Effects And Mechanisms Of Nitric Oxide Synthase Inhibitors On Acute Lung Injury Induced By Lipopolysaccharide In Rats

Acute lung injury (ALI) is acute, progressive, anoxic injury of alveolar -capillary membrane by causative agents of lung interior and exterior that not cardiogenic. ALI and acute respiratory distress syndrome (ARDS) are clinical syndromes and still remain a higher mortality of 40%～60% ,and a specific therapy is not available. Lipopolysaccharide(LPS) is the main composition of 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.NO is a highly reactive messenger molecule,and is synthesized from L-Arg by NO synthase(NOS). NO and NOS play a important role in LPS-induced acute lung injury,and have"dual effects"in ALI. It is very necessary to investigate the effects and mechanisms of NO and NOS inhibitors on LPS-induced acute lung injury.In the present study, the chronological changes of pulmonary apoptosis and the expression of iNOSmRNA,nNOSmRNA and eNOS mRNA were respectively measured and the effects of selective inducible NOS(iNOS) inhibitor AG,the nonselective NOS inhibitor L-NA on the expression of mediators of inflammation, pulmonary surfaetant(PS),Bcl-2, Bax,caspase-3 and pulmonary cell apoptosis were respectively observed in rats ALI induced by LPS.Part 1 Changes of pulmonary apoptosis and NOS mRNA on acute lung injury induced by lipopolysaccharide in ratsObjective: To observe the chronological changes of pulmonary apoptosis and the expression of iNOSmRNA,nNOSmRNA and eNOS mRNA in LPS-induced acute lung injury(ALI) and the relation of them,and investigate the mechanisms of ALI.Methods: Rats were randomly divided into 2 groups: group1: control; group2: LPS.The rats were injected with either saline or LPS and killed at 1, 3, 6,9 and 12h after administration of LPS.The expressions of iNOSmRNA, nNOSmRNA and eNOSmRNA in the lung tissue were respectively measured with RT-PCR methods;apoptotic rate, bcl-2 and bax were respectively determined by Flow Cytometry(FCM) and immunohistochemisty (IHC);the pathological changes of lung tissue were observed by light and electron microscope.Results:1. Gene expression of eNOS mRNA was detectable in the normal control group (0.288±0.017). Compared with that of the normal control group,the eNOSmRNA was significantly decreased at 3,6,9 and 12h after administration of LPS (P<0.05, P<0.01).2. Compared with that of the control group(0.081±0.002), the expression of iNOSmRNA was significantly increased at 3,6,9 and 12h(0.206±0.006,0.276±0.040, 0.360±0.007, 0.420±0.047) after administration of LPS (P<0.01);3. The nNOSmRNA has no significant change during the 12h after administration of LPS in LPS group.4. Apoptosis of lung tissues was determined by FCM. Significant DNA fragmentation was detected after lung injury. Remarkably high apoptotic percentages(24.350±3.845, 25.040±4.137,27.10±3.922,21.683±3.153)% were showed in the tissue from the LPS group compared with that of control group(11.373±3.088)% (P<0.01), and reached a peak of 27.103±3.922 at 9 hours,but decreased at 12 hours(21.683±3.153) after the start of LPS infusion,which was still obviously higher than that of control group (P < 0.01).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. The ratioes of Bcl-2 and Bax were lower in LPS 3h, LPS 6h, LPS 9h and LPS 12h group (0.615±0.185, 0.447±0.086, 0.327±0.067, 0.408±0.053) than that in control group(0.976±0.157) (P<0.01).5. Electronic microscope showed the microvillus and osmiophiIic multi1amellar body of alveolar typeⅡ(AT-Ⅱ)epithelial cells decreased; the mitochondria swelled, the cristae disrupted, dissolved or disappeared in LPS group. The above changes were not observed in sham group.Degree of ALI was gradually worsened after administration of LPSConclusion: It could be concluded that the expressions of iNOSmRNA, eNOSmRNA and nNOSmRNA were not consistent and NOS may regulate the apoptosis of pulmonary cell through affecting the balance of Bcl-2 and Bax in rats acute lung injury induced by LPS.Part 2 Effect of aminoguanidin on acute lung injury induced by lipopolysaccharide in ratsObjective:To investigate the effect and the possible mechanism of aminoguanidine (AG) on the lipopolysaccharide(LPS)-induced acute lung injury in rats.Methods: Male SD rats were randomly divided into 3 groups:①control group;②LPS group;③AG group. AG was administrated in AG group, saline was administrated in control group and LPS group.LPS group and AG group were further divided into 3 subgroups according to the duration of ALI:1h+3h group, 3h+3h group and 6h+3h group. In AG group and LPS group, LPS was administrated.Saline was administrated in control group. The pathological changes of lung injury were observed by light and electron microscopy.The concentrations of TNF-α, IL-6 in lung tissue were evaluated by radioimmunoassay, respectively.The expression of intercellular adhesion molecule 1(ICAM-1) and the translocation of NF-κB in the lung tissue were detected by immunohistochemistry. The SP-A mRNA was measured by in situ hybridization(ISH).Apoposis,the expression of caspase-3 protein,Bcl-2 and Bax were respectively detected with Flow Cytometry (FCM),Western blot analysis and IHC.Results:1. Electronic microscope showed the microvillus and osmiophiIic multilamellar body of alveolar typeⅡ(AT-Ⅱ) epithelial cells decreased; the mitochondria swelled, the cristae disrupted, dissolved or disappeared in LPS group. The above changes were not observed in control group.Degree of ALI was gradually worsened after administration of LPS. Administration of AG could ameliorate these pathological alterations.2. In the LPS group, NF-κB was significantly translocated from the cytoplasm into the nucleus. However, NF-κB remained in the cytoplasm in the control group. The expression of NF-κB in the nucleus was markedly higher in the LPS group than that of in the control group(P﹤0.01).The concentrations of TNF-α, IL-6 in lung tissue were significantly increased (P﹤0.01, P﹤0.05) in LPS group. In LPS group,the expressions of ICAM-1 showed buffy positive staining,and they were expressed in blood vessel endothelium ,alveolar and airway epithelial cells.Compared with those of LPS group, the NF-κB translocation into nucleus was significantly inhibited and the expression of NF-κB in the nuclei was attenuated (P﹤0.01), the concentrations of TNF-α, IL-6 and the expression of ICAM-1 in lung tissue were significantly decreased (P﹤0.01, P﹤0.05) in AG(1h+3h) and AG(3h+3h) group,respectively.3. Considerable gene expression of SP-A mRNA was detectable in the normal control group (0.113±0.208), and the expressions were markedly lower in LPS groups than that in control group(P﹤0.01). Compared with those of LPS group, the expression of SP-A mRNA was increased in group AG(1h+3h) (0.098±0.013)and AG(3h+3h()0.082±0.020(P﹤0.01, P﹤0.05),respectively.4. 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. The ratioes of Bcl-2 and Bax were lower in LPS4h, LPS6h, LPS9h group (0.595±0.121, 0.447±0.086, 0.327±0.067) than that in control group(0.976±0.157) (P<0.01). Compared with those of LPS group, the expression of Bcl-2 was increased(P<0.01),the expression of Bax was decreased (P<0.01),and the ratioes of Bcl-2 and Bax (0.828±0.144, 0.695±0.217, 0.443±0.092)were significantly increased in AG group (P<0.01).5. The results by Western blot showed that the expression of caspase-3 was remarkably higher in LPS group than that in control group(P<0.01),and the expression was lower in AG(1h+3h) and AG(3h+3h)group than that in LPS group(P<0.01).6. Apoptosis of lung cells was determined by FCM. DNA fragmentation was detected after lung injury. The tissue from the LPS group showed remarkably high apoptotic percentages with (24.350±3.845, 25.040±4.137,27.103±3.922,21.683±3.153)% compared with that of control group(11.373±3.088)% (P<0.01).The apoptotic index was markedly lower in AG(1h+3h) and AG(3h+3h)group than that of LPS group(P﹤0.01, P﹤0.05).Conclusion: Relatively early administration of AG could ameliorate LPS-induced acute lung injury in rats. The possible mechanism is that AG could inhibits NF-κB activation, subsequent leads to down-regulation of NF-κB-dependent inflammatory response in lung tissue, increase the expression of SP-A mRNA,reduce the expression of caspase-3 and Bax protein, increase the expression of bcl-2 protein, and regulate the balance between bcl-2 and Bax protein.Part 3 Effect of L-NA on acute lung injury induced by lipopolysacchari- de in ratsObjective:To investigate the effect and the possible mechanism of nonselective NOS inhibitor L-NA on the LPS-induced acute lung injury in rats.Methods: Male SD rats were randomly divided into 3 groups:①control group;②LPS group;③L-NA group. L-NA was administrated in L-NA group, saline was administrated in control group and LPS group.LPS group and L-NA group were further divided into 3 subgroups according to the duration of ALI:1h+3h group, 3h+3h group and 6h+3h group. In L-NA group and LPS group, LPS was administrated.Saline was administrated in control group. The pathological changes of lung injury were observed by light and electron microscopy. The concentrations of TNF-α, IL-6 in lung tissue were evaluated by radioimmunoassay, respectively.The expression of intercellular adhesion molecule 1(ICAM-1) and the translocation of NF-κB in the lung tissue were detected by immunohistochemistry. The SP-A mRNA was measured by in situ hybridization(ISH).Apoposis,the expression of caspase-3 protein,Bcl-2 and Bax were respectively detected with Flow Cytometry (FCM),Western blot analysis and IHC.Results:1. Electronic microscope showed the microvillus and osmiophiIic multi1amellar body of alveolar typeⅡ(AT-Ⅱ)epithelial cells decreased; the mitochondria swelled, the cristae disrupted, dissolved or disappeared in LPS group. The above changes were not observed in control group.Degree of ALI was gradually worsened after administration of LPS.Administration of L-NA could ameliorate these pathological alterations.2. In the LPS group, NF-κB was significantly translocated from the cytoplasm into the nucleus.However, NF-κB remained in the cytoplasm of lung tissue in the control group. The expression of NF-κB was markedly higher in the nucleus of LPS group than that of in the control group(P﹤0.01).The concentrations of TNF-α, IL-6 in lung tissue were significantly increased(P﹤0.01, P﹤0.05) in LPS group. In LPS group,the expressions of ICAM-1 showed buffy positive staining,and they were expressed in blood vessel endothelium,macrophage,alveolar and airway epithelial cells.Compared with those of LPS group, the NF-κB translocation into nucleus was significantly inhibited,and the expression of NF-κB in the nuclei(P﹤0.05, P﹤0.01) and the expression of ICAM-1 in lung tissue (P﹤0.05, P﹤0.01) was attenuated in L-NA(1h+3h) and L-NA(3h+3h) group, respectively, and the concentrations of TNF-α, IL-6 were not significantly altered in L-NA group.3. Considerable gene expression of SP-A mRNA was detectable in the normal control group (0.113±0.021), and the expressions were markedly lower in LPS groups than that in control group(P﹤0.01). Compared with those of LPS group, the expressions of SP-A mRNA were increased(P﹤0.05, P﹤0.01) in group L-NA(1h+3h) (0.096±0.013)and L-NA(3h+3h)(0.085±0.015)4. 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. The ratioes of Bcl-2 and Bax were lower in LPS4h, LPS 6h, LPS 9h group (0.595±0.121, 0.447±0.086, 0.327±0.067) than that in control group(0.976±0.157) (P<0.01). Compared with those of LPS group, the expression of Bax and the ratio of Bcl-2 and Bax were not significantly altered in L-NA group.5. The results by Western blot showed that the expression of caspase-3 was remarkably higher in LPS group than that in control group(P<0.01). There was no significant differences between L-NA group and the LPS group (P>0.05)6. Apoptosis of lung cells was determined by FCM. DNA fragmentation was detected after lung injury. The tissue from the LPS group showed remarkably high apoptotic percentages (23.96±3.10, 25.040±4.137,27.103±3.922)% compared with that of control group(11.373±3.088)% (P<0.01). The apoptotic index in L-NA(3h+3h) and L-NA(6h+3h) group had no significant differences compared with LPS group.Conclusion: L-NA could lighten LPS-induced acute lung injury to some degree in rats by inhibitting NF-κB activation and reducing the expression of ICAM-1, but has no association with significant alterations in the expression of caspase-3 and Bax protein and regulating the balance between bcl-2 and Bax protein and the apoptosis.Part4 Effect of AG and L-Arg on the lipopolysaccharide-induced alveolar macrophagesObjective: To investigate the effect of nitric oxide (NO) on the lipopolysaccharide-induced alveolar macrophages injury.Methods: Alveolar macrophages were isolated from bronchoalveolar lavage (BAL) fluid in rats,and were divided into four groups:①normal group;②LPS group: cells were treated with 10μg/ml LPS for 3h;③AG group: cells were stimultaneously treated with 2mmol/L AG and 10μg/ml LPS for 3h;④L-Arg group: cells were stimultaneously treated with 2mmol/L L-Arg and 10μg/ml LPS for 3h. The activity of lactate dehydrogenase(LDH) and the content of NO in culture medium was detected by colorimetric assay; Mitochondrial activity was determined by methylthiazolyl tetrazolium(MTT) test;Tumor necrosis factor-a (TNF-a) and Interleukin-6(IL-6) in the culture supernatants were respectively measured by radioimmunoassay.Results:1.In 10μg/ml LPS treated group, LDH activity(181.54±22.81), the contents of NO (765.69±32.14), TNF-a(542.52±70.11 )and IL-6(162.12±33.14) in culture medium were significantly increased compared with that of normal group(P﹤0.01);the mitochondial activity(0.450±0.041) was significantly decreased compared with that of normal group(0.574±0.043) ( P﹤0.01). The LDH activity(133.97±35.99)(P﹤0.05), the contents of NO (588.63±97.43),TNF-a(306.13±44.14) and IL-6(133.08±13.73) were decreased,and the mitochondial activity (0.571±0.099)was increased in AG group compared with that of LPS group.2. Compared with LPS group, the LDH activity(136.93±30.41) was decreased (P﹤0.05),the activity of mitochondria(0.556±0.039)was enhanced(P﹤0.01), the contents of TNF-(a277.57±40.50) and IL-(6130.73±16.61)were decreased in L-Arg group(P﹤0.01, P﹤0.05).The content of NO was not significantly altered in L-Arg group.Conclusion: The selective inhibiting reagent of iNOS AG can regulate the over-responses of inflamm action through depressing endogenous nitric oxide;L-Arg can also inhibit the secretion of inflammatory mediators induced by LPS.