patrina villosa Juss,PJF)对实验性急性肺损伤(acute lung injury,ALI)模型大鼠的肺保护作用及其潜在机制。方法:用气管内滴注5 mg/kg脂多糖(lipopolysaccharide,LPS)构建ALI大鼠模型。将60只雄性SD大鼠随机分为对照组、LPS组、LPS+低剂量(100 mg/kg)PJF组和LPS+高剂量(300 mg/kg)PJF组(后2组在ALI造模前1 h给予PJF灌胃),每组15只。造模后24 h,收集支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)和肺组织。HE染色显示肺组织形态;干湿称重法测量肺组织的湿/干重比;伊文思蓝染色评估肺组织中上皮屏障的通透性;ELISA检测BALF中炎症因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)和IL-6含量,以及肺组织中氧化应激指标超氧化物歧化酶(superoxide dismutase,SOD)、髓过氧化物酶(myeloperoxidase,MPO)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性及丙二醛(malondialdehyde,MDA)含量;免疫印迹法检测肺组织中C/EBP同源蛋白(C/EBP homologous protein,CHOP)、葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)和X框结合蛋白1(X-box binding protein 1,XBP1)蛋白表达。结果:与对照组比较,LPS组大鼠肺组织呈现肺泡结构不清和大量炎症细胞浸润,ALI评分和肺组织的湿/干重比显著升高(P<0.05),BALF中IL-6、IL-1β和TNF-α水平,以及肺组织中MDA含量、MPO活性及CHOP、GRP78和XBP1蛋白表达水平显著升高(P<0.05),肺组织中SOD和GSH-Px活性显著降低(P<0.05)。与LPS组比较,PJF干预组肺组织形态改善,ALI评分和肺组织的湿/干重比显著下降(P<0.05),BALF中IL-6、IL-1β和TNF-α水平,以及肺组织中MDA含量、MPO活性及CHOP、GRP78和XBP1蛋白表达水平显著降低(P<0.05),肺组织中SOD和GSH-Px活性显著升高(P<0.05);且高剂量组的效果明显优于低剂量组。结论:PJF对ALI大鼠具有肺保护作用,其机制可能与抑制炎症反应、氧化应激和内质网应激有关。

Correlation of eNOS gene G894T locus polymorphism with the oxidative and inflammatory endothelial function injury in patients with myocardial infarction

Objective:To study the correlation of endothelial nitric oxide synthase (eNOS) gene G894T locus polymorphism with the oxidative and inflammatory endothelial function injury in patients with myocardial infarction.Methods:87 patients with acute myocardial infarction treated in our hospital between May 2012 and December 2015 were selected as acute myocardial infarction (AMI) group and 90 healthy volunteers receiving physical examination during the same period were selected as control group. Peripheral arterial blood was collected to extract genomic DNA and then determine eNOS gene G894T locus polymorphism;peripheral venous blood was collected to separate serum and then determine endothelial injury, oxidative stress and inflammatory reaction indexes.Results:GG genotype proportion and G allele frequency of eNOS gene G894T locus of AMI group were significantly lower than those of control group (P<0.05) while the GT genotype and TT genotype proportion as well as T allele frequency were significantly higher than those of control group (P<0.05);serum nitric oxide (NO), SOD and GSH content of patients with GG genotype were significantly higher than those of patients with GT genotype and TT genotype (P<0.05) while vWF, ET-1, ox-LDL, MDA, -COOH, NF-κB, MCP-1, IL-6 and IL-18 content were significantly lower than those of patients with GT genotype and TT genotype (P<0.05).Conclusions: The proportion of eNOS gene G894T locus G mutation into T significantly increases in patients with myocardial infarction, and G894T locus G mutation into T can aggravate the endothelial injury caused by oxidative stress and inflammation.

槲皮素预处理ALI大鼠肺组织损伤、炎症/氧化应激反应、铁死亡及Nrf2/HO-1信号通路激活情况观察

目的观察槲皮素灌胃预处理的LPS诱导急性肺损伤(ALI)大鼠肺组织损伤、炎症反应、氧化应激反应、铁死亡及Nrf2/HO-1信号通路激活情况,以探讨槲皮素对ALI的预防作用及机制。方法24只SD大鼠分为槲皮素低、中、高剂量组和阳性对照组(地塞米松)、模型组、正常对照组。槲皮素低、中、高剂量组以25、50、100 mg/kg槲皮素灌胃,1次/天,连续7 d;槲皮素灌胃处理的第4天在大鼠气管内滴注5 mg/kg LPS;阳性对照组以地塞米松1.04 mg/kg灌胃,其余处理同槲皮素组;模型组以生理盐水灌胃,1次/天,连续7天,其余处理同槲皮素组;正常对照组以生理盐水连续灌胃7 d。末次灌注给药后,观察各组肺组织损伤(肺功能及肺组织病理改变、纤维组织阳性表达率、肺泡上皮细胞凋亡率)、炎症反应(肺泡灌洗液TNF-α、IL-6、IL-1β)、氧化应激反应(肺泡灌洗液SOD、GSH、MDA,肺组织ROS)、铁死亡(Fe^(2+)水平)及Nrf2/HO-1信号通路激活[肺组织核因子红细胞2相关因子2(Nfr2)、血红素加氧酶1(HO-1)、超氧化物歧化酶2(SOD2)、过氧化氢酶(CAT)蛋白]情况。结果与正常对照组比较,模型组PaCO_(2)水平升高,PaO_(2)、SaO_(2)水平降低(P均<0.01);肺组织可见肺泡上皮细胞变性坏死,纤维组织阳性表达率高;肺泡上皮细胞凋亡率高;肺泡灌洗液中TNF-α、IL-6、IL-1β水平均升高,SOD、GSH水平降低,MDA水平升高,肺组织ROS表达水平升高,肺泡灌洗液中Fe^(2+)水平升高(P均<0.05)。与模型组相比,槲皮素中、高剂量组和阳性对照组中PaCO_(2)均降低(P均<0.05),槲皮素高剂量组PaO_(2)和SaO_(2)水平升高(P均<0.05);槲皮素高剂量组与阳性对照组肺组织炎性浸润与纤维增生明显减少,肺泡恢复正常生理结构;槲皮素低、中、高剂量组和阳性对照组肺纤维组织阳性表达率均下降(P均<0.05),其中槲皮素高剂量组肺纤维组织阳性表达率最低;槲皮素低、中、高剂量组和阳性对照组肺泡上皮细胞凋亡率均降低(P均<0.01);槲皮素低、中、高剂量组和阳性对照组肺泡灌洗液中TNF-α、IL-6、IL-1β水平均降低、SOD、GSH水平升高、MDA水平降低,肺组织ROS表达水平降低(P均<0.01),肺泡灌洗液中Fe^(2+)水平降低。与正常对照组比较,模型组肺组织中CAT、HO-1、Nrf2、SOD2蛋白表达水平低(P均<0.01);与模型组比较,槲皮素中、高剂量组和阳性对照组肺组织中CAT、HO-1、Nrf2、SOD2蛋白表达水平高(P均<0.05)。结论槲皮素灌胃预处理的ALI大鼠肺组织损伤、炎症反应、氧化应激反应、铁死亡情况减轻,Nfr2/HO-1信号通路激活;槲皮素灌胃预处理可预防LPS诱导的大鼠ALI,可能通过抑制炎症反应、氧化应激反应及铁死亡而起作用;槲皮素可能通过上调Nfr2/HO-1信号通路而抑制炎症反应、氧化应激反应及铁死亡;50、100 mg/kg槲皮素均对LPS诱导的大鼠ALI起预防作用,以100 mg/kg槲皮素的作用效果更佳。