The Experimental Research About The Roles Of Na~+/H~+ Exchanger 1 In Endotoxin-induced Acute Lung Injury

PartⅠExpression of Na+/H+ exchanger 1 in endotoxin-induced acute lung injury in ratsObjective To build the model of the acute lung injury(ALI) induced by lipopolysaccharide(LPS) in rats in vivo and to observe the changes of Na+/H+ exchanger 1(NHE1) mRNA and protein expressions in the injurious lungs.Methods Forty Specific pathogen-free male Sprague-Dawley rats weighing 250-350 g were randomly divided into the following experimental groups (10 rats in each group):control group (C group):rats received iv. injection of normal saline and sacrificed by carotid artery bleeding 2 h after the administration of saline; LPS-2 h group(L-2 h group):rats received iv. injection of 6 mg/kg LPS and sacrificed by carotid artery bleeding 2 h after the administration of LPS; LPS-4 h group(L-4 h group); rats received iv. injection of 6 mg/kg LPS and sacrificed by carotid artery bleeding 4 h after the administration of LPS; LPS-6 h group(L-6 h group):rats received iv. injection of 6 mg/kg LPS and sacrificed by carotid artery bleeding 6 h after the administration of LPS. The samples of pulmonary tissue and lung lavage fluid were collected after experiments. The basic vital signs of rats were monitored and recorded. The pathological changes in lung tissue were examined with optical microscopy. The acute lung injury score and lung tissue wet / dry weight ratio were calculated. The total protein, tumor necrosis factor-a and macrophage inflammatory protein concentration in bronchoalveolar lavage fluid were detected. The myeloperoxidase(MPO) activity of lung tissue were measured. The nuclear factor-KB (NF-κB) activity of lung tissue were assayed with electrophoretic mobility shift assay (EMSA). The NHE1 mRNA and protein expressions in lung tissue were detected by immunohistochemistry,reverse transcriptase-polymerase chain reaction (RT-PCR) and Western Blot.Results (1) Compared with the control group, the Mean Arterial Pressure(MAP) of rats in LPS-2 h group,LPS-4 h group and LPS-6 h group were decreased significantly (all P<0.05)% Heart Rate (HR) and Respiratory Frequency(RF) of rats in LPS-2 h group,LPS-4 h group and LPS-6 h group were significantly elevated(all P<0.05); Arterial partial pressure of oxygen(PaO2) of rats in LPS-4 h group and LPS-6 h group were decreased significantly(all P <0.05). (2) The lung tissue in control group rats showed normal alveolar septum and alveolar spaces without exudation; the lung tissue of rats in LPS-2h,LPS-4 h group and LPS-6 h group showed high levels of intra-alveolar exudates, hyaline membrane formation, inflammatory cell infiltration, intra-alveolar hemorrhage, and interstitial edema. Compared with the control group, the lung tissue ALI scores of rats in LPS-2 h,LPS-4 h group and LPS-6 h group were significantly elevated(all P<0.05), the overall differences between groups were statistically significant (P<0.05). (3) Compared with the control group, the lung tissue wet/ dry weight ratio of rats in LPS-2 h group,LPS-4 h group and LPS-6h group were significantly elevated(all P<0.05), the overall differences between groups were statistically significant (P<0.05); the total protein(TP), tumor necrosis factor-α(TNF-α) and macrophage inflammatory protein(MIP-2) concentration in bronchoalveolar lavage fluid of rats in LPS-2 h group,LPS-4 h group and LPS-6 h group were significantly elevated(all P<0.05), the overall differences between groups were statistically significant (all P<0.05). (4) Compared with the control group, the MPO activity of lung tissue in LPS-2 h group,LPS-4 h group and LPS-6 h group were significantly elevated(all P<0.05), the overall differences between groups were statistically significant(P<0.05). (5) Compared with the control group, the NF-κB activity of lung tissue in LPS-2 h group,LPS-4 h group and LPS-6 h group were significantly elevated(all P<0.05); the overall differences between groups were statistically significant(P<0.05). (6) Compared with the control group, the NHE1 protein immunohistochemistry expressions of lung tissue in LPS-2 h group,LPS-4 h group and LPS-6 h group were significantly elevated(all P<0.05); the overall differences between groups were statistically significant(P<0.05). (7) Compared with the control group, the NHE1 mRNA expressions of lung tissue in LPS-2 h group,LPS-4 h group and LPS-6 h group were significantly elevated(all P<0.05); the overall differences between groups were statistically significant(P<0.05). (8) Compared with the control group, the NHE1 protein expressions of lung tissue in LPS-2 h group,LPS-4 h group and LPS-6 h group were significantly elevated (all P<0.05); the overall differences between groups were statistically significant(P<0.05).Conclusion Rats produced significant acute lung injury after intravenous injection of LPS 4 h with 6mg/kg, which includes endotoxin shock symptoms,histological inflammation in rats gross specimen pathology,lung tissue water content increased,inflammatory cytokines in lung tissue and bronchoalveolar lavage fluid increased; at the same time, the NHE1 mRNA and protein expressions of lung tissue in each LPS group were significantly elevated and remarkably increased with the duration of acute lung inflammatory response. Objective To investigate the effects of amiloride pretreatment on the acute lung injury(ALI) induced by lipopolysaccharide(LPS) in rats and the underlying mechanism.Methods Forty Specific pathogen-free male Sprague-Dawley rats weighing 250-350g were randomly divided into the following experimental groups (10 rats in each group):control group (C group):rats received iv. injection of normal saline; amiloride group(A group):rats received iv. injection of 10 mg/kg amiloride; LPS group(L group):rats received iv. injection of 6mg/kg LPS; amiloride pretreatment group(AL group):rats received iv. injection of 10 mg/kg amiloride 30 min before the administration of LPS. Degree of ALI were assessed by wet/dry weight ratio(W/D) and lung histological examination. Concentrations of total protien in bronchoalveolar lavage fluid(BALF) were determined. The activity of myeloperoxidase(MPO),the concentrations of malondialdehyde(MDA) and the Vitality of Superoxide dismutase(SOD) in lung tissue were also measured. The contents of nitrotyrosine(NT) in lung tissue were determined by semi-quantitative immunohistochemistry. The Na+/H+ exchanger 1 (NHE1) mRNA expressions in lung tissue were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of NHE1,extracellular signal regulated kinase (ERK) and mitogen-activated protein kinase(MAPK) p38 in lung tissue were also determined by Western blot analysis.Results Lung tissue ALI scores,W/D ratio,MPO activity,MDA concentrations,SOD vitality and NT contents, concentrations of total protienin BALF, the expression of NHE1, MAPK p38 and ERK increased significantly in LPS group as compared with control group(all P<0.05). These increases were significantly reduced in amiloride pretreatment group(all P<0.05) except MAPK p38 expression.Conclusion NHE1 inhibitor amiloride pretreatment can attenuate ALI induced by LPS in rats. This effect may be related to inhibition of ERK activation.Part IIIRoles of mitogen-activated protein kinases in the protection effects of amiloride on endotoxin-induced acute lung injury in ratsObjective To investigate the roles of mitogen-activated protein kinases(MAPKs) in the protection effects of amiloride pretreatment on the acute lung injury(ALI) induced by lipopolysaccharide(LPS) in rats.Methods Seventy specific pathogen-free male Sprague-Dawley rats weighing 250-350g were randomly divided into the following experimental groups (10 rats in each group):control group (C group):rats received iv. injection of normal saline; LPS group(L group):rats received iv. injection of 6mg/kg LPS; amiloride pretreatment group(AL group):rats received iv. injection of 10 mg/kg amiloride 30 mins before the administration of LPS; PD098059 pretreatment group(PL group):rats received iv. injection of 0.3mg/kg PD098059 30 mins before the administration of LPS; SB203580 pretreatment group(SL group):rats received iv. injection of 10mg/kg SB203580 30 mins before the administration of LPS; amiloride combined PD098059 pretreatment group(APL group):rats received iv. injection of 10 mg/kg amiloride and 0.3mg/kg PD098059 30 mins before the administration of LPS; amiloride combined SB203580 pretreatment group(ASL group):rats received iv. injection of 10 mg/kg amiloride and 10mg/kg SB203580 30 mins before the administration of LPS. All rats were sacrificed by carotid artery bleeding 6h after the experiment starting. The samples of pulmonary tissue and lung lavage fluid were collected after experiments. The pathological changes in lung tissue were examined with optical microscopy. The acute lung injury scores and lung tissue wet/dry weight ratio were calculated. The total protein, tumor necrosis factor-a and macrophage inflammatory protein concentration in bronchoalveolar lavage fluid were detected. The myeloperoxidase(MPO) activity of lung tissue were also measured; the NHE1 mRNA and protein expressions in lung tissue were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western Blot analysis.Results (1) The lung tissues in control group rats showed normal alveolar septum and alveolar spaces without exudation; the lung tissues of rats in the other groups showed high levels of intra-alveolar exudates, hyaline membrane formation, inflammatory cell infiltration, intra-alveolar hemorrhage, and interstitial edema. (2) Compared with the control group, concentrations of total protien,tumor necrosis factor-αand macrophage inflammatory protein in bronchoalveolar lavage fluid, lung tissue ALI scores,W/D ratio,MPO activity and NHE1 mRNA and protein expressions in the other groups were significantly increased (all P<0.05), the indicators above-mentioned in LPS group were the most significantly increased (all P<0.05); compared with the LPS group, the indicators above-mentioned in amiloride pretreatment group were decreased significantly (all P<0.05); compared with the amiloride pretreatment group, the indicators above-mentioned in PD098059 pretreatment group,SB203580 pretreatment group and amiloride combined PD098059 pretreatment group were decreased significantly (all P<0.05) however; compared with these three groups, the indicators above-mentioned in amiloride combined SB203580 pretreatment group were significantly decreased (all P<0.05) yet.Conclusion NHE1 inhibitor amiloride pretreatment can attenuate ALI induced by LPS in rats; extracellular signal-regulated kinase inhibitor can completely abolish this kind of protective effect of amiloride, which illustrated that this effect may be related to inhibition of extracellular signal-regulated kinase activation. Part IV Expressions of Na+/H+ exchanger 1 in lipopolysaccharide-activated mouse macrophage-like cells and the pretreatment effect of amilorideObjective To observe the changes of Na+/H+ exchanger 1(NHE1) mRNA and protein expressions in the lipopolysaccharide(LPS)-activated mouse macrophage-like cells and the pretreatment effect of amiloride.Methods The mouse macrophage-like cell line (RAW 264.7) cells were seeded in 6 well plate with the density of 1×106/ml and cultured in RPMI 1640 medium at 37℃in 5% CO2 incubator for 48 h in vitro, then all cells were randomly divided into 4 groups(n=12):group C:control; amiloride group:RAW 264.7 cells were co-incubated with amiloride 300μM (final concentration); LPS group:RAW 264.7 cells were co-incubated with LPS 1μg/ml (final concentration); amiloride pretreatment group:RAW 264.7 cells were co-incubated with amiloride 300μM (final concentration) for 30 mins before co-incubated with LPS 1μg/ml (final concentration). The concentrations of tumor necrosis factor-α(TNF-α) and the levels of nitric oxide(NO) in cell culture supernatants in each group were detected with enzyme linked immunosorbent assay(ELISA) and the Griess method 24 h after the start of the trial. The levels of intracellular reactive oxygen species(ROS) in each group were detected with the ROS sensitivity DCFH-DA fluorescent probe method 24 h after the start of the trial. The expressions of NHE1 mRNA in each group were measured by reverse transcription-polymerase chain reaction (RT-PCR) 3 h after the start of the trial. The levels of NHE1 protein expressions in each group were also detected with Western blot analysis 6 h after the start of the trial.Results Compared with the control group, the concentrations of TNF-a and the levels of NO in cell culture supernatants,the levels of intracellular ROS,the expressions of NHE1 mRNA and the levels of NHE1 protein expressions in amiloride group have no statistical difference(all P>0.05); but the indicators above-mentioned in LPS group and amiloride pretreatment group were significantly increased(all P<0.05). Compared with the LPS group, the indicators above-mentioned in amiloride pretreatment group were significantly decreased (all P<0.05).Conclusion NHE1 can be rapidly activated and the expressions of NHE1 were increased in the LPS-activated mouse macrophage-like cells; pretreatment intervention with NHE 1 inhibitor amiloride can restrain the changes.