Effects And Mechanisms Of Hydrogen Sulfide On Acute Lung Injury Induced By Lipopolysaccharide In Rats

Acute lung injury (ALI) is acute, progressive and hypoxia respiratory failure by endopathic and exopathic cause of lung rather than by cardiogenic one. The major clinical manifestations of ALI are obstinate dyspnea, hypoxemia, lung compliance decreased and so on. Lipopolysaccharide (LPS) is the main composition of gramnegative bacterium (G-) and is the most common cause of this syndrome. So it is one of the common ways that cause ALI by LPS in scientific research.Endogenous hydrogen sulfide (H2S) is produced from cysteine by cystathionine-β-synthase (CBS) and/or cystathionine-γ-lyase (CSE). It was reported as a neuromodulator in the brain because of its regulation to hippocampus, and it also can regulate the tesion of smooth muscle in gastrointestinal tract and blood vessel. According to many studies, H2S was suggested that it may have effects on many respiratory diseases and be a new endogenous signaling gasotransmitter after nitric oxide (NO) and carbon monoxide (CO). In the present study, we observed the chronological changes of H2S/CSE system in LPS-induced ALI in rats, investigated the effects of H2S on hyperoxidation, inflammatory factor, apoptosis and pulmoalverolar surfactant, and explored the possible mechanisms.Part 1 Change of endogenous hydrogen sulfide/cystathionine-γ-lyase system while acute lung injury induced by lipopolysaccharide in ratsObjective: To observe the chronological changes of interleukin-1β(IL-1β), interleukin-10 (IL-10) and H2S/CSE system in LPS-induced acute lung injury in rats, and to investigate the mechanisms of ALI.Methods: Eighty male rats were randomly divided into six groups.ⅠControl group;ⅡLPS 1 h group;ⅢLPS 3 h group;ⅣLPS 6 h group;ⅤLPS 9 h group;ⅥLPS 12 h group. There were fourty rats in control group (There were eight rats in per time point). The ALI model were induced by LPS in rats. The rats were respectively killed at 1, 3, 6, 9 or 12 hour after administration of LPS. The lung coefficient and wet-to-dry weight ratio (W/D) were respectively measured. The level of H2S in plasma, the activity of CSE in lung tissue and the contents of IL-1βand IL-10 in serum were respectively detected. The morphological changes of lung tissue were observed by light and electron mircroscope.Results:1. In control group, the lung coefficient and W/D were not altered from 1 hour to 12 hour. The lung coefficient and W/D were not altered at 1 hour after administration of LPS compared with those of control group (0.47±0.02 VS 0.46±0.03, 4.65±0.34 VS 4.41±0.33, P > 0.05). The lung coefficient and W/D were significantly increased at 3 hour after administration of LPS compared with those of control group (0.52±0.03 VS 0.47±0.02, 5.24±0.45 VS 4.43±0.21, P < 0.05). During 6 hour to 12 hour after administration of LPS, the lung coefficient and W/D were significantly increased compared with those of control group (P < 0.05 or P < 0.01).2. In control group, the contents of IL-1βand IL-10 were not altered from 1 hour to 12 hour. The content of IL-1βwas significantly increased at 1 hour after administration of LPS compared with that of control group (11.18±0.59 VS 9.26±1.37, P < 0.01). During 3 hour to 12 hour after administration of LPS, the content of IL-1βwas decreased compared with that of LPS 1 h group while was increased compared with that of control group (P < 0.05 or P < 0.01). The content of IL-10 was significantly increased at 1 hour after administration of LPS compared with that of control group (213.46±12.84 VS 11.84±1.25, P < 0.01). The content of IL-10 was significantly increased at 3 hour after administration of LPS compared with that of LPS 1 h group and control group. During 6 hour to 12 hour after administration of LPS, the content of IL-10 was decreased compared with that of LPS 3 h group while was significantly increased compared with control group (P < 0.01). 3. In control group, the level of H2S in plasma and the activity of CSE in lung tissue were not altered from 1 hour to 12 hour. The level of H2S in plasma and the activity of CSE in lung tissue were not altered at 1 hour after administration of LPS compared with those of control group (35.10±3.81 VS 37.22±4.48, 4.61±0.75 VS 4.68±0.63, P > 0.05). The level of H2S in plasma and the activity of CSE in lung tissue were significantly decreased at 3 hour after administration of LPS compared with those of control group (24.90±2.21 VS 36.67±5.35, 3.35±0.98 VS 4.61±0.68, P < 0.01). During 6 hour to 12 hour after administration of LPS, the level of H2S in plasma and the activity of CSE in lung tissue were significantly decreased compared with those of control group (P < 0.01).4. The structure of pulmonary alveoli were intact in control group. The structure of pulmonary alveoli were not altered at 1 hour after administration of LPS compared with control group. In LPS 3 h, 6 h, 9 h and 12 h groups, the lung tissues were injuried, the pulmonary alveoli were damaged, much exudate in the alveolar sacs and mang inflammatory cells infiltrated. The microvillus of typeⅡalveolar cells were orderly arranged, and the structure of mitochondria, the rough surfaced endoplasmic reticulums and the osmiophilic lamellar bodies were intact in control group. The structure of typeⅡalveolar cells were not altered at 1 hour after administration of LPS compared with those of control group. In LPS 3 h, 6 h, 9 h and 12 h groups, the microvillus of typeⅡalveolar cells were fusion, the mitochondrial swelling, the rough surfaced endoplasmic reticulums were degranulated and the osmiophilic lamellar bodies were fusion or disappear.Conlunsion:The IL-1βand IL-10 were increased while the lung tissue was not significantly injuried. During 3 hour to 12 hour after administration of LPS, the contents of IL-1βand IL-10 were increased, while the level of H2S in plasma and the activity of CSE in lung tissue were markedly decreased. It could be concluded that IL-1β, IL-10 and H2S/CSE system may be play a role in the physiopathologic process of ALI induced by LPS.Part 2 Effects of hydrogen sulfide on hyperoxidation in acute lung injury induced by lipopolysaccharide in ratsObjective: To study the effect of H2S on hyperoxidation in LPS-induced acute lung injury rats and explore the possible mechanism.Methods: Fourty-eight male rats were randomly divided into six groups (n=8).ⅠControl group;ⅡLPS group;ⅢLPS+NaHS Low dose group;ⅣLPS+NaHS Middle dose group;ⅤLPS+NaHS High dose group;ⅥLPS+PPG group. LPS was administrated in LPS group. Saline was administrated in control group. In LPS+NaHS Low, Middle and High dose groups or LPS+PPG group, sodium hydrosulfide (NaHS) or DL- propargylglycine (PPG) were respectively administrated at 3 hour after administration of LPS. The rats were respectively killed at 6 hour after administration of LPS or saline. The lung coefficient, W/D, the content of H2S in plasma and the activity of CSE in lung tissue were respectively detected. The content of malondialdehyde (MDA), and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in lung tissue were respectively measured. The morphological changes of lung tissue were respectively observed by light and electron microscopes.Results:1. The lung coefficient and W/D were significantly increased in LPS group compared with those of control group (P < 0.01). Compared with LPS group, the lung coefficient and W/D were respectively decreased in LPS+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01), while were significantly increased in LPS+PPG group (P < 0.05 or P < 0.01).2. The level of H2S in plasma and the activity of CSE in lung tissue were significantly decreased in LPS group compared with those of control group (P < 0.01). Compared with LPS group, the level of H2S in plasma and the activity of CSE in lung tissue were significantly increased in the LPS+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01), while were decreased in the LPS+PPG group (P < 0.05).3. Compared with control group, the content of MDA was significantly increased (4.04±1.21 VS 8.86±1.36, P < 0.01), and the activities of SOD and GSH-PX were significantly decreased (28.35±1.94 VS 16.38±4.42,21.45±2.99 VS 11.45±2.39, P < 0.01) in lung tissue in LPS group. Compared with LPS group, the content of MDA was significantly decreased, and the activities of SOD and GSH-PX were significantly increased in LPS+NaHS Low, Middle and High dose groups(P < 0.05 or P < 0.01). Compared with LPS group, the content of MDA was increased, the activity of SOD was decreased and the activity of GSH-PX was not altered in LPS+PPG group (P < 0.05 or P < 0.01).4. The structure of pulmonary alveoli were intact in control group. In LPS group, the lung tissue was injuried, the pulmonary alveoli were damaged, much exudate in the alveolar sacs and mang inflammatory cells infiltrated. In LPS+NaHS Low, Middile and High dose groups, the injury of the lung tissue was markedly ameliorated compared with that of LPS group. In LPS + PPG group, the injury of the lung tissue was markedly aggragated compared with that of LPS group. The microvillus of typeⅡalveolar cells were orderly arranged, and structure of the mitochondria, the rough surfaced endoplasmic reticulums and the osmiophilic lamellar bodies were intact in control group. In LPS group, the microvillus of typeⅡalveolar cells were fusion, the mitochondrial swelling, the rough surfaced endoplasmic reticulums were degranulated and the osmiophilic lamellar bodies were fusion or disappear. In LPS + NaHS Low, Middle and High dose groups, the injury of the lung tissue was ameliorated compared with that of LPS group. In LPS + PPG group, the injury of the lung tissue was markedly aggragated compared with LPS group. Conclusion: Relatively early administration of NaHS could ameliorate LPS-induced ALI in rats. The possible mechanism is that the H2S synthesis, and the activities of CSE, SOD and GSH-PX were increased, while the lung coefficient, W/D and the MDA synthesis were decreased after administration of NaHS. But relatively early administration of PPG could aggragate ALI. The possible mechanism is that the activities of CSE and SOD, and the H2S synthesis were decreased, while the lung coefficient, W/D and the MDA synthesis were increased after administration of PPG.Part 3 Effects of hydrogen sulfide on inflammatory factor in acute lung injury induced by lipopolysaccharide in ratsObjective: To study the effect of H2S on inflammatory factor in LPS-induced acute lung injury rats and explore the possible mechanism. Methods: Fourty-eight male rats were randomly divided into six groups (n=8).ⅠControl group;ⅡLPS group;ⅢLPS+NaHS Low dose group;ⅣLPS+NaHS Middle dose group;ⅤLPS+NaHS High dose group;ⅥLPS+PPG group. LPS was administrated in LPS group. Saline was administrated in control group. In LPS+NaHS Low, Middle and High dose groups or LPS+PPG group, NaHS or PPG were respectively administrated at3 hour after administration of LPS. The rats were respectively killed at 6 hour after administration of LPS or saline. The contents of interleukin-1β(IL-1β) and interleukin-10 (IL-10) in serum, and their mRNA expression in lung tissue were respectively measured. The expression of intercellular adhesion molecule-1 (ICAM-1) mRNA and the nuclear factor-κB (NF-кBp65) activity in lung tissue were respectively detected.Results:1. Compared with control group, the contents of IL-1βand IL-10 in serum were significantly increased in LPS group. Compared with LPS group, the content of IL-1βin serum was significantly deceased in LPS+NaHS Low, Middle and High dose groups (13.96±0.71 VS 15.01±0.81,12.45±1.35 VS 15.01±0.81,12.24±0.90 VS 15.01±0.81,P < 0.05 or P < 0.01), and the content of IL-10 in serum was significantly increased in LPS+NaHS Middle and High dose groups (137.68±6.27 VS 127.04±5.30, 138.56±6.29 VS 127.04±5.30, P < 0.05). Compared with LPS group, the content of IL-1βin serum was significantly increased (21.58±3.29 VS 15.01±0.81, P < 0.05) and the content of IL-10 in serum was significantly decreased (113.28±9.68 VS 127.04±5.30, P < 0.05) in LPS+PPG group.2. The expression of IL-1β, IL-10 and ICAM-1mRNA, and the activity of NF-кBp65 in lung tissue were detectable in control group. Compared with control group, the expression of IL-1β, IL-10 and ICAM-1 mRNA, and the activity of NF-кBp65 in lung tissue were significantly increased in LPS group. Compared with LPS group, the expression of IL-1βmRNA and the activity of NF-кBp65 in lung tissue were significantly decreased in LPS+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01), the expression of ICAM-1 mRNA was significantly decreased and the expression of IL-10 mRNA was significantly increased in LPS+NaHS Middle and High dose groups (P < 0.05 or P < 0.01). Compared with LPS group, the expression of IL-1βand ICAM-1 mRNA, and the activity of NF-кBp65 in lung tissue were significantly increased, while the expression of IL-10 mRNA was significantly decreased in the LPS+PPG group (P < 0.05 or P < 0.01).Conclusion: Relatively early administration of NaHS could ameliorate LPS-induced ALI in rats. The possible mechanism is that the content of IL-1β, the expression of IL-1βand ICAM-1 mRNA, and the activity of NF-κBp65 were decreased, while the content of IL-10 and the expression of IL-10 mRNA were increased after administration of NaHS. But relatively early administration of PPG could aggragate ALI. The possible mechanism is that the content of IL-1β, the expression of IL-1βand ICAM-1 mRNA, and the activity of NF-κBp65 were increased, while the content of IL-10 and the expression of IL-10 mRNA were decreased after administration of PPG. Part 4 Effects of hydrogen sulfide on apoptosis in acute lung injury induced by lipopolysaccharide in ratsObjective: To study the effect of H2S on apoptosis in LPS-induced acute lung injury rats and explore the possible mechanism.Methods: Fourty-eight male rats were randomly divided into six groups (n=8).ⅠControl group;ⅡLPS group;ⅢLPS+NaHS Low dose group;ⅣLPS+NaHS Middle dose group;ⅤLPS+NaHS High dose group;ⅥLPS+PPG group. In LPS group, LPS was administrated. Saline was administrated in control group. In LPS+NaHS Low, Middle and High dose groups or LPS + PPG group, NaHS or PPG were respectively administrated at 3 hour after administration of LPS. The rats were respectively killed at 6 hour after administration of LPS or saline. The apoptosis of lung cells was detected by FCM. The expressions of Bcl-2 and Bax were respectively detected by immunohistochemisty (IHC). The expressions of Caspase-3 and Caspase-9 were respectively analysised by Western blotting.Results:1. The rate of apoptosis was significantly increased in LPS group compared with that of control group (P < 0.01). Compared with LPS group, the rate of apoptosis was significantly decreased in LPS+NaHS Middle and High dose groups (P < 0.05), and was significantly increased in the LPS+PPG group (P < 0.01).2. The expressions of Bcl-2 and Bax showed buffy positive staining, and the Bcl-2 and Bax were expressed in endochylema of alveolar and airway epithelial cells in control group. In LPS group, a few positive expression of Bcl-2 and considerable positive expression of Bax were observed. In LPS group the expression of Bcl-2 and the ratio of Bcl-2 and Bax were significantly decreased, while Bax was significantly increased (P < 0.01). Compared with LPS group, the expression of Bcl-2 was significantly increased in LPS+NaHS High dose group (P < 0.05), the expression of Bax was significantly decreased in LPS+NaHS Middle and High dose groups (P < 0.05), the ratio of Bcl-2 and Bax was significantly increased in LPS+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01). Compared with LPS group, the expression of Bax was significantly increased (P < 0.05), while the expression of Bcl-2 and the ratio of Bcl-2 and Bax were significantly decreased in LPS+PPG group (P < 0.05 or P < 0.01).3. The results by Western blotting showed that the expression of Caspase-3 and Caspase-9 were markedly increased in LPS group compared with those of control group (P < 0.05). Compared with LPS group, the expression of Caspase-3 and Caspase-9 were significantly decreased in LPS+NaHS High dose group and were significantly increased in LPS+PPG group (P < 0.05).Conlusion: Relatively early administration of NaHS could ameliorate LPS-induced ALI in rats. The possible mechanism is that the rate of apoptosis was decreased, the expression of Bax, Caspase-3 and Caspase-9 were decreased, while the expression of Bcl-2 was increased after administration of NaHS. But relatively early administration of PPG could aggragate ALI. The possible mechanism is that the rate of apoptosis was increased, the expression of Bax, Caspase-3 and Caspase-9 were increased, while the expression of Bcl-2 was decreased after administration of PPG.Part 5 Effects of hydrogen sulfide on pulmoalverolar surfactant in acute lung injury induced by lipopolysaccharide in ratsObjective: To study the effect of H2S on pulmoalverolar surfactant (PS) in LPS-induced acute lung injury rats and explore the possible mechanism. Methods: Fourty-eight male rats were randomly divided into six groups (n=8).ⅠControl group;ⅡLPS group;ⅢLPS+NaHS Low dose group;ⅣLPS+NaHS Middle dose group;ⅤLPS+NaHS High dose group;ⅥLPS+PPG group. In LPS group, LPS was administrated. Saline was administrated in control group. In LPS+NaHS Low, Middle and High dose groups or LPS+PPG group, NaHS or PPG were respectively administrated at 3 hour after administration of LPS. The rats were respectively killed at 6 hour after administration of LPS or saline. The expression of SP-A, SP-B and SP-C mRNA in lung tissue were respectively analysised. The contents of total protein (TP) and total phospholipids (TPL) in bronchoalveolar lavage fluid (BLAF) were respectively measured.Results:1. The expression of SP-A, SP-B and SP-C mRNA in the lung tissue were significantly decreased in LPS group compared with those of control group (0.93±0.05 VS 1.35±0.11, 0.82±0.08 VS 1.12±0.10, 0.72±0.05 VS 0.81±0.08, P < 0.05 or P < 0.01). Compared with LPS group, the expression of SP-A and SP-B mRNA in lung tissue were significantly increased in LPS+NaHS Middle and High dose groups (P < 0.05 or P < 0.01), and the expression of SP-C mRNA was not altered in LPS+NaHS Low, Middle and High dose groups. Compared with LPS group, the expression of SP-A, SP-B and SP-C mRNA were significantly decreased in LPS+PPG group (0.85±0.08 VS 0.93±0.05, 0.74±0.06 VS 0.82±0.08, 0.66±0.05 VS 0.72±0.05, P < 0.05). 2. The content of TP was increased and TPL was decreased in BLAF in LPS group compared with those of control group (0.39±0.33 VS 0.24±0.05,0.29±0.04 VS 0.38±0.06, P < 0.05 or P < 0.01). Compared with LPS group, the content of TP in BLAF was significantly decreased in LPS+NaHS High dose group (P < 0.05), and the content of TPL in BLAF was significantly increased in LPS+NaHS Middle and High dose groups (P < 0.05). Compared with LPS group, the contents of TP was significantly increased and TPL was significantly decreased in BLAF in LPS+PPG group (0.43±0.04 VS 0.24±0.05,0.24±0.03 VS 0.29±0.04, P < 0.05).Conclusion: Relatively early administration of NaHS could ameliorate LPS-induced ALI in rats. The possible mechanism is that the expression of SP-A,SP-B and SP-C mRNA were increased, the content of TP was decreased and the content of TPL in BALF was increased after administration of NaHS. But relatively early administration of PPG could aggragate ALI. The possible mechanism is that the expression of SP-A,SP-B and SP-C mRNA were decreased, the content of TP was increased and the content of TPL was decreased in BALF after administration of PPG.