Mechanisms Of Mitogen-activated Protein Kinases (MAPK) In Ventilation-induced Lung Injury

Part 1 The model of ventilation induced lung injury in rats in vivoObjective To build the model of ventilation associated lung injury in rats in vivo and to investigate the pathophysiological changes and the mechanisms of the injurious lungs induced by ventilation.Methods 48 specified-pathogens free male Sprague-Dawley rats were randomized into three groups (n=16): GroupC: control group, with non-ventilation ; Group LV , with low Vt ventilation(Vt = 6ml/kg , 40 breathes /min, PEEP = 0 and FiO2 = 21%); Group HV, an injurious strategy, using a high Vt and no PEEP(42ml/kg, 40 breathes /min, PEEP = 0 and FiO2 = 21% ) . After 4h of ventilation, the rats were killed by exanguination and the samples were obtained. Arterial gas analysis, lung permeability index (LPI), lung wet/dry weight ratio(W/D) and the numbers of inflammatory cells in the bronchoalveolar lavage fluid (BALF) were performmed . Cytokine concentration of TNF-α and MIP-2 in lung tissues, BALF and plasma were detected by enzyme-linked immunosorbent assay(ELISA). The changes of histopathology with H&E staining and the ultramicrostructure of the pulmonary cells were also observed by light and transmission electron microscope(TEM).Results In the current study, the rats treated with high Vt ventilation for 4h had developed severe lung injury with respiratory failure .That was evidenced by arterial gas analysis , increases of lung injury parameters (all P < 0.01 versus the control group) and the pathological changes. In HV group, the concentrations of TNF-α and MIP-2 in the lung homogenate, BALF and plasma were increased significantly as compared with the groups of C and LV (P<0.01). Total lung inflammatory cells like neutrophils , macrophage and lymph cells in BALF , the lung injurious scores , even the percent of neutrophils in the HV group were increased significantly as compared with groups of C and LV too ; Otherwise , the neutrophils , macrophage and lymph cells which infiltrated in the perivascular , peribronchial and alveolar were increased significantly. The exudates, hemorrhage and hyaline member forming also presented in the alveoli and thichening of alveoar walls occurred. The I and II alveolar epithelial cells were severe injuried in HV group as compared with the other groups detected by light microscope and TEM (P<0.05).Conclusion These results suggested that the high tidal volume ventilation could cause the pulmonary structure destroy , lung edema and also caused the severe inflammatory response represented by the release of cytokines largely and infiltration of PMN , macrophage and lymphocyte into lungs . That is to say ventilation associated lung injury is not only a mechanical effect, but also is accompanied with a inflammatory damage which has been called the biotrauma , and this may be the essence of the pathophysiological changes for VILI. Part 2The changes of the early response genes expression in lungs due to high tidal volume ventilation in ratsObjective To investigate the expression changes of Egr-1, IL-1β and C-jun mRNA and protein in the lung injuried by high tidal volume ventilation and to find the relationship between the lung injury and these genes expression.Methods Forty male SD rats weighing 250-320 g were randomly divided into 5 groups (n=8 each ): group A received no mechanical ventilation; group B-E received mechanical ventilation for 30(B), 60(C), 90(D) and 120(E) minutes. The animals were anesthetized with intraperitoneal 3% pentobarbital sodium 35 mg/kg , tracheostomized and mechanically ventilated ( Vt = 42 ml/kg, RR= 40 bmp, I:E=1:2, FiO₂=21%, PEEP = 0 cmH₂O ) with a ventilator for small animals. Arterial blood samples were taken for blood gas analysis. The lungs were removed for microscopic examination using HE straining .The expression of Egr-1, C-jun and IL-1β mRNA and proteins was detected by RT-PCR and immunohistochemical technique, respectively.Results There was no significant dfference in PaO₂ and SaO₂ among five groups . PaCO₂ was signifcantly decreased in group B and C compared to group A while it was increased in group E as compared with group B and C . The expression of Egr-1 , C-jun and IL-1β mRNA and protein was significantly increased by mechanical ventilation in a duration-dependent manner .Hitological studies demanstrated that the damage to the lung was correlated with the duration of mechanical ventilation in terms of perivascular inflammatory cell infiltration , exudates and hemorrhage in the alveoli and thichening of alveoar walls.Conclusion The current data suggested that mechanical ventilation activated and upregulated the early response genes in the lung tissues which might be involved in the underling mechanism of lung damage induced by mechanical ventilation. Part 3The effects of mitogen-activated protein kinases pathway on ventilation induced lung injury in rats in vivoObjective Both clinical and basic research have demonstrated that injurious ventilation strategies mighgt induce the lung injury, but the precise mechanism was unknown up to now .This study aimed to investigate the effects of the mitogen-activated protein kinases (MAPK) on ventilation induced lung injury in rats in vivo. Methods Anesthetized male SD rats were randomized into 12 groups(n=6 each): control group(C) received no mechanical ventilation; Low Vt ventilation group(LV) received mechanical ventilation with Vt = 6 ml/kg, RR= 40 bmp, I:E=1:2, FiO₂=21%, positive end-expiratory pressure = 0 cmH₂O and high Vt ventilation group (HV)received mechanical ventilation with the same respiratory parameters as the LV group but the Vt was 42 ml/kg and the following groups of the inhibitors of MAPK involving SP600125, PD98059 and SB203580 pretreated the animals in each group respectively 30 minutes before ventilation; After 4h ventilation, the levels of activation and protein expression of JNK/p-JKN, ERK/p-ERK and p38/p-p38 were analysised by western blot assay; Cytokine concentration of TNF-α and MIP-2 in the lung homogenate, bronchoalveolar lavage fluid (BALF) and plasma were detected by ELISA and the lung injury parameters were observed as the first part of this study did. Results In adult rats, the low and high Vt ventilation might significantly elevate the levels of phosphorylation of ERK-1/2, JNK and p38(just in HV group), as well as the concentration of TNF-α and MIP-2 in the lung homogenate, BALF and plasma , in particular, the lung injury in HVgroup was more severe as compared with the groups of C and LV (P<0.01). The MAPK inhibitors of SP600125, SB203580 and PD98059 attenuated the activation of JNK , ERK-1/2 and p38 induced by high Vt ventilation and reduced the concentrations of TNF-α and MIP-2 remarkably too (P<0.05 or 0.01).On the other hand, The lung injury was attenuated by these inhibitors from the evidence of lung injury parameters : the decrease of W/D, LPI and inflammatory cells in the lungs and even, the lung injury scores in the groups of LV and HV as compared with the unpretreatment groups , but they were not restored to the status of the control group.Conclusion The results of our study show that mechanical ventilation triggers specific signalling pathways, such as the mitogen-activated protein kinase pathways, which may contribute to the lung histology damage and pulmonary inflammation with the more recruitment and cytokines release .Part 4The effects of mitogen-activated protein kinases pathway on the activation of NF-κB induced by mechanical ventilation in rats in vivoObjective To investigate the effects of mitogen-activated protein kinase pathway on the activation of NF-κB induced by mechanical ventilation , exporing the possible mechanism of ventilation- induced lung injury.Methods Anesthetized male SD rats were randomized into 12 groups(n=6 each): control group(C) received no mechanical ventilation, groups LV and HV which received the low (V_t = 6 ml/kg) or high (V_T = 42 ml/kg) tidal volume ventilation with RR = 40 bmp, I:E =1:2, FiO₂ = 21% and no PEEP in both groups , and even the following groups of SP600125, PD98059 and SB203580 pretreated the animals in each group respectively 30 minutes before ventilation. The expression level of NF-κB p65 was detected by Western blotting. Immunohistochemical test were obtained to detect the expression and location of NF-κB and I-κBβ in the lung cells. Electrophoresis mobility shift assay was used to analyze the activation of NF-κB in the lungs of groups.Results The levels of NF-κB p65 were elevated significantly in HV and LV groups than that of group C (P<0.05), but it was more higher in group HV (P<0.01). NF-κB was activated in both of LV and HVgroups and displayed stronger DNA-binding activity in the HVgroup as comparied with the groups of C and LV(P<0.05); The number of positive cells located in the nuclear was increased significantly in HVgroup too; However , the expression of I-κBβ in the HV group was decreased significantly as compared with the other groups; But the activation and expression level of NF-κB were attenuated by the inhibitors of MAPK involving SP600125 , PD98059 and SB203580 respectively as the activation of JNK , ERK and p38 were suppressed at the same time. Surprisingly, the expression of I-κBβ was properly converse to that of NF-κB in the same time (P<0.05).Conclusions This result showed that NF-kB were activated during the lung injury induced by ventilation and the inhibitors of MAPK could inhibit the activation of NF-κB which might be through the attenuation the degradation of I-κBβ , All of these means that MAPK, at least in part, may participate in the activation of NF-kB induced by ventilation which may be involved in the underlying mecahnism of lung damage induced by mechanical ventilation . Part 5The effects of MAPK on the genes expression of the characteristic proteins from I and II alveolar epithelial cells in ventilation induced lung injuryObjective To investigate the effect of MAPK on the genes expression of the characteristic proteins from I and II alveolar epithelial cells during ventilation induced lung injury in rats.Methods Anesthetized male SD rats were randomized into six groups(n = 6 each): control group(C) received no mechanical ventilation, groups LV and HV with low (Vt= 6 ml/kg) or high (Vt = 42 ml/kg) tidal volume ventilation groups which received mechanical ventilation with RR = 40 bmp, I:E=1:2, FiO₂=21%, positive end-expiratory pressure = 0 cmH₂O for both groups , and even followed with the groups of the inhibitors of MAPK involving SP60125, PD98059 and SB203580 pretreated animals just in HV group. After 4h ventilation , the animals in each groups were killed and the lungs were removed for detect the expression of SP-A, B, C and RTI40 mRNA by RT-PCR.Results There was no significant difference in the expression of SP-A, B, C and RTI40 mRNA between C and LV groups while the expression of SP-A and SP-C mRNA were decreased but RTI40 mRNA increased significantly in group HV as compared with groups C and LV (P< 0.05 or < 0.01) .The expression of SP-B was no significant difference among six groups (P> 0.05) ; However, the expression of SP-A and C was increased in the pretreated groups with the three inhibitors as compared with the unpretreated HV group while more stronger were found in PD98059+HV group than the other pretreatment groups (P < 0.05) . There was no significance difference in the expression of SP-B and RTI40 mRNA among three pretreatment groups . Conclusion The results of our study show that mechanical ventilation may inhibit and downregulate the expression of the SP-A and C but upregulate the expression of RTI40 mRNA.The inhibitors of MAPK can attenuate the changes of SP-A,C and RTI40 mRNA during the process of high tidal volume ventilation , which indicates that MAPK ,at least in part, may play a role in the functions of I and II alveolar epithelial cells in the lung injury induced by mechanical ventilaiton. Part 6The mechanism of cyclic stretch-induced intcrleukin-8 production in A549Objective To observe the mechanism and the effects of c-jun NH2-terminal kinase(JNK) on the the gene and protein expressions of IL-8 induced by cyclic stretch in A549 cells.Methods To examine the effects of stretch at the cellular level, we used a cell stretch device that applies uniform biaxial strain to flexible cell culture membranes. A549 cells, a type II alveolar cell line, were cultured in DMEM for 48h. After that the cells were grown on fibronectin-coated silicone elastomeric membranes for 48h . And then , the cells were exposed to cell stretch at 15% strain at 30cycles per minute for 0, 15, 30, 60 and 120 minutes respectively. In the pretreatment group we used the pharmacologic inhibitor (SP600125)incubation the cells 30 min before being stretched in order to inhibit JNK and to find the changes of the expression of IL-8 mRNA and protein induced by cyclic stretch in the A549 cells. The total RNA and proteins were obtained from three tests for each time point in cells . The concentration of IL-8 protein in cell supernatants was determined by enzyme-linked immunosorbent assay. Total messenger RNA (mRNA) in cells was extracted and detected for IL-8 mRNA by RT-PCR. Meanwhile, Kinase activity of JNK was determined by western blot assays. Results Fifteen percent strain upregulated the expression of IL-8 mRNA at being stretched for 30 minutes , but at 60 and 120 minutes strain did more comparied with that of stretched for 0 and 15 minutes (P<0.05, P<0.01) in the control group. There were -significant increase of the expression of IL-8 mRNA with the stretch for 30, 60, 120min than those in the pretreatment group (P<0.05) . Levels of IL-8 protein in cell supernatant were increased after 15% strain for 30, 60 and 120 mins which was similar to the manner of the gene expression did. Phospho -JNK activity was increased after 15% strain at 30, 60 and 120 minutes as comparing with that of 0 and 15 minutes in control group. In contrast, incubated with SP600125, the expression mRNA and protein of IL-8 were decreased significantly as well as the activation of JNK were inhibited at 15, 30, 60 and 120 minutes as comparied with that of the control group. Conclusion Stretch-induced IL-8 protein production in A549, a type alveolar II cell line were dependent, at least in part, on the activation of JNK. Stretch-induced IL-8 production and MAP kinase activation may be one of the important mechanisms in the mechanical stretch-induced biotrauma.