Endotoxin-induced Lung Injury In Rats Research Articles

Effect of lidocaine on Rho/ROCK signaling pathway during endotoxin-induced acute lung injury in rats

Objective To evaluate the effect of lidocaine on Ras homologue(Rho)/Rho kinase(ROCK)signaling pathway during endotoxin-induced lung injury(LI) in rats. Methods Forty SPF male Wistar rats, aged 5-8 weeks, weighing 200-250 g, were divided into 5 groups(n=8 each)using a random number table method: control group(group C), lipopolysaccharide(LPS)group(group LPS)and lidocaine at 3 different doses groups(L1-3 groups). LI was induced by intraperitoneal LPS 5 mg/kg(0.1 ml). The equal volume of normal saline was given intraperitoneally in group C. Lidocaine 2, 4 and 8 mg/kg was intraperitoneally injected at 1 h before LPS administration in L1-3 groups, respectively.The animals were sacrificed at 6 h after LPS or normal saline administration.Broncho-alveolar lavage fluid(BALF)was collected for determination of concentrations of interleukin-1 beta(IL-1β), IL-6 and tumor necrosis factor-alpha(TNF-α)(by enzyme-linked immunosorbent assay). The lung tissues were obtained for examination of the pathological changes which were scored and for measurement of the wet/dry weight ratio(W/D ratio)and activities of myeloperoxidase(MPO)and for determination of the expression of Rho, ROCK1, ROCK2, myosin phosphatase target protein 1(MYPT1), phosphorylated MYPT1(p-MYPT1)and ZO-1(by Western blot). The phosphorylation of MYPT1 was calculated. Results Compared with group C, the activity of MPO, lung injury score, W/D ratio and concentrations of IL-1β, IL-6 and TNF-α in BALF were significantly increased, the expression of Rho, ROCK1 and ROCK2 was up-regulated, the phosphorylation of MYPT-1 was increased, and the expression of ZO-1 was down-regulated in the other four groups(P<0.05). Compared with group LPS, the activity of MPO, lung injury score, W/D ratio and concentrations of IL-1β, IL-6 and TNF-α in BALF were significantly decreased, the expression of Rho, ROCK1 and ROCK2 was down-regulated, the phosphorylation of MYPT-1 was decreased, and the expression of ZO-1 was up-regulated in L1-L3 groups(P<0.05). Conclusion Lidocaine can inhibit activation of Rho/ROCK signaling pathway during endotoxin-induced LI in rats, and the effect may be related to the anti-inflammatory mechanism of lidocaine. Key words: Lidocaine; Rho factor; rho-Associated kinases; Endotoxemia; Respiratory distress syndrome, adult

Effect of curcumin on phosphorylation of TAK1 during endotoxin-induced lung injury in rats

Objective To evaluate the effect of curcumin on the phosphorylation of transforming growth factor-β-activated kinase 1 (TAK1) during endotoxin-induced lung injury in rats. Methods Eighteen pathogen-free healthy male Sprague-Dawley rats, weighing 180-220 g, were divided into 3 groups (n=6 each) using a random number table: control group (group C), lung injury group (group LI) and curcumin group (group CUR). Lung injury was induced by intra-tracheal instillation of lipopolysaccharide 5 mg/kg in 1 ml of normal saline in LI and CUR groups.Curcumin 200 mg/kg was injected intraperitoneally at 30 min before establishment of the model in group CUR, while the equal volume of dimethyl sulfoxide was given instead in group LI.The rats were sacrificed at 12 h after administration of lipopolysaccharide or normal saline, and lungs were removed for determination of the expression of TAK1 and phosphorylated TAK1 (p-TAK1) in lung tissues.The right upper lobe of the lung was removed for determination of wet/dry weight ratio (W/D ratio). Results Compared with group C, the W/D ratio was significantly increased, and the expression of TAK1 and p-TAK1 was up-regulated in group LI (P<0.05). Compared with group LI, the W/D ratio was significantly decreased, and the expression of TAK1 and p-TAK1 was down-regulated in group CUR (P<0.05). Conclusion The mechanism by which curcumin attenuates endotoxin-induced lung injury is related to inhibition of the phosphorylation of TAK1 in lung tissues of rats. Key words: Curcumin; Endotoxemia; Respiratory distress syndrome, adult; Transforming growth factor beta

Prophylactic inhalation of L-alanyl-L-glutamine enhances heat shock protein 72 and attenuates endotoxin-induced lung injury in rats.

Studies have demonstrated that heat shock protein 70 (HSP70) plays an important role in the protection of stressed organisms. The development of strategies for enhancing HSPs expression may provide novel means of minimizing inflammatory lung conditions, such as acute lung injury. This study aimed to examine the effect of L-alanyl-L-glutamine (GLN) inhalation in enhancing pulmonary HSP72 (inducible HSP70) expression and attenuating lung damage in a model of acute lung injury induced by lipopolysaccharide (LPS) inhalation. The experimental rats were randomly assigned to one of four experimental groups: (1) NS: saline inhalation; (2) NS-LPS: pretreatment by saline inhalation 12 h before LPS inhalation; (3) GLN: glutamine inhalation; (4) GLN-LPS: pretreatment by glutamine inhalation 12 h before LPS inhalation. The results show that GLN compared with saline administration, led to significant increase in lung HSP72 both in non LPS-treated rats and LPS-treated rats. In LPS-treated rats, pretreatment by GLN inhalation produced less lung injury as evidenced by the decrease in lung injury score and dramatic decrease in lactate dehydrogenase (LDH) activity and polymorphonuclear leukocyte cell differentiation counts (PMN %) in the bronchoalveolar lavage fluid. The study indicates that prophylactic glutamine inhalation associated with the enhancement of HSP72 synthesis attenuates tissue damage in experimental lung injury.

Effect of heparin upon inflammatory reaction of endotoxin-induced acute lung injury in rat

To investigate the effects of heparin upon inflammatory reaction and associated mechanism of endotoxin-induced acute lung injury (ALI) in rat. Thirty-six male Sprague-Dawley rats were randomly divided into three equal groups namely: ALI group, heparin treatment group and normal control group. The ALI rats were induced by injecting endotoxin intravenously and sacrificed at 4 h after model establishment. The lung histology was scored by a modification of Smith technique. The albumin permeability of pulmonary microvascular (P(alb)) was measured by single nuclide tracer technique. Tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and von Willebrand factor (vWF) levels of serum were determined using commercial enzyme-linked immunosorbent assay kits. The expressions of lung tissue extacellular signal-regulated kinases (ERK)-1/2, P38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinases (JNK) were determined by Western blotting. The Smith lung injury score in heparin treatment group and ALI group were (5.00 +/- 1.26) and (8.00 +/- 1.09) respectively. The values were significantly higher than that of normal control group (0.67 +/- 0.52, both P < 0.01). However, the Smith lung injury score in heparin treatment group was significantly lower than that of ALI group (P < 0.01). The P(alb), TNF-alpha, IL-6 and vWF of heparin treatment group were (0.28 +/- 0.04), (1.92 +/- 0.35) microg/L, (1.22 +/- 0.13) ng/ml and (24.9 +/- 4.0) U/L respectively. The values were significantly higher than those of normal control group [0.20 +/- 0.02, (0.51 +/- 0.09) microg/L, (0.23 +/- 0.05) ng/ml and (14.0 +/- 3.0) U/L respectively, all P < 0.01] but significantly lower than those of ALI group [(0.38 +/- 0.04), (2.77 +/- 0.37) microg/L, (1.62 +/- 0.13) ng/ml and (31.8 +/- 7.5) U/L respectively, all P < 0.01]. The lung tissue levels of phospho-ERK1/2 and phospho-P38 MAPK expressions of heparin treatment group were markedly higher than those of normal control group, whereas markedly lower than those of ALI group. There was no marked difference of phospho-JNK expression in all three groups. Heparin markedly inhibits the expressions of phospho-ERK1/2 and phospho-P38 MAPK, down-regulates the inflammatory reaction, attenuates the endothelial permeability of pulmonary vasculature and significantly improves endotoxin-induced lung injury in rats.

The role of angiotensin-converting enzyme inhibition in endotoxin-induced lung injury in rats*

Cardiovascular Research Institute; University of California; San Francisco, CA (Neyrinck) Cardiovascular Research Institute; University of California; San Francisco, CA (Matthay) Departments of Anesthesia and Medicine; University of California; San Francisco, CA (Matthay) The authors have not disclosed any potential conflicts of interest.

Effects of caffeic acid phenethyl ester on lipopolysaccharide-induced lung injury in rats

Extracts of propolis, a natural beehive product, have been known for centuries to have a variety of beneficial medical properties, among which their anti-inflammatory effect is a major one. Caffeic acid phenethyl ester (CAPE), an active propolis component, has antimicrobial, anti-inflammatory, antioxidant, carcinostatic and immunomodulatory properties. In this study, we aimed to investigate the efficacy of CAPE in endotoxin-induced lung injury in rats. Lung injury was induced by a footpad injection of lipopolysaccharide (LPS). In the treatment group, 10 μmol kg −1 CAPE was injected intraperitoneally immediately after LPS injection. At 24 h after LPS and/or CAPE injection, blood and lung tissue specimens were collected. MDA levels and MPO activity in serum and lung tissue, serum total antioxidant levels, lung tissue Na +/K + ATP-ase activity and histopathological evaluation were determined to assess the efficacy of CAPE treatment. CAPE was found to be efficient in reducing inflammation and lung tissue damage induced by LPS in rats.

Hyperoxia-induced emphysematous changes in subacute phase of endotoxin-induced lung injury in rats.

We examined the effects of prolonged hyperoxia (75% O(2)) on lung structure and collagen metabolism in the subacute phase of lung injury induced by continuous infusion of endotoxin (LPS) in a rat model. Experimental groups included control, endotoxin alone, endotoxin plus hyperoxia, and hyperoxia alone. Endotoxin-treated rats received a bolus of LPS (10 mg/kg i.v.) followed by 500 microg.kg(-1).day(-1) in continuous infusion for 10 days. The bronchoalveolar lavage (BAL) fluid/plasma albumin concentration ratio, an index of capillary permeability, and neutrophil and macrophage counts in BAL fluid were highest in the endotoxin plus hyperoxia group. On pathological examination, prolonged hyperoxia exacerbated destruction of the alveolar wall and caused most prominent emphysematous changes in the endotoxin plus hyperoxia group. Lung tissue hydroxyproline concentration was significantly decreased in the hyperoxia group and increased in the endotoxin group. The latent forms of MMP-2 and MMP-9 increased in BAL fluid of the endotoxin- and/or hyperoxia-treated groups, whereas the activities of collagenase and gelatinase, and the active form of MMP-2 were all increased in the hyperoxia-treated groups. Added to endotoxin, prolonged hyperoxia degraded collagen, the major structural component of basement membranes, and caused emphysematous changes associated with activation of collagenase and MMP-2. Our observations suggest that, in the subacute phase of endotoxin-induced lung injury, prolonged hyperoxia causes pulmonary emphysematous changes with persistent injury to the alveolar capillary barrier. Collagenase and MMP-2 activated by hyperoxia, together with MMP-9, may play prominent roles in disruption of the alveolar basement membranes and degradation of collagen lining the alveolar walls.

Recombinant thrombomodulin prevents endotoxin-induced lung injury in rats by inhibiting leukocyte activation

Acute respiratory distress syndrome (ARDS) is a serious complication of sepsis. Thrombomodulin, an important endothelial anticoagulant, binds thrombin to generate activated protein C (APC). We have previously demonstrated that APC prevents endotoxin (ET)-induced pulmonary vascular injury by inhibiting activated leukocytes. We therefore examined whether recombinant human soluble thrombomodulin (rhs-TM) prevents activated leukocyte-induced pulmonary vascular injury in rats receiving ET. Intravenous administration of rhs-TM prevented ET-induced pulmonary accumulation of leukocytes and increase in pulmonary vascular permeability, as well as ET-induced histological changes, such as leukocyte infiltration and pulmonary interstitial edema. Dansyl-Glu-Gly-Arg-chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, did not prevent these effects of ET. rhs-TM did not prevent ET-induced pulmonary accumulation of leukocytes and pulmonary vascular injury in rats pretreated with DEGR-Xa. These results suggest that rhs-TM prevents ET-induced pulmonary vascular injury by inhibiting pulmonary accumulation of leukocytes and that this effect may be mediated primarily by APC generation.

Dibutyryl-cAMP Blocks Endotoxin-induced Lung Injury in Rats

We investigated the effect of dibutyryl-cAMP pretreatment on endotoxin-induced hemodynamic changes and lung vascular injury in rats. In catheter-implanted, unanesthetized rats, intraperitoneal injection of Salmonella enteritidis endotoxin (2 mg/kg) decreased cardiac output and systemic blood pressure while increasing total pulmonary vascular resistance. Db-cAMP (1 mg given intraperitoneally every 30 min), although not significantly affecting cardiac output and systemic blood pressure, blocked the increase in total pulmonary resistance caused by endotoxin. Ninety minutes after intraperitoneal endotoxin injection, perfused lungs from endotoxin-treated rats exhibited increased pulmonary vascular permeability, as assessed by increased extravascular accumulation of 125I-albumin and water. Db-cAMP treatment in vivo markedly attenuated the increases in lung albumin leak index and wet-to-dry weight ratio caused by endotoxin without affecting lung microvascular pressures. This protective action of db-cAMP is not due to its effect on prostaglandin or leukotriene synthesis since endotoxin-stimulated increases in lung tissue 6-keto-prostaglandin F1 alpha, thromboxane B2 and leukotriene C4 were not inhibited. We conclude that db-cAMP blocks endotoxin-induced lung injury in the rat by a mechanism independent of eicosanoid products and speculate that agents that increase intracellular cAMP may be therapeutically useful in acute lung vascular injury.

Endotoxin-induced lung injury in rats: role of eicosanoids

We studied lung vascular injury and quantitated lung eicosanoids in rats after intraperitoneal injection of Salmonella enteritidis endotoxin. Within 40 min after endotoxin injection (20 mg/kg), lung tissue thromboxane B2 doubled, although 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) increased by 8- to 10-fold. Lung 5-hydroxyeicosatetraenoic acid and leukotriene C4 were variably increased by endotoxin. The levels of all eicosanoids returned to base line 6 h after endotoxin challenge. Lung vascular injury, as assessed by the extravascular accumulation of 125I-albumin and water in isolated perfused lungs, was observed 90 min after endotoxin injection (0.02-20 mg/kg) in vivo. Inhibition of the cyclooxygenase pathway with indomethacin and the lipoxygenase pathway with diethylcarbamazine and 2-(12-hydroxydodeca-5,10-dinyl)-3,5,6-trimethyl-1,4-benzoqui none failed to attenuate endotoxin-induced lung injury. In addition, essential fatty acid deficiency, which markedly reduced lung tissue levels of 6-keto-PGF1 alpha, thromboxane B2, and leukotriene C4, did not protect against endotoxin injury. We conclude that although lung eicosanoids are activated during endotoxemia, they do not play a crucial role in the development of acute lung vascular injury in rats.