| Objective: Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is the acute and ongoing respiratory failure caused by pulmonary and extrapulmonary besides cardiogenic insults. There are many causes for ALI/ARDS, which can be classified as pulmonary causes (direct causes, e.g. severe pneumonia), and extrapulmonary causes (indirect causes, e.g. sepsis). Inflammatory reaction, lung injury and fibrosis is the major pathogenesy and pathologic changes of ARDS, and glucocorticoids (GCs) has the function of restraining inflammatory reaction, lightening cytokines'damage to tissues, and retarding fibrosis. However, such is not the case. Clinical research shows that ALI patients with different triggering causes react quite differently to the hormone therapy. Thus the different levels of GR expression can affect the curative effect of GCs.Therefore, our experiment established two ALI animal models with Lipopolysaccharide (LPS) and Escherichia coli (E.coli), compared differences in cytokine TNF-α,IL-1βcontent,expression of NF-κB P65,GR and the efficacy of GCs to explore the inflammatory mechanism and GCs curative efficacy of ALI induced by different causes and potential mechanism . The result of this research will provide theoretical basis for the clinical application of GCs in the treatment of ARDS caused by different motivations.Methods: 48 male SD rats were divided randomly into six groups:①NS group: 0.9% saline (1ml) was injected intravenously via a tail vein,②LPS group: LPS (8mg/kg) was injected intravenously via a tail vein,③E.coli group: E.coli (5.0×109cfu/ml, 3ml/kg) was instilled into trachea,④NS+Dex group,⑤LPS+Dex group,⑥E.coli+Dex group.④⑤⑥three groups were intraperitoneally injected with Dexamethasone (2mg/kg) immediately after replicating model.LPS, NS groups are sample to determine corresponding index at 4 hours after replicating model, and E.coli group did it at 24 hours after replicating model.SD rats were anaesthetized by intraperitoneal injection 4% chloralhydrate (3ml/kg). Blood was collected by arteria cruralis bloodletting and centrifuged (3000rpm, 4℃, 10 min). Serum was stored in -80℃and used to measure the concentration of TNF-αand IL-1β. The medius lobe of the right lung was placed in 10% neutral formalin fluid, embedded in paraffin, stained with hematoxylin-eosin and examined changes of pathomorphology in the lung and scored under a light microscope. The rest of lung embedded in paraffin stained with immunohistochemical method to examine the expression level of NF-κB P65 and GR positive cells. The posterior lobe was stored in liquid nitrogen to measure Western Blotting. A trachea catheter was used for bronchoalveloar lavage in left lung. BALF was collected and centrifuged (1200rpm, 4℃, 10 min). Supernatant was stored in -80℃and was used to measure the concentration of TNF-α, IL-1β. Cellular sediment was suspended in 0.9% saline 200μl to be counted the total leukocytes.Results: (1) In both groups of LPS and E.coli, the total leukocytes in BALF were higher than that of NS group (respectively 36.22±5.60, 366.97±31.63 vs 13.06±1.94, P<0.01), and the total leukocytes in BALF of E.coli was higher than that of LPS (P<0.01). The total leukocytes in BALF in both groups of LPS+Dex and E.coli+Dex were lower than those in both groups of LPS and E.coli (respectively 19.34±2.61 vs 36.22±5.60 and 247.25±22.16 vs 366.97±31.63, P<0.01). (2) In groups of LPS and E.coli, histopathologic grade were remarkably higher than that in NS group (P<0.05). However, there was no difference between the histological grade of the two ALI model groups (15.38±1.06 vs 15.88±1.73, P>0.05). The grade in the group of LPS+Dex was decreased than that in the LPS group (12.13±0.99 vs 15.38±1.06, P<0.01). But there was no difference in the histological grade between the E.coli and E.coli+Dex group (14.25±1.75 vs 15.88±1.73, P>0.05). (3) In both groups of LPS and E.coli, the expression of NF-κB P65 positive cells were higher than those of NS group (respectively 34.08±3.82, 36.33±4.05 vs 3.11±1.10, P<0.01). Compared with LPS group, the staining of LPS+Dex group became lighter evidently with the positive reaction decreasing (26.14±3.91 vs 34.08±3.82, P<0.01). But there was no difference in staining and positive reaction between the E.coli and E.coli+Dex group (35.10±4.22 vs 36.33±4.05, P>0.05). In both groups of LPS and E.coli, the expression of GR positive cells were lower than those of NS group (respectively 20.80±0.88, 18.06±1.67 vs 28.86±3.14, P<0.01). Compared with LPS group, the positive reaction was more obviously increased in LPS+Dex group (24.58±2.67 vs 20.80±0.88, P<0.01). But there was no difference in staining and positive reaction between the E.coli and E.coli+Dex group (17.49±2.69 vs 18.06±1.67, P>0.05). (4)The western blot results demonstrated that the color of NF-κB P65 bar was deeper in both groups of LPS and E.coli than those of NS group. Compared with LPS group, the color of NF-κB P65 bar was lighter in LPS+Dex group. But there was no difference between the E.coli and E.coli+Dex group. The color of GR bar was lighter in both groups of LPS and E.coli than those of NS group. Compared with LPS group, the color of GR bar was deeper in LPS+Dex group. But there was no difference between E.coli and E.coli+Dex group. (5)In both groups of LPS and E.coli, the concentrations of TNF-α, IL-1βin serum and BALF were higher than those of NS group (P<0.01). The above parameters in LPS+Dex group were decreased when compared to LPS group (P<0.05). However, there was no difference between the E.coli and E.coli+Dex group (P>0.05).Conclusion: 1 The ALI models induced by pulmonary direct infection were replicated successfully by instilling Escherichia coli (E.coli) into the rat's trachea.2 The level of NF-κB P65, GR and the content of cytokines in pulmonary and extrapulmonary ALI/ARDS models are not consistent. It may be elucidate that inflammation mechanism of ALI/ARDS is related to different etiological factors.3 The curative effect of hormonal therapy is different between pulmonary and extrapulmonary ALI/ARDS. Dexamethasone alleviates the pulmonary inflammatory injury caused by Lipopolysaccharide (LPS), but little useful to that of caused by Escherichia coli (E.coli). So the treatment effect of glucocorticoid is associated with the etiological factor and pathogenesis of ALI/ARDS. |
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