| Nitric oxide (NO) is endothelium-derived relaxing factor (EDRF) which takes a part in the signal conduction in cell, has the effect of relaxing artery and decreasing viscosity of platele,and has anti-inflammatory action. But excess NO and it′s metabolic product may induce the organism damage. Inducible nitric oxide synthase (iNOS) which is only activated at the pathologic state is a type of nitric oxide synthase (NOS). The iNOS produces lots of NO that has persistent disadvantage to the organism. The NO synthesized by constitutive nitric oxide synthase (cNOS) is existed in normal organism which has physiological functions.Acute lung injury (ALI) is a clinical syndrome with distinctively pathological changes induced by all kinds of factors in lung tissue.Severity ALI is designated acute respiratory distress syndrome(ARDS).Many animal experiments and clinic studies have showed that infection, wound and shock were the common causes inducing ALI and lots of factors and systems taken part in the pathological processes of ALI and ARDS. Internal investigation of the acquired pneumonia in hospital shows that Gram-Negative bacillus accounts for majority of pulmonary infective patients(>60%). Infection induced by Gram-negative bacteria becames the frequent cause of lung injury, and bacterial endotoxin takes an important effect in the process of lung injury. Mitochondria as the energy subunit of cell, lead to disturbance of producing energy when they are damaged. Studies showed that NO participates in the pathological processes of ALI. However, when ALI occurs, whether NO and NOS participate in the pathological processes of ALI, and whether NOS inhibitor ameliorate the injury of mitochondria were unknown. In this experiment, we established model of ALI by injection LPS in rats to investigate the change of NO level and the relationship with NOS in this pathological processes, observe of the effect of NO in lung mitochondria in ALI and the effect of NOS inhibitor on mitochondria injury in ALI rats.Part I Research of Methods for Isolation of Mitochondria from Lung in RatsObjective: To establish a simple and feasible method for separation of mitochondria from lung in rats.Methods: Using different isolation mediums, rat pulmonary mitochondrial pellet was collected by differential centrifugation. Then Accumulated mitochondria were fixed in the fixtive. Lastly, mitochondrial ultrastructure was observed by transmission electronic microscope. Results: The results of different separating mediums used to isolate mitochondria of rat lung showed that pulmonary mitochondria isolated in kalium chloratum were swollen, autolytic or broken, but the overwhelming majority of mitochondria separated by STE solution were intact.Conclusions: The method of STE isolation medium is simple, and reliable. Mitochondria can be intactly accumulated in this way.Part II Effect of Aminoguanidine on Mitochondria from Acute Lung Injury Induced by LPS in RatsObjective: The purpose of the present study was to examine the effect of selective inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG), on mitochondria from acute lung injury induced by LPS in rats.Methods: Twenty-four male SD rats were randomly divided into three groups (n=8 in each group):①Sham control group;②LPS injury group;③LPS + AG treatment group. The model of acute lung injury was prepared with injection of LPS in rat. AG was respectively administrated through intraperitioneal injection at 3h after injury induced by LPS, and rats were killed at 6h after injury and the mitochondria of lung were isolated by differential centrifugation. The activities of T-NOS, iNOS, ATPase, SOD and GSH-Px, and the contents of NO and MDA from mitochondria were respectively measured. Ultrastructure changes of lung mitochondria were examined by Electronic microscope after injury and AG treatment.Results1 Compared with Sham group the activities of ATPase,SOD and GSH-Px in lung mitochondria were significantly decreased(P<0.01), and the contents of MDA was markedly increased in LPS group(P<0.01). Compared with LPS group, the activities of ATPase, SOD and GSH-Px were significantly enhanced (P<0.01), and the contents of MDA were decreased in AG group (P<0.01).2 Compared with Sham group the activities of T-NOS and iNOS were markedly enhanced respectively (P<0.01), and the contents of NO were significantly increased in LPS group (P<0.01). Compared with LPS group, the activities of T-NOS and iNOS were decreased (P<0.01), and the contents of NO were also decreased in AG group (P<0.01). However, there was no significantly change in the activity of cNOS in LPS group and AG group (P>0.05).3 Compared with Sham group the swelling of mitochondria was markedly increased with the decreased OD values at 540nm after lung injury(P<0.01); and the activity of mitochondria was decreased with the decreased OD values at 570nm after lung injury(P<0.01); and membrane fluidity was decreased with increased theηvalues(P<0.01).Compared with LPS group, mitochondrial swelling was significantly decreased, membrane fluidity and mitochondrial activity were markedly increased (P<0.01).4 The study examined by Transmission Electronic Microscope showed that lung cytoplasm and the mitochondria of alveolar type II cell swelled, the cristae were disrupted, dissolved or disappeared after acute lung injury. AG could ameliorate these injury induced by LPS in rats.Conclusions: It could be concluded that AG could inhibit the activity of iNOS and the production of NO, beneficially improve the energy pump of mitochondria, increase the activity of mitochondria, and ameliorate oxidative injury after actue lung injury, and then can effectively protect lung tissue against damage induced by LPS.Part III Effect of NG-Nitro-L-Arginine on Mitochondria from Acute Lung Injury Induced by LPS in RatsObjective: The purpose of the present study was to examine the effect of nonselective nitric oxide synthase inhibitor, NG-Nitro-L-Arginine (L-NA), on mitochondria from acute lung injury induced by LPS in rats.Methods: Twenty-four male SD rats were randomly divided into three groups (n=8 in each group):①Sham control group;②LPS injury group;③LPS + L-NA treatment group. The model of acute lung injury was prepared with injection of LPS in rat. L-NA was respectively administrated through intraperitioneal injection at 3h after injury induced by LPS, and rats were killed at 6h after injury and the mitochondria of lung were isolated by differential centrifugation. The activities of T-NOS, iNOS, ATPase, SOD and GSH-Px, and the contents of NO and MDA from mitochondria were respectively measured. Ultrastructure changes of lung mitochondria were examined by Electronic microscope after injury and L-NA treatment.Results1 Compared with Sham group the activities of ATPase,SOD and GSH-Px in lung mitochondria were significantly decreased (P<0.01), and the contents of MDA was markedly increased in LPS group (P<0.01). Compared with LPS group, the activities of ATPase, SOD and GSH-Px were significantly enhanced (P<0.01), and the contents of MDA were decreased in L-NA group (P<0.01).2 Compared with Sham group the activities of T-NOS and iNOS were markedly enhanced respectively (P<0.01), and the contents of NO were significantly increased in LPS group (P<0.01). However, there was no significantly change in the activity of cNOS in LPS group (P>0.05). Compared with LPS group, the activities of T-NOS, iNOS and cNOS were significantly decreased (P<0.01), and the contents of NO were decreased in L-NA group (P<0.01).3 Compared with Sham group the swelling of mitochondria was markedly increased with the decreased OD values at 540nm after lung injury(P<0.01); and the activity of mitochondria was decreased with the decreased OD values at 570nm after lung injury(P<0.01); and membrane fluidity was decreased with increased theηvalues(P<0.01).Compared with LPS group, mitochondrial swelling was significantly decreased, membrane fluidity was increased (P<0.05) and mitochondrial activity was increased (P<0.01).4 The study examined by Transmission Electronic Microscope showed that lung cytoplasm and the mitochondria of alveolar type II cell swelled, the cristae were disrupted, dissolved or disappeared after acute lung injury. L-NA could ameliorate these injury induced by LPS in rats.Conclusions: It could be concluded that L-NA could inhibit the activity of NOS and the production of NO, beneficially improve the energy pump of mitochondria, increase the activity of mitochondria, and ameliorate oxidative injury after actue lung injury, and then can effectively protect lung tissue against damage induced by LPS.
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