| ObjectiveAcute lung injury, particularly its most severe form, acute respiratory distress syndrome, is a critical complication of stress situations. Although the exact pathogenetic mechanisms remain poorly understood, multiple risk factors for acute lung injury have been identified, with bacterial sepsis representing a common predisposing condition. Intraperitoneal administration of LPS, a glycolipid of the outermost membrane of gram-negative bacteria, is a well accepted experimental model of sepsis-induced acute lung injury. Recent studies have shown that ceramide has marked proapoptotic and proinflammatory properties through serving as a second messenger in the signal transduction pathway triggered by several agents of stress and extracellular stimuli such as LPS. Moreover, acidic sphingomyelinase (ASMase), one of SMases, is often associated with ceramide increase in stress response. Several in vivo findings suggest that inhibition of ASMase with pharmacological agents can attenuate the lung edema induced by PAF, and reduced lethality caused by sepsis and hepatic ischemia-reperfusion injury. Thus, we hypothesized that inhibition of ASMase can be a possible therapeutic approach for acute lung injury. In this study, we investigated whether imipramine, one of pharmacological inhibitors of ASMase could exert protective effects in endotoxin-induced acute lung injury.MethodsMice were randomly divided into four groups:LPS+imipramine, LPS+vehicle, sham+imipramine and sham+vehicle. Mice were administered intraperitoneally with LPS at 20mg/kg or an equal volume of the vehicle. Mice were treated with imipramine at 25mg/kg or an equal volume of the vehicle 30 min prior to LPS administration. Two sets of experiments were performed. In the first set of experiments, mice were humanely killed at 6 h,12 h and 18 h after LPS administration. Bronchoalveolar lavage fluid, serum and lung tissues were collected for further analysis. In the second set of experiments, survival rates of the mice were checked every 12 h during 6 days for survival analysis. Results1. Intraperitoneal administration of LPS is a well accepted experimental model of sepsis-induced acute lung injury. Morphological examination of lung sections after LPS administration displayed infiltration of inflammatory cells, alveolar congestion and haemorrage and marked swelling of the alveolar walls showing that acute lung injury model was successfully induced in our experiment.2. Administration of imipramine at a dose of 25 mg/kg 30 min before LPS challenge significantly increased the survival rate of mice administrated with LPS.3. The lung tissue histopathological changes of the mice treated with imipramine before LPS injection was significantly attenuated and the total severity scores of lung injury were significantly reduced at 12h (9.65±0.43VS11.9±0.80, P<0.05) and18h (11.05±0.73VS14.3±0.39, P<0.05) in the imipramine pretreated group, compared to the group where LPS alone was used.4. Wet/dry ratio was dramatically elevated in LPS-administrated mice and reached a peak of 5.24±0.11 at 12h.Imipramine pretreatment showed a significant reduction in the wet/dry ratio at 6h (4.80±0.04VS5.16±0.05, P< 0.05),12h (4.87±0.11VS5.24±0.11, P<0.05)and 18h(4.77±0.07VS5.14±0.03, P<0.05).5. LPS administration boosted neutrophil sequestration. We found that MPO activity increased sharply after LPS challenge and reached a peak of 37.55±5.67 Units/min/mg protein at 18h, and MPO activity was significantly reduced at 12h (10.10±1.28 Units/min/mg protein VS28.48±7.53 Units/min/mg protein, P< 0.05) and 18h (11.39±1.36 Units/min/mg protein VS37.55±5.67 Units/min/mg protein, P<0.05) in the LPS+imipramine group as compared to the LPS+vehicle group.6. We detected the pro-inflammatory cytokine TNF-αand anti-inflammatory cytokine IL-10 by ELISA. Basal levels of TNF-αand IL-10 in bronchoalveolar lavage fluid at 6h were 218.50±22.11pg/ml,83.02±15.33pg/ml, respectively. The levels of TNF-a and IL-10 in bronchoalveolar lavage fluid were elevated in LPS-treated mice. TNF-a level reached a peak of 3860.31±274.80pg/ml at 6h, while IL-10 level reached a peak of 2809.00±143.70pg/ml at 6h. Pretreatment with imipramine significantly decreased TNF-α(6h: 2204.00±242.30VS3860.31±274.80,12h:2236.00±853.90VS3612.00±412.70, P<0.05) and IL-10 (6h:1645.00±222.50VS2809.00±143.70,12h: 1463.00±137.80VS2117.00±117.20, P<0.05) level at 6h and 12h.7. TNF-a and IL-10 level in serum after LPS challenge followed the same pattern in the bronchoalveolar lavage fluid findings noted above. Basal levels of TNF-a and IL-10 in serum at 6h were 228.90±54.72pg/ml,236.55±45.92pg/ml, respectively. TNF-a level reached a peak of 3619.76±376.30pg/ml at 6h, while IL-10 level reached a peak of 3261.16±484.70pg/ml at 6h. Pretreatment with imipramine significantly decreased TNF-α(6h:1871.00±425.70VS3619.76±376.30,12h: 1250.00±329.40VS2832.00±201.10, P<0.05) and IL-10 (6h: 1755.00±281.50VS3261.16±484.70,12h:1283.00±153.80VS2221.00±330.00, P<0.05) level in serum at 6h and 12h.8. TNF-a mRNA levels at 6 h and 12 h, IL-6 mRNA levels at 18h and IL-10 mRNA level at 6h were significantly reduced after administration of imipramine.9. LPS administration induced a significant increase of NF-κB DNA binding activity. However, treatment with imipramine 30 min before LPS challenge significantly inhibited the up-regulation of NF-κB DNA binding activity and NF-κB p65 level as compared to the LPS+vehicle group.10. Treatment with imipramine 30 min before LPS challenge significantly inhibited the increase of Bax protein and the reduction of Bcl-2 protein, compared to the LPS+vehicle group.11. Compared with the group where LPS alone was used, imipramine treatment shows marked reduction in the level of pp38, pASK1 and Txnip.Conclusions1. Imipramine improves survival in mice challenged with lethal dose LPS. 2. The anti-inflammatory effects of imipramine in the LPS-induced ALI was due to the suppression of NF-κB DNA binding activity.3. The anti-apoptosis effects of imipramine in the LPS-induced ALI were due to the suppression of ceramide-Txnip-pASKl-pp38 signal transduction pathway. |
PDF Full Text Request