Research On Expression Of Neutrophil Gelatinase–associated Lipocalin (NGAL) In Mouse Model Of Ventilator-associated Lung Injury

Backgroud:Mechanical ventilation is known to induce and aggravate lung injury, despite its lifesaving effects. One of the underlying mechanisms is biotrauma, in which an inflammation and innate immunity response play a crucial role. Biological markers of ventilator associated lung injury(VALI) is important for early diagnosis and prognostic of VALI, and provide new insight into the mechanisms of VALI. To date the biomarkers of VALI reported almost are the key mediators in the pathway of inflammatory and immunological network, but the precise function of each mediators has not been completely understood. Currently, there have been no studies to validate the sensitivity or specificity of any of biological markers which target for early diagnosis and treatment for VALI. So more studies of identifying novel, senstive and specific biological markers are very needed. Neutrophil (polymorphonuclear leukocytes, PMN) has been shown to play an important role in the progression of VALI, which contribute to the development of VALI, and may lie at the centre of a positive feedback loop perpetuating lung injury. Therefore PMN or key mediators in the PMN regulatory pathway are likely to be the pomising molecular candidates for VALI. Neutrophil gelatinase–associated lipocalin (NGAL) is produced mainly by neutrophil and also secreted by alveolar epithelial cell. And the functional role of NGAL involved in the regulation of alveolar epithelial cell has not been elucidated. The literature of NGAL associated VILI are rare. Moreover, status of activation of neutrophil can be reflected by the measurement of NGAL, which extensively participate in the progress of inflammation and immunity. As an acute phase and a secreatory phase protein, NGAL are easily detected in the serum and bronchoalveolar lavage fluid (BAL). Based on the above biological characters of NGAL as biomarker, we proposed that NGAL could be an important target of VALI for more study, which as sensor of mechanical force and inflammatory stimulus play an crucial role in the pathogenesis of VALIObjective:Mice with health or injurious lungs were subjected in different strategy of mechanical ventilation to construct murine model of acute lung injury. Our purpose were to elucidate the effects of mechanical ventilation on expression of NGAL in murine models, in hope of identifying NGAL as a novel and potential biomarker for VALI. NGAL maybe emerge as powerful tools for the early prediction of patients with high risk of VALI and provide mechanistic insights and new therory of better understand the mechanisms of VALI.Contents and methods:Part 1: Male C57BL/6 mice with health lung were ventilated with low-PIP(PIP=15cmH2O2, PEEP=0 cmH2O2), high-PIP(PIP=50cmH2O2, PEEP=2.5 cmH2O2 ) , high-volume ( Vt=30ml/kg , PEEP=0cmH2O2 ) and low-volume(Vt=6ml/kg, PEEP=5cmH2O2)for 2h, respectively. We detected the changes of expression of NGAL, MMP-9 and IL-1βmRNA by the methods of semi-quantitive RT-PCR and realtime RT-PCR. At the same time, we observed the NGAL protein levels in murine lung tissue, serum and BAL by the means of Western blot analysis. In addition, levels of MMP-9 and IL-1βin serum and BAL were measured by ELISA. To evaluate spatial localization of NGAL expression, we performed triple immunohistochemical staining in murine lung tissue samples from the murine injurious ventilation models.Part 2: Health mice were subjected to high-volume ( Vt=30ml/kg , PEEP=0cmH2O2)or low -volume(Vt=6ml/kg, PEEP=5cmH2O2)mechanical ventilation for 2h or 4h. We study whether ventilation time frame could affect the expression of NGAL, MMP-9 and IL-1β. (Methods see the above) .Part 3: Mice were received intratracheal of LPS at a dose of either 2.5mg/kg or 5 mg/kg in 200μl of saline solution. After 24h of first insult of LPS, mice were randomized to mechanical ventilation for 2 h with high-volume(Vt=30ml/kg, PEEP=0cmH2O2)or low-volume(Vt=6ml/kg, PEEP=5cmH2O2).We evaluated NGAL, MMP-9 and IL-1βchanges of expression in inflamed lungs of mouse after LPS- pretreated at different ventilation strategy. (Methods see the above) .Results:Part 1: NGAL expression were increased in different ventilation group, and the level were significant elevated in injurious ventilation groups. The higher level of NGAL expression were observed in the high-PIP and high-volume group, 41.9 and 3.29-fold increase, respectively. In the same way, the amount of NGAL protein in BAL increased almost 4 and 1.8-fold in this two groups. In addition, NGAL protein in serum and lung tissue sample increased in different degree. Otherwise, We found strong murine NGAL expression in the vascular endothelium, lung epithelium cells and infiltrating neutrophils. These data implied important role for NGAL as sensor of mechanical stimulus and involving in the pathogenesis of VALI. On the other side, MMP-9 and IL-1βexpression increased in parallel with the expression of NGAL in high-volume ventilation group.Part 2: Dramatically up-regulation of NGAL was also detected in high-volume ventilation for 2h or 4h (3.29 and 8.75-fold increase, respectively), indicating that NGAL expression maybe positively related with the time of ventilation in injurious ventilation groups. But in the low-volume group, expression of NGAL were no significant changes, keeping at low expression levels, which suggest NGAL were powerful tools for the early prediction of VALI.Part 3: There was significant increase in NGAL expression in LPS-injuryed lungs in mice 24h after receiving intratracheal LPS treatment. In the murine LPS combined low-volume group, the NGAL gene and protein in BAL were the highest levels of expression (119.43 and 6.1-fold increase) than only LPS and ventilation group. Moreover, MMP-9 and IL-1βexpression also increased obviously in BAL and serum. These results indicated mechanical ventilation and LPS synergistically induce NGAL production and release. Non-injouris ventilation strategy combined LPS were enough to induced lung injury.Clonclusions:1. NGAL expression were increased in different ventilation strategy, and its level were significant elevated in injurious ventilation groups.2. NGAL expression positively related with the time of ventilation in high-volume ventilation strategy.3. LPS could stimulate the up-regulation of NGAL expression. And the function of LPS and mechanical ventilation induced NGAL production and release were synergistical.4: NGAL could be as a potential and novel biomarker for the early prediction of VALI. And monitoring NGAL changes in serum and BAL were useful tool to diagnose patients with high risk factor of VALI. .