| Objective: acute lung injury (ALI) / acute respiratory distress syndrome (ARDS) is a disordered inflammatory response. It is characterized of the extensive damage of alveolar epithelial cells and pulmonary capillary endothelial cells induced by a lot of serious diseases, such as infection, trauma and shock. The basic pathophysiological changes of ALI/ARDS are non-cardiogenic pulmonary edema induced by increased pulmonary vascular permeability,pulmonary hyaline membrane formation, as well as microatelectasis which cause acute dyspnea, refractory hypoxemia, decreased pulmonary compliance. ALI/ARDS is associated with several clinical causes including endo-pulmonary injury from aspiration, pneumonia, drowning and lung trauma as well as exo-pulmonary injury from sepsis, multiple injury and severe pancreatitis. The survey shows that sepsis is the top risk factor of ALI/ARDS, about 40%～50% ARDS patients are related to the infection or sepsis. Aspiration of gastric contents is about 30% ARDS contributer in clinical practice. Usually, two and more etiological factors are included in an ARDS patient at the same time. Although the etiopathogenisis of ALI and ARDS is complicated, they are just different serious stages in the course of systemic inflammatory reaction caused by various diseases. Another means, ARDS is severe ALI period.The serious injury can induce systemic inflammatory response syndrome (SIRS), which can lead to multiple organ dysfunction syndrome (MODS). Lung is the first target organ. ALI or ARDS firstly occurs, and is the important part of MODS. "Two-hit" model explained SIRS how to develop into MODS.ALI is actually an inflammatory reaction. Enhanced expression of inflammatory mediators plays an important role in the pathogenesis of pulmonary inflammation. Inflammatory cells, cytokines and inflammatory mediators involved in inflammation constitute "cell network" and "cytokine network" in inflammatory response and immune regulation of ALI, and play an important role in the pathogenesis. NF-κB, as a widespread transcription factor, has close relation with acute lung injury. NF-κB is the neck of controlling the inflammatory mediators and cytokines gene expression. Its activation and overexpression of cytokines result in the network imbalance, which cause inflammatory cascade efficacy.Because glucocorticoids (GCs) have powerful anti- inflammatory effect, its application in ALI/ARDS has received extensive attention. But at present, the effect of glucocorticoid anti-inflammatory is still unclear. The timing, dosage and course of treatment of glucocorticoids remains huge different in academic. It is still unclear that whether there is different effect of anti-inflammatory with different doses of GC. At the same time, it is different in the response of GCs in ALI patients induced by different risk factors, and its specific mechanism needs to be further studied.Therefore, in our study, we established three kinds of ALI animal models: Lipopolysaccharide (LPS) or Hydrochloric acid (HCl) one hit and LPS-HCl two hit model. By comparing the level of proinflammatory factor (tumor necrosis factor, TNF-α), anti-inflammation factor (interleukin-10, IL-10) and nuclear transcription factor (NF-kB) respectively, we explored the effect and mechanism of different doses of dexamethasone in ALI induced by different risk factors.Methods: 72 male SD rats, body weight 250～300g, were divided randomly into twelve groups:⑴LPS group,⑵LPS+ Dex1 group,⑶LPS+Dex8 group,⑷HCl group,⑸HCl+Dex1 group,⑹HCl+Dex8 group,⑺LPS-HCl group,⑻LPS-HCl+ Dex1 group,⑼LPS-HCl+Dex8 group,⑽NS group,⑾NS+ Dex1 group,⑿NS+Dex8 group,Each group has 6 animals.⑴⑵⑶groups were intravenously injected with LPS (8mg/kg) via a tail vein;⑷⑸⑹groups were instilled with HCl(pH 1.8, 2ml/kg) into the trachea;⑺⑻⑼groups were intravenously injected with LPS (4mg/kg) via a tail vein, then HCl(pH 1.8, 0.5ml/kg) was instilled into the trachea after 4 hours;⑵⑸⑻⑾groups were intraperitoneally injected with Dexamethasone (1mg/kg) immediately after replicating model;⑶⑹⑼⑿ groups were intraperitoneally injected with Dexamethasone (8mg/kg) immediately after replicating model;⑽⑾⑿groups were 0.9% saline was injected intravenously via a tail vein.SD rats were anaesthetized with intraperitoneal injection 4% chloral hydrate (4ml/kg) at 4 hours after replicating model. Blood was obtained by puncturing heart, collected and centrifuged (3000rpm, 4℃, 10min). Serum was stored in -80℃and used to measure the concentration of TNF-αand IL-10. The anterior lobe of the right lung, weight 100mg, was used to extract nucleoprotein. The relative activity of NF-κB in lung was measured by electrophoretic mobility shift assay (EMSA). The lower lobe of the right lung was placed in 10% neutral formalin fluid, embedded in paraffin, stained with hematoxylin-eosin to observe the changes of pathomorphology for lung tissue, and to detect the expression of NF-κB P65 in lung by immunohistochemistry. The wet/dry was determined with the posterior lobe and accessory lobe of the right lung (80℃, 48h). The broncho-alveolar lavage was performed in left lung through a trachea catheter with 0.9% ice-saline, and the bronchoalveolar lavage fluid (BALF) was collected and centrifuged (1200rpm, 4℃, 10min). Supernatant was stored in -80℃and used to measure the concentration of TNF-α, IL-10 and total protein. The cellular sediment was suspended in 0.9% saline 200μl to be counted the total leukocytes.Results: (1) Pathologic scores: There were no significant difference among the groups of LPS, HCl and LPS-HCl (P>0.05). After administration of different doses of Dex, the scores in the groups of LPS and LPS-HCl were decreased than that in the corresponding model groups (P<0.05). The score in HCl+Dex1 group was remarkably higher than that in LPS+Dex1 group and LPS-HCl+Dex1 group (P<0.05). (2) Compared with NS group, the total leukocytes in BALF further increased in the groups of LPS, HCl and LPS-HCl (P<0.01). After injecting different doses of Dex, the parameters in LPS group both were significantly decreased (P<0.05). The parameters in the groups of HCl+Dex1 and LPS-HCl+Dex1 both were higher than LPS+Dex1 group (P<0.05). (3) Compared with NS group, protein concentration in BALF increased in all the groups of LPS, HCl and LPS-HCl (P<0.01). The parameters in the groups of HCl and LPS-HCl both were higher than that in LPS group (P<0.01, P<0.05). After giving different doses of Dex, the parameters in LPS group and LPS-HCl group were decreased (P<0.05). The parameters in HCl+Dex1 group was significantly decreased than that in HCl group (P<0.05). (4) Compared with NS group, W/D increased in the groups of LPS, HCl and LPS-HCl (P<0.01). Compared with LPS group, W/D in LPS+Dex1 group were decreased (P<0.05), so was the parameter in LPS+Dex8 group. W/D in the group of LPS-HCl+Dex1 was lower than that in the group of LPS-HCl (P<0.05). (5) In groups of LPS, HCl and LPS-HCl, the concentrations of TNF-αin serum and in BALF were markedly higher than those in NS group (P<0.01). After administration of different doses of Dex, the above parameters in LPS group and LPS-HCl group were decreased (P<0.05), and there was no significant difference between low-dose and high-dose of Dex (P>0.05). (6) In serum and BALF of LPS group, HCl group and LPS-HCl group, the concentrations of IL-10 were remarkably higher than those in NS group (P<0.01), and the content of IL-10 in BALF in HCl group was higher than that in LPS group and LPS-HCl group (P<0.05). After Dex injecting, the concentrations of IL-10 in either serum or BALF in LPS group and LPS-HCl group were increased (P<0.05). The contents of IL-10 in BALF in the groups of HCl+Dex1 and HCl+Dex8 both were remarkably higher than that in HCl group (P<0.01). (7) The effect of immuno- histochemistry: The expression and distribution of NF-κB P65 in the groups of LPS, HCl and LPS-HCl were obviously increased than that in NS group (P<0.01). The expression of NF-κB P65 in the groups of LPS and LPS-HCl were higher than that in HCl group(P<0.01), NF-κB P65 positive reaction product shew the movement of NF-κB P65 from kytoplasm to nucleus. After administration of Dex, the expression of NF-κB P65 in LPS group and LPS-HCl group were markedly decreased (P<0.05), and the positive reaction further lessened. (8) The effect of EMSA: The level of NF-κB in LPS group, HCl group and LPS-HCl group were significantly higher than that in NS group (P<0.01). The level of NF-κB in LPS group was obviously higher than those in HCl group and LPS-HCl group (P<0.01). After administration of Dex, the level of NF-κB in LPS group and LPS-HCl group were obviously decreased (P<0.01), but there was no significant difference between low-dose and high-dose of Dex (P>0.05). The level of NF-κB in LPS+Dex8 group were lower than that in LPS-HCl+Dex8 group (P<0.05).Conclusion: (1) Inflammatory reaction mediated by cytokines plays a major role in ALI/ARDS. The level of cytokines in ALI/ARDS of different etiopathogenisis is different, inflammatory reaction may be related to the etiopathogenisis.(2) "One hit" and "two hit" causing ALI/ARDS both can activate NF-κB. NF-κB activation may play an improtant role in development and progression of ALI/ARDS. The pathogenesy of ALI may be due to the imbalance of inflammatory reaction which is related to the elevation of NF-κB.(3) Glucocorticoid could show anti-inflammatory effect and relieve the lung injury in ALI, which was contributed to the regulatory roles of GC on the NF-κB activation and cytokines. But the therapeutic efficacy of GC is different on different type of ALI/ARDS. It could associate with the differences in pathogenesis. More importantly, the effect could be related to the activity of NF-κB, as well as the number and affinity of GR.(4) It was found that GC has efficacy on NF-κB activation and cytokine regulation, but the dose-effect relationship was not found in our study.
|
PDF Full Text Request