| Objective: Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized of hypoxemia respiratory failure as a result of the loss of barrier function of the alveolar epithelial and pulmonary capillary endothelial cells induced by different predisposing factors. The development of ALI/ARDS is associated with several clinical disorders including direct pulmonary injury from pneumonia and aspiration as well as indirect pulmonary injury from trauma, sepsis. Severe sepsis and aspiration are the most serious causes of ARDS. The development of ALI/ARDS in human being is caused by complex risk factors but rarely single instigating factor. Compared to one hit model, two hit model of ALI should reflect comorbidities and risk factors found in patients more accurately. It is necessary to investigate the mechanism of ALI with two hit animal model for elucidating pathogenesis of ALI and evaluating efficacy of intervention.The complex network of inflammatory cytokines and chemokines play a major role in mediating, amplifying, and perpetuating the lung injury process. Simultaneous production of anti-inflammatory cytokines can potentially counteract pro-inflammatory cytokine effects and modify the intensity of the inflammatory process. Understanding the balance of pro- and anti-inflammatory cytokines in the lungs of patients with ARDS is an important step in unraveling the pathogenesis of this devastating clinical syndrome. Although a large number of studies have confirmed inflammation played an important role in ALI, we do not have enough information about the differences of the inflammatory mechanism of ALI induced by different risk factors.Because glucocorticoids (GCs) have powerful anti-inflammatory property, GCs have been used in ARDS for many years. Clinical physicians have found that there were differences in the response of ALI patients induced by different risk factors to GCs, but the mechanism need to be further studied. GCs exert most of their effects through specific, ubiquitously distributed intracellular glucocorticoid receptor (GR). GR can be synthesized through GRmRNA translation, so GRmRNA level should be related with the level of GR. Finally it can influence GCs curative effect. This may be one of the mechanisms of differences in GCs curative efficacy.Therefore, our experiment established two ALI animal models with Lipopolysaccharide (LPS) one hit and Lipopolysaccharide-Hydrochloric acid (HCl) two hit respectively. We compared differences in cytokine content, GRmRNA level and the efficacy of GCs to explore the inflammatory mechanism and GCs curative efficacy of ALI induced by different causes and potential mechanism.Methods: 48 male SD rats were divided randomly into six groups:①NS group: 0.9% saline (0.5ml/kg) was instilled into the trachea at 4 hours after intravenous injection of 0.9% saline (1.2ml/kg),②LPS group: LPS (8mg/kg) was injected intravenously via a tail vein,③LPS-HCl group: pH1.8 HCl (0.5ml/kg) was instilled into the trachea at 4 hours after intravenous injection of LPS (4mg/kg),④NS+Dex group,⑤LPS+Dex group,⑥LPS-HCl+Dex group.④⑤⑥three groups were intraperitoneally injected with Dexamethasone (2mg/kg) immediately after replicating model.SD rats were anaesthetized by intraperitoneal injection 10% chloral hydrate (4ml/kg) at 4 hours after replicating model. Blood was collected by puncturing heart and centrifugated (3000rpm, 4℃, 10min). Serum was stored in -80℃and be used to measure the concentration of TNF-α, IL-1β, IL-4 and IL-10. The anterior lobe of the right lung was used to extract total RNA. The relative content of GRmRNA in lung was determined by RT-PCR method. The lower lobe of the right lung was placed in 10% neutral formalin fluid, embedded in paraffin, stained with hematoxylin-eosin and examined under a light microscope. The wet/dry was measured with the posterior lobe and accessory lobe of the right lung (80℃, 48h). A trachea catheter was used for bronchoalveloar lavage of left lung. BALF was collected and centrifugated (1200rpm, 4℃, 10min). Supernatant was stored in -80℃and be used to measure the concentration of TNF-α, IL-1β, IL-4, IL-10 and protein. Cellular sediment was suspended in 0.9% saline 200ul to be counted the total leukocytes.Results: (1) In both groups of LPS and LPS-HCl, W/D, the total leukocytes and protein concentration in BALF were higher than those of NS group (P<0.05). When compared between the two ALI model groups, there was no obvious difference in the above parameters except the protein concentration in BALF (P>0.05). These parameters in LPS+Dex group were lower than those in LPS groups (P<0.05). W/D in LPS-HCl+Dex group were decreased when compared to LPS-HCl group (P<0.05). (2) In groups of LPS and LPS+HCl, histological 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 (P>0.05). The grade in the groups of LPS+Dex and LPS-HCl+Dex were lower than that in the corresponding model groups(P<0.01). When compared to LPS+Dex group, the histological grade of LPS-HCl+Dex group was significantly increased (P<0.01). (3) The content of IL-1βin serum of every group was undetected. In both groups of LPS and LPS-HCl, the concentrations of TNF-αin serum and the concentrations of TNF-α, IL-1βin 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). In LPS-HCl+Dex group, the concentrations of TNF-αin serum and BALF were lower than those in LPS-HCl group (P<0.05). (4) In serum and BALF of LPS group and LPS-HCl group, the concentrations of IL-4 and IL-10 were remarkably higher than those in NS group (P<0.05). In both LPS+Dex and LPS-HCl+Dex groups, the concentrations of IL-10 in ether serum or BALF were higher than those in the corresponding model groups (P<0.05). (5) The level of GRmRNA in LPS group and LPS-HCl group were significantly lower than that in NS group (P<0.01). Dex significantly increased the level of GRmRNA in the two model groups (P<0.05). The level of GRmRNA in LPS+Dex group was increased when compared to LPS-HCl+Dex group (P<0.01).Conclusion: (1) The level of GRmRNA in lung tissues of ALI/ARDS models decreased, which led to an insufficient function of adrenal cortex from the level of GR and weakened anti-inflammation mechanism. This may be an important step in the development of ALI/ARDS.(2) The content of cytokines in one hit and two hit ALI/ARDS models are not consistent. It can be concluded that inflammation mechanism of ALI/ARDS is related to different risk factors.(3) The efficacy of GCs therapy on one hit and two hit ALI/ARDS is different. GCs can alleviate the pulmonary injury in two model groups, but the efficacy on LPS group is better than LPS-HCl group. It may be related to the number and function situation of GR and the differences in pathogenesis of two model groups.(4) It is different in the effect of Dex on the level of GRmRNA between control group and ALI model groups, which may be associated with different functional situation of GCs and GRmRNA in every group. |
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