| Objective:Inhalation injury is one of main reasons of death in the early stage of burn patients, the pathophysiological mechanisms of Inhalation injury is closely related to apoptosis. In this experiment, the experimental subjects was rats, we observed the pathological changes of lung tissue after smoke inhalation injury, and identify the time changes of the expression of the Nitric oxide and inducible Nitric-oxide synthase iNOS, and identify the apoptosis and the expression of Bax and Bcl-2 after smoke inhalation injury using Aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, to find the protection mechanisms of AG for inhalation injury and provide theoretical basis for the clinical treatment of inhalation injury.Methods:Part one:36 healthy SD rats were randomly divided into two groups, thirty rats were subjected to inhalation of 5 minutes, five times, then were euthanized at 2,6,12, 24, and 48 hrs postinjury (injured group). Additional sheep received sham injury and were euthanized after 24 hrs (sham group). Lung tissue was obtained at the respective time points for the measurement of NO, inducible NOS protein expression, and scored the histological changes under the optical microscope after stained with hematoxylin and eosin.Part two:66 healthy SD rats were randomly divided into there groups: noninjured and nontreated (sham group; n=6), injured but nontreated (control group, n=30), and injured but treated with 100 mg/kg AG (treated group, n=30). control group and treated group were euthanized at 2,6,12,24, and 48 hrs postinjury. Lung tissue was obtained at the respective time points for pathological examination, immunohistochemistry to observe the expression of Bcl-2 and Bax changes, TUNEL (TdT-mediated dUTP Nick-End Labeling) to detect the apoptosis of lung cells in rats, and calculated apoptotic index (AI).Results:Part one:1.Gross observation:the bronchus'mucous congestion and swelling, mucus or foam-like secretions can be seen in the bronchus after injured, lung surface was wet, there were scattered subpleural bleeding spots, lung congestion and swelling, dark red, at 2h after injury the changes more limited,12h when the changes are most obvious.2. HE staining and optical microscopy lung injury score:compare with sham group, all levels of the bronchial wall edema, pulmonary interstitial edema, alveolar septal thickening, alveolars collapse or even completely blocked, part of the alveolar hemorrhage after injured. Lung injury score began to increase at 2h,after 24h had statistical significantly differences(P<0.01), the fastest change of pathological injury 12h to 24h after injury.3. NO, iNOS:Compared with the sham group, NO levels are elevated after 2h in the injured group, peaked at 12h, and iNOS content in lung homogenates increased began to appear at 2h,6h to 12h between the fastest rising, iNOS levels peaked at 12h, then 12h to 24h was significantly decreased.Part two:1. HE staining:comparison with the injury group, lung congestion and edema was lighting, alveolar wall smooth, visible in a few alveolar neutrophils, lymphocytes, mild lung edema and congestion in the treated group.2. Immunohistochemistry of Bax and Bcl-2 changes:compared with the control group, expression of Bax significantly increased after injured, progressively increased to maximum at 24 hrs, after which its expression decreased, but still significant higher than the control group. The treat group compared with the injured, from the beginning at 6h and after each time point shows Bax expression decreased (P<0.05). Compared with the control group, the expression of Bcl-2 is relatively constant in injured group, while the treat group increased the level of Bcl-2.3. apoptosis of lung cells:TUNEL assay showed that, compared with the control group, apoptotic index increased (P <0.01) in injured group at all time points after 2h.6h and 24h the treat group AI lower than injured group (all P<0.05),12h and 48h, the two groups was no significant difference in AI (P>0.05).Conclusion:The expression of NO,iNOS were increased in lung tissue in rats after smoke inhalation injury. iNOS,NO and apoptosis-related protein Bcl-2, bax may be closely related to the damage of lung after smoke inhalation injury, apoptosis plays an important regulatory role. Inducible nitric oxide synthase inhibitors inhibit the excessive NO production, then to regulation of apoptosis, thereby protecting smoke inhalation lung injury. |
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