| Part 1 The model of ventilation induced lung injury in rats in vivoObjective To build the model of ventilator induced lung injury in rats in vivo and to investigate the pathophysiological changes and the mechanisms of the injurious lungs induced by ventilation.Methods Forty Sprague-Dawley rats weighing 300–350g were randomly divided into the following experimental groups (10 rats in each group): (1) A group: rats were unventilated; (2) B group: rats were ventilated with 40 ml/kg tidal volume room air for one hours; (3) C group: rats were ventilated with 40ml/kg tidal volume room air for two hours; (4) D group: rats were ventilated with 40ml/kg tidal volume room air for four hours. The samples of pulmonary tissue and lung lavage fluid were collected after experiments. The expression of MIP-2,ICAM-1,TNF-a,p22phox mRNA in lung tissue was measured by reverse transcriptase-polymerase chain reaction (RT-PCR); Apoptosis of the lung cells was assayed with terminal deoxynucleodityl transferase-mediated nick-end labeling (TUNEL) method; NF-κB activity was assayed with EMSA; NF-κB p65 was detected with Western Blot; Lung pathological changes were examined with optical microscopy; Total protein, wet/dry ratios (W/D), MDA,SOD,myeloperoxidase (MPO) activity and neutrophil counts of the lung tissue or lavage fluid were measured with corresponding methods.Results A group has no significant ALI findings. After mechanical ventilation, the B,C,D group showed high levels of intra-alveolar exudates, hyaline membrane formation, inflammatory cell infiltration, intra-alveolar hemorrhage, and interstitial edema. The degree of ALI in B,C,D group was remarkably aggravated with the duration of mechanical ventilation. Compared with A group, ALI scores in B,C,D group was significanty elevated(P<0.001), and remarkably increased with the duration of mechanical ventilation(P<0.01); Compared with A group, W/D ratios,MPO,total proteins,WBC,neutrophil counts,AI was significantly elevated in B,C,D group(P<0.01 or 0.001), and remarkably increased with the duration of mechanical ventilation(P<0.01 or 0.01 or 0.001);. Compared with A group, SOD in B,C,D group was significantly increased(P<0.05 or 0.001); Compared with B group, SOD in C group has no remarkably difference and D group shows a significant decreases(P<0.05); Compared with C group, D group shows a significant decreased SOD(P<0.05). Compared with A group, MDA in B,C,D group was significantly increased(P< 0.001); Compared with B group, MDA in C group has no remarkably difference and D group shows a significant increases(P<0.01); Compared with C group, D group shows a remarkable increased MDA(P<0.01). Compared with A group, the expression of MIP-2,ICAM-1,TNF-a,p22phox mRNA in B,C,D group was significanty elevated(P<0.001), and remarkably increased with the duration of mechanical ventilation(P<0.05 or 0.01); Compared with A group, NF-κB activity and NF-κB p65 in B,C,D group was significanty elevated(P<0.01), and remarkably increased with the duration of mechanical ventilation(P<0.05 or 0.01).Conclusion High volume ventilator produced significant acute lung injury in rat, which includes lung oedema,Leukocytic infiltrate,acute inflammatory reaction,oxidative stress and extensive lung cell apoptosiss. Part 2 The activation of RAS system on ventilator induced lung injury in ratObjective To investigate the activation of ROS system on ventilator induced lung injury in rat.Methods The study was divided into two parts. (一)twenty-four Sprague-Dawley rats weighing 300–350g were randomly divided into the following experimental groups (6 rats in each group): (1) B1 group: rats were unventilated; (2) B2 group: rats were ventilated with 40 ml/kg tidal volume room air for one hours; (3) B3 group: rats were ventilated with 40ml/kg tidal volume room air for two hours; (4) B4 group: rats were ventilated with 40ml/kg tidal volume room air for four hours. (二)twenty-four Sprague-Dawley rats weighing 300–350g were randomly divided into the following experimental groups (6 rats in each group): (1) C1 group: rats were unventilated; (2) C2 group: rats were ventilated with 10 ml/kg tidal volume room air for one hours; (3) C3 group: rats were ventilated with 20ml/kg tidal volume room air for two hours; (4) C4 group: rats were ventilated with 40ml/kg tidal volume room air for four hours. The samples of pulmonary tissue and lung lavage fluid were collected after experiments. The expression of ANG,AT1,ACE in lung tissue was measured by reverse transcriptase-polymerase chain reaction (RT-PCR); ANG II in lung tissues was detected with ELISA; ACE protein in lung tissues was measured by Western Blot; Lung pathological changes were examined with optical microscopy; Total protein, wet/dry ratios (W/D),MPO activity and neutrophil counts of the lung tissue or lavage fluid were measured with corresponding methods.Results B1 group has no significant ALI findings. After mechanical ventilation, the B2,B3,B4 group showed high levels of intra-alveolar exudates, hyaline membrane formation, inflammatory cell infiltration, intra-alveolar hemorrhage, and interstitial edema. Compared with B1 group, ALI scores in B2,B3,B4 group was significanty elevated(P<0.001), and remarkably increased with the duration of mechanical ventilation(P<0.01); There was no significant ALI findings in C1,C2 group. C3,C4 showed significant ALI findings. Compared with C1,C2 group, ALI scores in C3, C4 group was significanty increased(P<0.01); Compared with C3 group, ALI scores in C4 group was significanty increased(P<0.01). Compared with B1 group, W/D ratios,MPO,total proteins,neutrophil counts was significantly elevated in B2,B3,B4 group(P<0.01), and remarkably increased with the duration of mechanical ventilation(P<0.05 or 0.01); Compared with C1,C2 group , W/D ratios,MPO,total proteins,neutrophil counts in C3,C4 group was significanty increased(P<0.05 or 0.01). Compared with C3 group, W/D ratios,MPO,total proteins,neutrophil counts in C4 group was significanty increased(P<0.05 or 0.01); Compared with B1 group,ANG,AT1,ACE,ANG II in B2,B3,B4 group was significanty elevated(P<0.01), and remarkably increased with the duration of mechanical ventilation(P<0.05 or 0.01); There was no significant difference about ANG,AT1,ACE,ANG II in C1,C2 group; Compared with C1,C2 group, ANG,AT1,ACE,ANG II in C3,C4 group was significanty increased(P<0.05 or 0.01); Compared with C3 group, ANG,AT1,ACE,ANG II in C4 group was significanty increased(P<0.05 or 0.01).Conclusion High volume ventilation produced significant VILI and remarkably activated the RAS system, and significantly increased the level of ANG II and AT1 in lung tissue, which probably play an important role in VILI.Part 3 ANG II upregulate proinflammatory cytokine expression in A549 cells0bjective This experiment is aimed to study the efects of angiotensinⅡon NF-κB and IL-8 in A549 cells.Methods A549 cells were cultured in vitro and treated with ANG II at a 10-6mmol/L concentration for 0,1,2,4h. NF-κB activity was measured by EMSA and NF-κB p65 was detected with Western Blot. IL-8 mRNA was detected with RT-PCR.Results Compared with D1 group, D2,D3,D4 group shows significant increased NF-κB activity(P<0.01); Compared with D2 group, D3,D4 group significantly increased NF-κB activity(P<0.01); Compared with D3 group, D4 group shows significant increased NF-κB activity(P<0.01). Compared with D1 group, D2,D3,D4 group shows significant increased NF-κB p65 expression(P<0.01); Compared with D2 group, D3,D4 group significantly increased NF-κB p65 expression(P<0.05 or 0.01); Compared with D3 group, D4 group shows significant increased NF-κB p65 expression(P<0.01). Compared with D1 group, D2,D3,D4 group shows significant increased IL-8 mRNA expression(P<0.01); Compared with D2 group, D3,D4 group significantly increased IL-8 mRNA expression(P<0.05 or 0.01); Compared with D3 group, D4 group shows significant increased IL-8 mRNA expression(P<0.01).Conclusion ANG II significantly increased NF-κB activity and IL-8 mRNA expression in A549 cells, which probably play an important role in VILI.Part 4 Losartan attenuates Ventilator-induced lung injuryObjective Forty Sprague-Dawley rats weighing 300–350g were randomly divided into the following experimental groups (10 rats in each group): (1) a group: rats were unventilated; (2) b group: rats were ventilated with 8 ml/kg tidal volume room air for two hours; (3) c group: rats were ventilated with 40ml/kg tidal volume room air for two hours; (4) d group: rats were pretreated with Losartan (30mg/kg, i.p.) prior to high volume ventilation. The samples of pulmonary tissue and lung lavage fluid were collected after experiments. The expression of angiotensinogen and AT1 receptor mRNA in lung tissue was measured by reverse transcriptase-polymerase chain reaction (RT-PCR); MIP-2,ANG II in lung was detected with ELISA; Apoptosis of the lung cells was assayed with terminal deoxynucleodityl transferase-mediated nick-end labeling (TUNEL) method; NF-κB activity was measured by EMSA and NF-κB p65 was detected with Western Blot.Lung pathological changes were examined with optical microscopy; Total protein, wet/dry ratios (W/D), MDA,SOD,myeloperoxidase (MPO) activity and neutrophil counts of the lung tissue or lavage fluid were measured with corresponding methods.Results Compared with control or low volume ventilation, high volume ventilation caused significant ventilator-induced lung injury and increased the expression of ANG II,angiotensinogen and AT1 receptor mRNA in the lung. Compared with LVT or control group, total protein, the number of apoptotic cells, MIP-2,MDA,W/D ratio, MPO activity and neutrophil counts were significantly higher in c group. NF-κB activity and NF-κB p65 were also significantly increased in c group than a,b group. however, SOD in lung tissue was significantly decreased in c group than a,b group. Pretreatment with Losartan attenuated ventilator-induced lung injury, and prevented the increase in total protein , NF-κB activity,NF-κB p65,MIP-2,the number of apoptotic cells, W/D ratio, MPO and neutrophil counts caused by high volume ventilation.Conclusion Our study indicates that high volume ventilation causes remarkable lung injury and upregulates angiotensinogen and AT1 receptor expression of in the lung, and that Losartan , a selective inhibitor of subtype AT1 receptors for angiotensin II , can relieves acute lung injury caused by high volume ventilation. |
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