Cellular changes in bronchoalveolar lavage fluid in hyperoxia-induced lung injury

It is well known that high concentration oxygen exposure is a model of acute lung injury (ALI). However, controversy exists over the mechanism. This study was designed to clarify the cellular characteristics in bronchoalveolar lavage fluid (BALF) and body weight loss of rats exposed to oxygen(> 90%). Young male Wistar rats, aged 6 weeks, were divided into three groups: (1) room air group (exposed to room air, n = 22); (2) hyperoxia < 48 h group (exposed to over 90% oxygen for less than 48 h, n = 18); (3) hyperoxia 66–72 h group (exposed to over 90% oxygen for 66–72 h group, n = 7). Compared to the room air group, the total cell counts in the hyperoxia 66–72 h group decreased, whereas the neutrophils increased significantly. The body weights of the rats exposed to room air continued to increase. However, the body weights of oxygen-exposed rats increased slightly on the first day and weight loss was seen from the second day. All rats were noted to have bilateral pleural effusion in the hyperoxia 66–72 h group. The data suggests that (1) an increase in neutrophil count is an evident feature of hyperoxia-induced lung injury; (2) high concentration oxygen exposure can give rise to anorexia and malnutrition, which may play a role in hyperoxia-induced lung injury. Blocking neutrophil influx into lung tissue in the early phase and improving malnutrition are two effective methods to reduce hyperoxic lung injury.     

新生大鼠高氧性肺损伤肺组织内源性谷氨酸释放的变化

目的探讨谷氨酸在新生大鼠高氧性肺损伤中的作用。方法SD新生大鼠,出生后12h内随机分为空气对照组和高氧组。高氧组维持氧浓度≥95%,分别在1,3和7d后每组处死5只大鼠,取肺脏,测定肺组织湿重/干重(W/D)比值,HE染色观察肺组织病理变化;另取小鼠,进行支气管肺泡灌洗,制备支气管肺泡灌洗液(BALF),用血细胞计数板进行白细胞计数,全自动生化分析仪测定乳酸脱氢酶(LDH)活性,Lowry法检测总蛋白含量,高效液相色谱法检测谷氨酸含量。结果与空气对照组比较,持续高氧暴露1d新生大鼠肺组织W/D比值无明显变化,暴露3和7dW/D比值明显增加。HE染色可见,持续高氧暴露3d肺泡腔内少量炎症细胞渗出,暴露7d肺泡内红细胞和炎症细胞进一步增多,肺组织结构紊乱,肺泡数量减少。持续高氧暴露1d新生大鼠BALF中LDH活性明显增加,白细胞计数和总蛋白含量无明显变化,暴露3和7dBALF中LDH活性、总蛋白含量和白细胞计数均高于空气对照组。持续高氧暴露1和3dBALF中谷氨酸含量亦明显高于空气对照组。结论高浓度氧可引起新生大鼠急性肺损伤,诱导肺组织内源性谷氨酸的释放,提示谷氨酸在高氧性肺损伤中发挥重要作用。     

Mitochondrial thiol status in the liver is altered by exposure to hyperoxia

Patients with poorly functioning lungs often require treatment with high concentrations of supplemental oxygen, which, although often necessary to sustain life, can cause lung injury. The mechanisms responsible for hyperoxic lung injury have been investigated intensely and most probably involve oxidant stress responses, but the details are not well understood. In the present studies, we exposed adult male C57/Bl6 mice to >95% O2 for up to 72 h and obtained lung and liver samples for assessment of lung injury, measurements of tissue concentrations of coenzyme A (CoASH) and the corresponding mixed disulfide with glutathione (CoASSG), as possible biomarkers of intramitochondrial thiol redox status. Subcellular fractions were prepared from both tissues for determination of glutathione reductase (GR) activities. Lung injury in the hyperoxic mice was demonstrated by increases in lung weight to body weight ratios at 48 h and by increases in bronchoalveolar lavage protein concentrations at 72 h. Lung CoASH concentrations declined in the hyperoxic mice, but CoASSG concentrations were not increased nor were CoASH/CoASSG ratios decreased, as would be expected for an oxidant shift in mitochondrial thiol-disulfide status. Interestingly, CoASSG concentrations increased (from 6.72+/-0.54 to 14.10+/-1.10 nmol/g of liver in air-breathing controls and 72 h of hyperoxia, respectively, P<0.05), and CoASH/CoASSG ratios decreased in the livers of mice exposed to hyperoxia. Some apparent effects of duration of hyperoxia on GR activities in lung or liver cytosolic, mitochondrial, or nuclear fractions were observed, but the changes were not consistent or progressive. Yields of isolated hepatic nuclear protein were decreased in the hyperoxic mice within 24 h of exposure, and by 72 h of hyperoxia, protein recoveries in purified nuclear fractions had declined from 41.8 to 14.8 mg of protein/g animal body weight. Concentrations of 10-formyltetrahydrofolate dehydrogenase were diminished in hepatic mitochondria of hyperoxic mice. A second protein in hepatic mitochondria of approximately 25 kDa showed apparent decreases in thiol content, as determined by fluorescence intensities of monobromobimane derivatives separated by SDS-PAGE. The mechanisms responsible for the observed effects and the possible implications for the adverse effects of hyperoxic therapies are not known and need to be investigated.     

谷氨酰胺诱导热休克蛋白的表达及对肺损伤的保护作用的研究

目的 通过利用谷氨酰胺(Gln)诱导大鼠热休克蛋白70(HSP70)表达以探讨HSP70对急性肺损伤的保护作用.方法 雄性SD大鼠42只随机分成三组:O组为油酸损伤组(经尾静脉按25mL/kg量注入生理盐水后注入油酸);G组为干预组(经尾静脉按25mL/kg量注入3%Gln溶液后注入油酸);C组为正常对照组(经尾静脉注...     

高浓度氧诱发急性肺损伤发病机制的探讨

目的:探讨高浓度氧造成急性肺损伤的发生机制。方法:将雄性6周龄Wistar鼠30只分为对照组(空气中饲养,n=20)和实验组(暴露在90%～95%氧气中72h,n=10)。结果:实验组动物外周血中的红细胞(RBC)、血红蛋白(Hb)及红细胞比容(HCT)计数高于对照组(P<0.05);实验组动物外周血RBC、Hb计数随高浓度氧暴露时间的延长逐渐升高;对照组中动物体质量持续增长,而实验组动物体质量首日轻度增加,第2天开始呈现逐渐下降的趋势;对照组气管肺泡清洗液(BALF)中以巨噬细胞为主,而实验组BALF中可见大量中性粒细胞浸润;实验组动物均出现双侧胸水。结论:中性粒细胞动员并进入肺组织是高浓度氧诱发急性肺损伤的明显特征;营养不良可能是诱发急性肺损伤的因素之一,早期抑制中性粒细胞进入肺组织和纠正营养不良是治疗高浓度氧诱发急性肺损伤的重要途径。     

The fate of antioxidant enzymes in bronchoalveolar lavage fluid over 7 days in mice with acute lung injury

Characterization of lung injury is important if timely therapeutic intervention is to be used properly and successfully. In this study, lung injury was defined as the progressive formation of pulmonary edema. Our model gas was phosgene, a pulmonary edemagenic compound. Phosgene, widely used in industry, can produce life-threatening pulmonary edema within hours of exposure. Four groups of 40 CD-1 male mice were exposed whole-body to either air or a concentration x time (c x t) amount of 32-42 mg/m(3) (8-11 ppm) phosgene for 20 min (640-840 mg x min/m(3)). Groups of air- or phosgene-exposed mice were euthanized 1, 4, 8, 12, 24, 48, or 72 h or 7 days postexposure. The trachea was excised, and 800 micro l saline was instilled into the lungs and washed back and forth 5 times to collect bronchoalveolar lavage fluid (BALF). The antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), total glutathione (GSH), and protein were determined at each time point. Phosgene exposure significantly enhanced both GPx and GR in phosgene-exposed mice compared with air-exposed mice from 4 to 72 h, p < or = 0.01 and p < or = 0.005, respectively. BALF GSH was also significantly increased, p < or = 0.01, from 4 to 24 h after exposure, in comparison with air-exposed. BALF protein, an indicator of air/blood barrier integrity, was significantly higher than in air-exposed mice 4 h to 7 days after exposure. In contrast, BALF SOD was reduced by phosgene exposure from 4 to 24 h, p < or = 0.01, versus air-exposed mice. Except for protein, all parameters returned to control levels by 7 days postexposure. These data indicate that the lung has the capacity to repair itself within 24-48 h after exposure by reestablishing a functional GSH redox system despite increased protein leakage. SOD reduction during increased leakage may indicate that barrier integrity is affected by superoxide anion production.     

The Fate of Antioxidant Enzymes in Bronchoalveolar Lavage Fluid Over 7 Days in Mice with Acute Lung Injury

Characterization of lung injury is important if timely therapeutic intervention is to be used properly and successfully. In this study, lung injury was defined as the progressive formation of pulmonary edema. Our model gas was phosgene, a pulmonary edemagenic compound. Phosgene, widely used in industry, can produce life-threatening pulmonary edema within hours of exposure. Four groups of 40 CD-1 male mice were exposed whole-body to either air or a concentration × time (c × t) amount of 32-42 mg/m 3 (8-11 ppm) phosgene for 20 min (640-840 mg·min/m 3) . Groups of air- or phosgene-exposed mice were euthanized 1, 4, 8, 12, 24, 48, or 72 h or 7 days postexposure. The trachea was excised, and 800 µl saline was instilled into the lungs and washed back and forth 5 times to collect bronchoalveolar lavage fluid (BALF). The antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), total glutathione (GSH), and protein were determined at each time point. Phosgene exposure significantly enhanced both GPx and GR in phosgene-exposed mice compared with air-exposed mice from 4 to 72 h, p ≤ .01 and p ≤ .005, respectively. BALF GSH was also significantly increased, p ≤ .01, from 4 to 24 h after exposure, in comparison with air-exposed. BALF protein, an indicator of air/blood barrier integrity, was significantly higher than in air-exposed mice 4 h to 7 days after exposure. In contrast, BALF SOD was reduced by phosgene exposure from 4 to 24 h, p ≤ .01, versus air-exposed mice. Except for protein, all parameters returned to control levels by 7 days postexposure. These data indicate that the lung has the capacity to repair itself within 24-48 h after exposure by reestablishing a functional GSH redox system despite increased protein leakage. SOD reduction during increased leakage may indicate that barrier integrity is affected by superoxide anion production.     

Sex-specific differences in hyperoxic lung injury in mice: Implications for acute and chronic lung disease in humans

Sex-specific differences in pulmonary morbidity in humans are well documented. Hyperoxia contributes to lung injury in experimental animals and humans. The mechanisms responsible for sex differences in the susceptibility towards hyperoxic lung injury remain largely unknown. In this investigation, we tested the hypothesis that mice will display sex-specific differences in hyperoxic lung injury. Eight week-old male and female mice (C57BL/6J) were exposed to 72 h of hyperoxia (FiO₂ > 0.95). After exposure to hyperoxia, lung injury, levels of 8-iso-prostaglandin F₂ alpha (8-iso-PGF 2α) (LC–MS/MS), apoptosis (TUNEL) and inflammatory markers (suspension bead array) were determined. Cytochrome P450 (CYP)1A expression in the lung was assessed using immunohistochemistry and western blotting. After exposure to hyperoxia, males showed greater lung injury, neutrophil infiltration and apoptosis, compared to air-breathing controls than females. Pulmonary 8-iso-PGF 2α levels were higher in males than females after hyperoxia exposure. Sexually dimorphic increases in levels of IL-6 (F > M) and VEGF (M > F) in the lungs were also observed. CYP1A1 expression in the lung was higher in female mice compared to males under hyperoxic conditions. Overall, our results support the hypothesis that male mice are more susceptible than females to hyperoxic lung injury and that differences in inflammatory and oxidative stress markers contribute to these sex-specific dimorphic effects. In conclusion, this paper describes the establishment of an animal model that shows sex differences in hyperoxic lung injury in a temporal manner and thus has important implications for lung diseases mediated by hyperoxia in humans.     

The Temporal Profile of Cytokines in the Bronchoalveolar Lavage Fluid in Mice Exposed to the Industrial Gas Phosgene

Diagnosis of an exposure to airborne toxicants can be problematic. Phosgene is used widely in industry for the production of many synthetic products, such as polyfoam rubber, plastics, and dyes. Although nearly 100% of the gas is consumed during processing, there is the potential problem of accidental or even intentional exposure to this irritant/choking agent. Exposure to phosgene has been known to cause latent life-threatening pulmonary edema. A major problem is that there is a clinical latency phase from 3 to 24 h in people before irreversible acute lung injury occurs. Assessment of markers of acute lung injury after a suspected exposure would be useful in developing rational treatment strategies. These experiments were designed to assess bronchoalveolar lavage fluid (BALF) for the presence of the early markers of exposure to phosgene in mice from 1 to 72 h after exposure. Separate groups of 40 CD-1 male mice (Crl:CD-1(ICR)BR) weighing 29 ± 1 g were exposed whole-body to either air or a concentration × time (c × t) amount of 32 mg/m 3 (8 ppm) phosgene for 20 min (640 mg·min/m 3) . BALF from air- or phosgene-exposed mice was taken at 1, 4, 8, 12, 24, 48, and 72 h postexposure. After euthanasia, the trachea was excised, and 800 µl saline was instilled into the lungs and washed 5 ×. BALF was assessed for interleukin (IL)-4, IL-6, tumor necrosis factor (TNF)α, IL-1α, macrophage inflammatory protein (MIP)-2, and IL-10. At 4 h postexposure, IL-6 was 15-fold higher for phosgene-exposed mice than for the time-matched air-exposed control group. At 8 and 12 h, IL-6, IL-1β, MIP-2, and IL-10 were significantly higher in phosgene-exposed mice than in time-matched air-exposed controls, p ≤ .05 to p ≤ .001, whereas TNFα reached peak significance from 24 to 72 h. IL-4 was significantly lower in the phosgene-exposed mice than in the air-exposed mice from 4 to 8 h after exposure. These data show that BALF is an important tool in assessing pro- and anti-inflammatory markers of phosgene-induced acute lung injury and that knowledge of these temporal changes may allow for timely treatment strategies to be applied.     

银杏叶提取物对大鼠高氧肺损伤的影响

目的:明确银杏叶提取物(EGb)能否减轻高氧诱导的大鼠急性肺损伤。方法:SD大鼠随机分为EGb组、高氧组和对照组。EGb组和高氧组大鼠置于>95%氧箱中,对照组置于空气中。实验开始及高氧暴露24 h、48 h时,EGb组大鼠均管饲EGb100 mg/kg,另外两组大鼠予等量生理盐水。检测胸水含量、支气管肺泡灌洗液(BALF)中白细胞和中性粒细胞计数、总蛋白、细胞因子IL-6、IL-1β及TNF-α含量、肺组织丙二醛(MDA)含量以及病理学改变。结果:EGb组大鼠胸水含量和BALF中白细胞、中性粒细胞计数明显低于高氧组(P<0.05)。EGb组和高氧组总蛋白含量差异无统计学意义,均明显高于对照组(P<0.01)。EGb组BALF中IL-6含量显著高于另外两组(P<0.01)。三组大鼠BALF中均未检测到IL-1β和TNF-α,肺组织MDA含量差异无统计学意义。EGb组和高氧组肺组织学评分差异无统计学意义(P>0.05)。结论:EGb可部分减轻高氧诱导的大鼠急性肺损伤,机制可能与促进肺组织IL-6产生有关。     

The temporal profile of cytokines in the bronchoalveolar lavage fluid in mice exposed to the industrial gas phosgene

Diagnosis of an exposure to airborne toxicants can be problematic. Phosgene is used widely in industry for the production of many synthetic products, such as polyfoam rubber, plastics, and dyes. Although nearly 100% of the gas is consumed during processing, there is the potential problem of accidental or even intentional exposure to this irritant/choking agent. Exposure to phosgene has been known to cause latent life-threatening pulmonary edema. A major problem is that there is a clinical latency phase from 3 to 24 h in people before irreversible acute lung injury occurs. Assessment of markers of acute lung injury after a suspected exposure would be useful in developing rational treatment strategies. These experiments were designed to assess bronchoalveolar lavage fluid (BALF) for the presence of the early markers of exposure to phosgene in mice from 1 to 72 h after exposure. Separate groups of 40 CD-1 male mice (Crl:CD-1(ICR)BR) weighing 29 +/- 1 g were exposed whole-body to either air or a concentration x time (c x t) amount of 32 mg/m(3) (8 ppm) phosgene for 20 min (640 mg x min/m(3)). BALF from air- or phosgene-exposed mice was taken at 1, 4, 8, 12, 24, 48, and 72 h postexposure. After euthanasia, the trachea was excised, and 800 micro l saline was instilled into the lungs and washed 5x. BALF was assessed for interleukin (IL)-4, IL-6, tumor necrosis factor (TNF) alpha, IL-1alpha, macrophage inflammatory protein (MIP)-2, and IL-10. At 4 h postexposure, IL-6 was 15-fold higher for phosgene-exposed mice than for the time-matched air-exposed control group. At 8 and 12 h, IL-6, IL-1beta, MIP-2, and IL-10 were significantly higher in phosgene-exposed mice than in time-matched air-exposed controls, p < or = 0.05 to p < or = 0.001, whereas TNF alpha reached peak significance from 24 to 72 h. IL-4 was significantly lower in the phosgene-exposed mice than in the air-exposed mice from 4 to 8 h after exposure. These data show that BALF is an important tool in assessing pro- and anti-inflammatory markers of phosgene-induced acute lung injury and that knowledge of these temporal changes may allow for timely treatment strategies to be applied.     

Chronological changes in electrolyte levels in arterial blood and bronchoalveolar lavage fluid in mice after exposure to an edemagenic gas

Detection of acute lung injury is important if therapeutic medical countermeasures are to be used to reduce toxicity in a timely manner. Indicators of injury may aid in the eventual treatment course and enhance the odds of a positive outcome following a toxic exposure. This study was designed to investigate the effects of a toxic exposure to the industrial irritant gas phosgene on the electrolyte levels in arterial blood and bronchoalveolar lavage fluid (BALF). Phosgene is a well-known chemical intermediate capable of producing life-threatening pulmonary edema within hours after exposure. Four groups of 40 Crl:CD-1(ICR)BR male mice were exposed whole-body to either air or phosgene at a concentration x time (c x t) amount of 32-42 mg/m(3) (8-11 ppm) phosgene for 20 min (640-840 mg x min/m(3)). BALF from air- or phosgene-exposed mice was taken at 1, 4, 8, 12, 24, 48, or 72 h postexposure. After euthanasia, the trachea was excised, and 800 microl saline was instilled into the lungs. The lungs were washed 5x. Eighty microliters of BALF was placed in a cartridge and inserted into a clinical i-STAT analyzer. Na(+), Cl(-), K(+), and ionized Ca(2+) were analyzed. Arterial blood electrolyte levels were also analyzed in four additional groups of air- or phosgene-exposed mice. The left lung was removed to determine wet weight (WW), an indicator of pulmonary edema. Na(+) was significantly higher in air than in phosgene-exposed mice at 4, 8, and 12 h postexposure. Temporal changes in BALF Cl(-) in phosgene mice were not statistically different from those in the air mice. Both Ca(2+) and K(+) were significantly higher than in the air-exposed mice over 72 h, p < or = 0.03 and p < or = 0.001 (two-way analysis of variance, ANOVA), respectively. Significant changes in BALF K(+) and Ca(2+) occurred as early as 4 h postexposure in phosgene, p < or = 0.005, versus air-exposed mice. Over time, there were no significant changes in arterial blood levels of Na(+), Cl(-), or Ca(2+) for animals exposed to air versus phosgene. However, arterial K(+) concentrations were significantly higher, p < or = 0.05, than in air-exposed mice across all time points, with the highest K(+) levels of 7 mmol/L occurring at 8 h and 24 h after exposure. Phosgene caused a time-dependent significant increase in WW from 4 to 12 h, p < or = 0.025, compared with air-exposed mice. These data demonstrate that measuring blood K(+) levels 1 h after exposure along with BALF Na(+), K(+), and Ca(2+) may serve as an alternate indicators of lung injury since both K(+) and Ca(2+) follow temporal increases in air-blood barrier permeability as measured by wet weight.     

Beta1-adrenergic agonist is a potent stimulator of alveolar fluid clearance in hyperoxic rat lungs

Because it was still uncertain whether a stimulation of beta1-adrenoceptors accelerated alveolar fluid clearance in hyperoxic lung injury, the effect of denopamine, a selective beta1-adrenergic agonist, on alveolar fluid clearance was determined in rats exposed to 93% oxygen for 48 and 56 h. Alveolar fluid clearance was measured by the progressive increase in the concentration of Evans blue labeled albumin instilled into the alveolar spaces over 1 h at 37 degrees C in isolated rat lungs. The principle results were as follows: 1) Although lung water volume increased in rats exposed to hyperoxia for 48 and 56 h, basal alveolar fluid clearance did not change for up to 56 h; 2) Denopamine increased alveolar fluid clearance in rats exposed to hyperoxia as well as in rats without exposure to hyperoxia; 3) Denopamine primarily increased amiloride-insensitive alveolar fluid clearance in rats exposed to hyperoxia; 4) The potency of denopmaine was similar to that of terbutaline, a selective beta2-adrenergic agonist. In summary, denopamine is a potent stimulator of alveolar fluid clearance in rats exposed to hyperoxia.     

Regulation of pulmonary and hepatic cytochrome P4501A expression in the rat by hyperoxia: implications for hyperoxic lung injury

Supplemental oxygen therapy is frequently used in the treatment of pulmonary insufficiency, as is encountered in premature infants, and in patients with acute respiratory distress syndrome. However, hyperoxia causes lung damage in experimental animals and may do so in humans. Cytochrome P4501A enzymes have been implicated in hyperoxic lung injury. In this study, we investigated the mechanisms of CYP1A1 regulation by hyperoxia and tested the hypothesis that aryl hydrocarbon receptor (AHR)-dependent mechanisms contribute to induction of CYP1A1 and that modulation of CYP1A by hyperoxia may have implications for lung injury. Exposure of adult male Sprague-Dawley rats to hyperoxia for 24 to 48 h led to increased expression of pulmonary CYP1A1 enzyme, which was preceded by enhancement of the corresponding mRNA, followed by decline of induction at 60 h, when the animals displayed severe respiratory distress and lung inflammation. Similarly, hepatic CYP1A1/1A2 mRNAs were markedly induced between 24 and 48 h of hyperoxia, with induction declining by 60 h. Electrophoretic mobility shift assays (EMSA) and experiments with AHR (-/-) mice indicated that AHR-dependent mechanisms contributed to CYP1A induction. The AHR (-/-) mice were refractory to CYP1A1 induction by hyperoxia and were more sensitive to lung injury than wild-type mice. Lungs of hyperoxic rats showed increase in the expression of CYP1A1 in airway epithelial cells, type II pneumocytes, and endothelial cells. In conclusion, our results suggest that induction of CYP1A1 by hyperoxia is mediated by AHR-dependent mechanisms and that modulation of CYP1A enzymes by hyperoxia may have implications for hyperoxic lung injury.     

西地那非对脂多糖诱导的小鼠急性肺损伤的作用

目的明确西地那非对急性肺损伤(ALI)的治疗作用及可能机制。方法采用脂多糖(LPS,4 mg·kg~(-1))气道滴入诱导的小鼠ALI模型。随机分为生理盐水组、LPS模型组、西地那非3,10及30 mg·kg~(-1)组、地塞米松5 mg·kg~(-1)组。测定肺干/湿重比值,常规细胞形态学检测支气管肺泡灌洗液(BALF)中白细胞,肺组织切片观察病理改变;测定肺组织匀浆髓过氧化酶(MPO)活性、NO含量、NOS活性及TNF-α含量。结果LPS诱导的ALI小鼠肺干/湿重比值明显下降;BALF中白细胞总数及中性粒细胞比例明显增加;肺毛细血管通透性明显增加;肺组织间隙大量中性粒细胞浸润和红细胞渗出;肺组织匀浆TNF-α含量和MPO活性明显增加,NO含量、总NOS活性及iNOS活性明显增加。同时腹腔注射西地那非可剂量依赖性地降低ALI小鼠肺干/湿重比值;减少BALF中的白细胞总数及中性粒细胞的比例;降低肺毛细血管通透性;改善肺组织病理变化;抑制肺组织匀浆TNF-α含量、MPO活性及NO含量、总NOS活性及iNOS活性的增加。结论西地那非对LPS诱导的ALI有保护作用,提示NO- cGMP途径可能在ALI中起重要作用。     

Genipin alleviates LPS-induced acute lung injury by inhibiting NF-κB and NLRP3 signaling pathways

Genipin has been reported to have anti-inflammatory effect. However, its role on lipopolysaccharide (LPS)-induced acute lung injury (ALI) has not been explored. This study aimed to evaluate the effect of genipin on murine model of acute lung injury induced by LPS. The mice were treated with genipin 1 h before LPS administration. 12 h later, the myeloperoxidase (MPO) in lung tissues and lung wet/dry ratio were detected. The levels of TNF-α, IL-1β and IL-6 in bronchoalveolar lavage fluid (BALF) were measured by ELISA. Apart from this, we use western blot to detect the protein expression in the NF-κB and NLRP3 signaling pathways. The results showed that the treatment of genipin markedly attenuated the lung wet/dry ratio and the MPO activity. Moreover, it also inhibited the levels of TNF-α, IL-1β, IL-6 in the BALF. In addition, genipin significantly inhibited LPS-induced NF-κB and NLRP3 activation. In conclusion, these results demonstrate that genipin protected against LPS-induced ALI through inhibiting NF-κB and NLRP3 signaling pathways.     

咯利普兰对脂多糖诱导的小鼠急性肺损伤的抑制作用

目的探讨磷酸二酯酶4(PDE4)抑制剂治疗急性肺损伤的作用机制。方法气道内滴入脂多糖3 mg.kg-1制备小鼠急性肺损伤模型,10 min后一次性ip不同剂量咯利普兰或地塞米松;同时设假手术和模型组。给药6 h后处死小鼠,观察肺湿重/干重比值和肺组织的病理改变;用细胞形态学方法计数支气管肺泡灌洗液(BALF)中白细胞和中性粒细胞;考马斯亮蓝法测定BALF总蛋白含量;髓过氧化物酶(MPO)活性测定试剂盒测定肺组织匀浆MPO活性;ELISA法测定肺组织匀浆肿瘤坏死因子α(TNF-α)含量;高效液相色谱法测定肺组织匀浆中cAMP-PDE和PDE4活性。结果小鼠气道内滴入脂多糖6 h后,与假手术组比较,模型组肺湿重/干重比值明显升高;肺组织病理观察可见肺血管和气道周围有大量中性粒细胞浸润;BALF中白细胞和中性粒细胞增多,蛋白含量增加;肺组织MPO活性、TNF-α水平、cAMP-PDE和PDE4活性升高。与模型组比较,咯利普兰(0.1,0.3及1.0 mg.kg-1)和地塞米松(0.5 mg.kg-1)可降低肺组织湿重/干重比值,降低BALF中白细胞总数、中性粒细胞数目和蛋白含量,改善肺组织病理变化,肺组织中MPO活性、TNF-α含量、cAMP-PDE和PDE4活性亦明显降低。结论咯利普兰治疗急性肺损伤的作用机制可能与抑制PDE4活性、抑制中性粒细胞黏附和趋化及降低TNF-α水平有关。     

Prenatal administration of the cytochrome P4501A inducer, Β-naphthoflavone (BNF), attenuates hyperoxic lung injury in newborn mice: implications for bronchopulmonary dysplasia (BPD) in premature infants

Supplemental oxygen contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. In this investigation, we tested the hypothesis that prenatal treatment of pregnant mice (C57BL/6J) with the cytochrome P450 (CYP)1A1 inducer, ß-napthoflavone (BNF), will lead to attenuation of lung injury in newborns (delivered from these dams) exposed to hyperoxia by mechanisms entailing transplacental induction of hepatic and pulmonary CYP1A enzymes. Pregnant mice were administered the vehicle corn oil (CO) or BNF (40 mg/kg), i.p., once daily for 3 days on gestational days (17-19), and newborns delivered from the mothers were either maintained in room air or exposed to hyperoxia (> 95% O₂) for 1-5 days. After 3-5 days of hyperoxia, the lungs of CO-treated mice showed neutrophil infiltration, pulmonary edema, and perivascular inflammation. On the other hand, BNF-pretreated neonatal mice showed decreased susceptibility to hyperoxic lung injury. These mice displayed marked induction of ethoxyresorufin O-deethylase (EROD) (CYP1A1) and methoxyresorufin O-demethylase (MROD) (CYP1A2) activities, and levels of the corresponding apoproteins and mRNA levels until PND 3 in liver, while CYP1A1 expression alone was augmented in the lung. Prenatal BNF did not significantly alter gene expression of pulmonary NAD(P)H quinone reductase (NQO1). Hyperoxia for 24-72 h resulted in increased pulmonary levels of the F₂-isoprostane 8-iso-PGF_2α, whose levels were decreased in mice prenatally exposed to BNF. In conclusion, our results suggest that prenatal BNF protects newborns against hyperoxic lung injury, presumably by detoxification of lipid hydroperoxides by CYP1A enzymes, a phenomenon that has implications for prevention of BPD in infants.     

N-乙酰半胱氨酸对成年大鼠高氧肺损伤氧化应激机制的影响

目的:研究N-乙酰半胱氨酸(NAC)对成年大鼠高氧肺损伤氧化应激机制的影响.方法:将48只健康成年雄性SD大鼠随机分为6组,其中5组置入动物实验氧舱内吸入100%浓度氧气,吸氧时间为24h、48h、72h、96h、96h、作为24h、48 h、72 h、96h组和NAC干预组,还有1组吸入空气0h作为0h组,即对照组;计算氧合指数,测定肺系数;测定肺组织和血清中MDA含量,SOD及T-AOC活性;观察肺脏组织病理学变化.结果:随高氧暴露时间延长,大鼠氧合指数逐渐下降,肺系数逐渐增加.肺组织和血清中MDA含量逐渐增加,96 h达到峰值,SOD、T-AOC活性逐渐下降,96 h达到最低值.与96h组比较,NAC干预组氧合改善,肺系数下降,MDA含量减少,SOD及T-AOC活性增加.结论:NAC通过减轻机体的氧化应激反应在一定程度上能够改善高氧所致成年大鼠急性肺损伤.     

Immunolocalization of Inhaled Recombinant Human Manganese Superoxide Dismutase Reveals a Prolonged Retention in Lung Surface Lining Fluids

Abstract

Oxygen-derived radicals play critical roles in many types of lung injuries involving environmental pollutants. Recently, intranasal insufflation of recombinant human manganese superoxide dismutase (rh-MnSOD) was reported to be efficacious against a hyperoxia model of oxidant lung injury in mice. We employed immunocytochemistry to examine the distribution, retention, and location of rh-MnSOD given to mice by intranasal insufflation. Mice were given a single dose (20 mg/kg) of either rh-MnSOD or bovine serum albumin and killed at 0.25, 4, 24, 48, or 120 h after treatment. Lungs were fixed by vascular perfusion. Lung sections from animals in the different time points and treatment groups were labeled with rabbit anti-rh-MnSOD antibody and studied at both light and electron microscopic levels. rh-MnSOD labeling was patchy but widely disseminated at the early time points and mainly localized in surface lining fluids and to a lesser extent on epithelial cell surfaces. The intensity of the labeling dropped off after 24 h and was virtually absent 120 h after treatment. There was little intracellular labeling of epithelial or interstitial cells or matrix labeling for rh-MnSOD. Alveolar macrophages had heavy labeling for rh-MnSOD in vesicles and scattered throughout their cytoplasm. rh-MnSOD appears to be cleared by a combination of mucociliary transport in the conducting airways and macrophage endocytosis in the gas exchange regions. rh-MnSOD given by intranasal insufflation delivered this protein to lung epithelial surfaces and protected against hyperoxia-induced injury, which suggests that oxygen-derived radicals in this compartment are important mediators of oxidant-induced lung injury.