| Acute lung injury（ALI） and its most severe manifestation, acute respiratory distress syndrome（ARDS）, is a clinical syndrome characterized by acute hypoxemic respiratory failure, bilateral pulmonary infiltrates on frontal chest radiograph consistent with edema, and normal cardiac filling pressures. The resulting lung damage can evoke lung failure and multiple organ dysfunctions associated with increased mortality. Sepsis（the presence of pus-forming bacteria or their toxins in the blood or tissues） is one of the most important causes of ALI/ARDS. Lipopolysaccharide（LPS） plays an important role in initiating inflammatory response and causing systemic inflammatory response syndrome（SIRS） and sepsis. LPS injection in vivo is a classic method to manufacture a sepsis-induced animal ALI model. The pathogenesis of ALI/ARDS is not certain. Studies show that apoptosis is involved in the pathogenesis of ALI/ARDS. H2S may participate the process of apoptosis in lung tissue in ALI, but so far there is no research related to the effects of H2S on lung apoptosis of lung tissue by mitochondria dependent mechanism in ALI in rats.Using transmission electron microscopy, Western blotting and gene detection technology, in the level of mitochondria, from morphology, protein expression and gene expression, this research observed the effects of H2S on lung tissue cell apoptosis in LPS induced ALI in rats, so as to explore the mechanism of H2S through the mitochondrial dependent pathway impact on lung tissue cell apoptosis in LPS induced ALI in rats, further to evaluate therapeutic effect of H2S on LPS induced by ALI and Na HS on the ALI/ARDS caused by serious infection. And then we want to provide new ideas for the treatment of ALI/ARDS. Part 1 Changes of endogenous H2S/cystathionine-γ-lyase system in acute lung injury induced by lipopolysaccharide in ratsObjective: To observe the changes of H2S/CSE system in acute lung injury induced by lipopolysaccharide in rats.Methods: Eighty male rats were randomly divided into five groups. ①Control group,②LPS 1 h group,③LPS 3 h group,④LPS 6 h group,⑤9 h group（n=8）. Rats were anaesthetized with an injection of 100 g/L chloral hydrate（3 ml/kg）. The ALI models were induced by LPS via sublingual vein injections. The rats were respectively killed at 1, 3, 6 or 9 hour after administration of LPS. The lung coefficient and wet-to-dry weight ratio（W/D） were respectively measured. The contents of IL-1β in serum were detected by ELISA. The content of H2S in plasma and the activity of CSE in lung tissue were respectively detected. The morphological changes of lung tissue were observed by electron microscope.Results:1 The lung coefficient was not significantly altered from 1 hour to 9 hour after administration of LPS in control group. Compared with those of control group, the lung coefficient was not significantly altered at 1 hour after administration of LPS（P > 0.05）, however, the lung coefficient was significantly increased at 3（P < 0.05）, 6（P < 0.01） and 9（P < 0.01） hour after administration of LPS.2 W/D was not significantly altered from 1 hour to 9 hour after administration of LPS in control group. Compared with those of control group, W/D was not significantly altered at 1 hour after administration of LPS（P > 0.05）, however, W/D was significantly increased at 3（P < 0.01）, 6（P < 0.05） and 9（P < 0.01） hour after administration of LPS.3 The content of IL-1β in serum was not significantly altered from 1 hour to 9 hour after administration of LPS in control group. Compared with those of control group, the content of IL-1β in serum was significantly increased at 1 hour after administration of LPS（P < 0.05）, however, the content of IL-1β in serum was significantly increased at 3（P < 0.01）, 6（P < 0.01） and 9（P < 0.01） hour after administration of LPS.4 The content of H2S in plasma was not significantly altered from 1 hour to 9 hour after administration of LPS in control group. Compared with those of control group, the content of H2S in plasma was not altered at 1 hour after administration of LPS（P >0.05）,however, the content of H2S in plasma was significantly decreased at 3（P < 0.01）, 6（P < 0.01） and 9（P < 0.01） hour after administration of LPS.5 The activity of CSE in lung tissue was not significantly altered from 1 hour to 9 hour after administration of LPS in control group. Compared with those of control group, the activity of CSE in lung tissue was not altered at 1 hour after administration of LPS（P >0.05）. The activity of CSE in lung tissue was significantly decreased at 3（P < 0.01）, 6（P < 0.01） and 9（P < 0.01） hour after administration of LPS.6 Transmission electron microscope showed that the microvillus of rat type Ⅱ alveolar cells were orderly arranged, and the structure of the rough surfaced endoplasmic reticulums,the osmiophilic lamellar bodies and mitochondria, were intact in control group. In LPS 3 h, 6 h and 9 h groups, mitochondrial were swollen with disrupted or disintegrated cristae.Some rough endoplasmic reticulum disintegrated cristae, visible part of the rough endoplasmic reticulum appeared deregulation of eosinophils.Osmiophilic lamellar bodies lamellar structure significantly had fused or disappeared.Conclusion:The lung tissue was not significantly injured at 1 hour after administration of LPS, but the content of IL-1β in serum was increased. During 3 h, 6h, 9h after administration of LPS, lung tissue was significantly injured, the content of IL-1β in serum was significantly increased, but the content of H2S in plasma and the activity of CSE were markedly decreased. It could be concluded that H2S/CSE system and IL-1β may be play a role in the pathophysiologic process of ALI induced by LPS. Part 2 Effects of H2S on Apoptosis of lung cells in acute lung injury induced by lipopolysaccharide in ratsObjective: To observe the effects H2S on apoptosis of lung cells in acute lung injury induced by lipopolysaccharide in rats.Methods: Eighty male rats were randomly divided into five groups. ①Control group,②LPS group,③LPS+low dose Na HS group,④ LPS+middle dose Na HS group,⑤LPS+high dose Na HS group. Rats were anaesthetized with an injection of 100 g/L chloral hydrate（3 mg/kg）. LPS（5 mg/kg） was administrated via sublingual vein injections in LPS group in rats. In LPS+ low, middle and high dose Na HS groups, Na HS（0.78 mg/kg, 1.56 mg/kg and 3.12 mg/kg） was respectively administrated at 3 hour after administration of LPS. Saline was administrated in control group via sublingual vein injections. The rats were respectively killed at 6 hour after administration of LPS or saline. Cells apoptosis were measured by flow cytometry.Results: Apoptosis rate of lung tissue cells in control group in rats was 21.81±2.17%. Compared with those of control group, the apoptosis rate of lung tissue cells were significantly increased in LPS group in rats（P < 0.01）; Compared with those of LPS group, the apoptosis rate of lung tissue cells were significantly decreased in LPS+ low, middle and high dose Na HS groups in rats（P < 0.01）.Conclusion:The cell apoptosis rate of lung tissue cells was significantly increased in ALI induced by LPS in rats. The apoptosis rate of lung tissue cells was significantly decreased after administration of Na HS in ALI induced by LPS in rats. Part 3 Effects of H2S on factors related apoptosis of lung cells in acute lung injury induced by lipopolysaccharide in ratsObjective: To observe the effects of H2S on factors related apoptosis of lung tissue cells in acute lung injury induced by lipopolysaccharide in rats.Methods: Eighty male rats were randomly divided into five groups. ①Control group,②LPS group,③LPS+low dose Na HS group,④ LPS+middle dose Na HS group,⑤LPS+high dose Na HS group. Rats were anaesthetized with an injection of 100 g/L chloral hydrate（3 ml/kg）. LPS（5 mg/kg） was administrated via sublingual vein injections in LPS group in rats. In LPS+ low, middle and high dose Na HS groups, Na HS（0.78 mg/kg,1.56 mg/kg and 3.12 mg/kg） was respectively administrated at 3 hour after administration of LPS. Saline was administrated in control group via sublingual vein injections. The rats were respectively killed at 6 hour after administration of LPS or saline. The expressions of caspase-3, caspase-9, bcl-2, bax and Smac/DIABLO were respectively analysised by Western blotting. The gene expression of caspase-3, caspase-9, bcl-2, bax and Smac/DIABLO were respectively analysised by real-time reverse transcription PCR.Results:1 The expression of the caspase-3 and caspase-9 in lung tissue by Western blottingCompared with those of control group, the expression of caspase-3 in lung tissue was markedly increased in LPS group in rats（P < 0.01）. Compared with those of LPS group, the expression of caspase-3 in lung tissue was significantly decreased in LPS+ low, middle and high dose Na HS groups（P < 0.01）. Compared with those of control group, the expression of caspase-9 in lung tissue was significantly increased in LPS group in rats（P < 0.01）. Compared with those of LPS group, the expression of caspase-9 in lung tissue was significantly decreased in LPS+ low, middle and high dose Na HS groups in rats（P < 0.01）.2 The expression of the bcl-2 and bax in lung tissue by Western blottingCompared with those of control group, the expression of bcl-2 in lung tissue was markedly increased in LPS group in rats（P < 0.01）. Compared with LPS group, the expression of bcl-2 in lung tissue was significantly increased in LPS+ low, middle and high dose Na HS groups in rats（P < 0.05 or P < 0.01）. Compared with those of control group, the expression of bax in lung tissue was markedly increased in LPS group in rats（P < 0.01）. Compared with LPS group, the expression of bax in lung tissues was significantly decreased in LPS+ low, middle and high dose Na HS groups in rats（P < 0.05 or P < 0.01）. Compared with those of control group, the bcl-2/bax in lung tissue was markedly decreased in LPS group in rats（P < 0.01）. Compared with those of LPS group, the bcl-2/bax in lung tissue was significantly increased in LPS+ low, middle and high dose Na HS groups in rats（P < 0.01）.3 The expression of the Smac/DIABLO in lung tissue by Western blottingCompared with those of control group, the expression of Smac/DIABLO in lung tissue was markedly increased in LPS group in rats（P < 0.01）. Compared with those of LPS group, the expression of Smac/DIABLO in lung tissue was significantly decreased in LPS+ low, middle and high dose Na HS groups in rats（P < 0.01）.4 The expression of the caspase-3 and caspase-9 m RNA in lung tissue by real-time reverse transcription PCRCompared with those of control group, the expression of Caspase-3 m RNA in lung tissue was markedly increased in LPS group in rats（P < 0.01）. Compared with those of LPS group, the expression of caspase-3 m RNA in lung tissue was significantly decreased in LPS+ low, middle and high dose Na HS groups in rats（P < 0.01）. Compared with those of control group, the expression of caspase-9 m RNA in lung tissue was markedly increased in LPS group in rats（P < 0.01）. Compared with those of LPS group, the expression of caspase-3 m RNA in lung tissue was not significantly altered in LPS+ low dose Na HS group, however, the expression of caspase-3 m RNA in lung tissue was significantly decreased in LPS+ middle and high dose Na HS groups in rats（P < 0.05 or P < 0.01）.5 The expression of the bcl-2 and bax m RNA in lung tissue by real-time reverse transcription PCRCompared with those of control group, the expression of bcl-2 m RNA in lung tissue was markedly increased in LPS group in rats（P < 0.05）. Compared those of with LPS group, the expression of bcl-2 m RNA in lung tissue was significantly increased in LPS+ low, middle and high dose Na HS groups in rats（P < 0.01）. Compared with those of control group, the expression of bax m RNA in lung tissue was markedly increased in LPS group in rats（P < 0.01）. Compared with those of LPS group, the expression of bax m RNA in lung tissue was significantly decreased in LPS+ low, middle and high dose Na HS groups in rats（P < 0.05 or P < 0.01）.6 The expression of the Smac/DIABLO m RNA in lung tissue by real-time reverse transcription PCRCompared with those of control group, the expression of Smac/DIABLO m RNA in lung tissue was markedly increased in LPS group in rats（P < 0.05）. Compared with those of LPS group, the expression of Smac/DIABLO m RNA in lung tissue was significantly decreased in LPS+ low, middle and high dose Na HS groups in rats（P < 0.01）.Conclusion:After administration of Na HS in ALI induced by LPS in rats, the expression of caspase-3, caspase-9, bax and Smac/DIABLO was decreased, the expression of bcl-2 was increased and the expression of caspase-3, caspase-9, bax and Smac/DIABLO m RNA were decreased; the expression of bcl-2 m RNA was increased. Part 4 Effects of H2S on mitochondrial pathway apoptosis of lung cells in acute lung injury induced by lipopolysaccharide in ratsObjective: To observe the effects of H2S on mitochondrial apoptosis pathway（the direct factors related apoptosis） of lung cells in acute lung injury induced by lipopolysaccharide in rats.Methods: Eighty male rats were randomly divided into five groups. ①Control group,②LPS group,③LPS+low dose Na HS group,④ LPS+middle dose Na HS group,⑤LPS+high dose Na HS group. Rats were anaesthetized with an injection of 100 g/L chloral hydrate（3 ml/kg）. In LPS group LPS（5 mg/kg）was administrated via sublingual vein injections. In LPS+ low, middle and high dose Na HS groups, Na HS（0.78 mg/kg, 1.56 mg/kg and 3.12 mg/kg） was respectively administrated at 3 hour after administration of LPS. Saline was administrated in control group via sublingual vein injections. The rats were respectively killed at 6 hour after administration of LPS or saline. The lung tissue was quickly excised and was homogenated. The cytoplasmic protein and mitochondrial protein were extracted. The lung mitochondrial and cytosol Cyt C and AIF protein expression were respectively analysised by Western blotting.Results:1 The lung mitochondrial and cytosol Cyt C protein expression by Western blottingThe Cyt C protein expression in the lung mitochondria was significantly decreased in the LPS injury group compared with those of the control group in rats（P < 0.05 or P < 0.01）. Compared with those of the LPS injury group, the Cyt C protein expression in the lung mitochondria was markedly increased in LPS+ low, middle and high dose Na HS groups in rats（P < 0.05 or P < 0.01）. The cytosol Cyt C protein expression was significantly increased in the LPS group compared with those of the control group in rats（P < 0.05 or P < 0.01）. In LPS+ low, middle and high dose Na HS groups, the cytosol Cyt C protein expression was markedly decreased compared with those of the LPS injury group in rats（P < 0.01）.2 The lung mitochondrial and cytosol AIF protein expression by Western blottingThe AIF protein expression in the lung mitochondria was significantly decreased in the LPS injury group compared with those of the control group in rats（P < 0.05 or P < 0.01）. In LPS+ low, middle and high dose Na HS groups, the AIF protein expression in the lung mitochondria was markedly increased compared with those of the LPS injury group in rats（P < 0.05 or P < 0.01）. The cytosol AIF protein expression was significantly increased in the LPS group compared with those of the control group in rats（P < 0.05 or P < 0.01）. In LPS+ low, middle and high dose Na HS groups, the cytosol AIF expression was markedly decreased compared with those of the LPS injury group in rats（P < 0.01）.Conclusion:The Cyt C and AIF protein expression in lung mitochondrial were significantly decreased, while the Cyt C and AIF protein expression in lung cytosol were significantly increased, and the rate of lung cells apoptosis was significantly decreased after administration of Na HS in ALI induced by LPS in rats. Part 5 Effects of H2S on mitochondria of lung cells in acute lung injury induced by lipopolysaccharide in ratsObjective: To observe the effects of H2S on structure and function of mitochondria of lung cells in acute lung injury induced by lipopolysaccharide in rats.Methods: Eighty male rats were randomly divided into five groups. ①Control group,②LPS group,③LPS+low dose Na HS group,④LPS+middle dose Na HS group,⑤LPS+high dose Na HS group. Rats were anaesthetized with an injection of 100 g/L chloral hydrate（3 ml/kg）. In LPS group LPS（5 mg/kg）was administrated via sublingual vein injections. In LPS+ low, middle and high dose Na HS groups, Na HS（0.78 mg/kg, 1.56 mg/kg and 3.12 mg/kg） was respectively administrated at 3 hour after administration of LPS. Saline was administrated in control group via sublingual vein injections. The rats were respectively killed at 6 hour after administration of LPS or saline. The lung tissue was quickly excised and was homogenated. The mitochondria are isolated with differential centrifugation. The content of malondialdehyde（MDA） and the activities of superoxide dismutase（SOD） and glutathione peroxidase（GSH-PX） in lung mitochondria and the mitochondrial swelling and activity were determined. The lung mitochondria ultrastructures were observed with an electron microscope.Results:1 Effects of H2S on mitochondrial ultrastructure in rat lung cellsTransmission electron microscope showed that there were significant differences between the mitochondrial ultrastructure in the control and LPS groups in rat lung cells. The mitochondria in rat lung cells was swollen with disrupted or disintegrated cristae, and the osmiophilic lamellar bodies had fused or disappeared in LPS injury group. Compared with those of LPS injury group, the mitochondrial damage was slightly mitigated in the LPS + low-dose Na HS group and significantly mitigated in the LPS + middle-dose and high-dose Na HS groups in rats.2 Effects of H2S on MDA content and mitochondrial ATPase, SOD, and GSH-PX activityCompared with those of control group, the MDA content was significantly increased（P < 0.01） and the SOD, GSH-PX, and ATPase activities significantly decreased（P < 0.01） in lung mitochondria in the LPS group in rats. Compared with those of the LPS group, the MDA content was significantly decreased and the SOD, GSH-PX, and ATPase activities were significantly increased in lung mitochondria in LPS+ low, middle and high dose Na HS groups in rats（P < 0.05 or P < 0.01）.3 Effects of H2S on lung mitochondria swelling and activityCompared with those of control group, the swelling of the lung mitochondria was significantly increased（the OD540 value was significantly decreased） and the activity of the lung mitochondria was significantly decreased in the LPS group in rats（P < 0.01）. In the LPS+ low, middle and high dose Na HS groups, the swelling of the lung mitochondria was markedly decreased（the OD540 value was significantly increased） and the activity of the lung mitochondria was markedly increased compared with those of the LPS group in rats（P < 0.05 or P < 0.01）Conclusion:It could be concluded that H2S have a beneficial effect against ALI induced by LPS with decreasing the mitochondrial lipid peroxidation level and protecting the cell structure and function. |
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