| Acute respiratory dystress syndrome(ARDS)is one of the life-threatening respiratory failure,which could be caused by various diseases.Pneumonia is the most common direct cause of ARDS.Pseudomonas Aeruginosa is the most common bacteria in Intensive Care Unit(ICU).Pneumonia with Pseudomonas could indirectly lead to lung injury or ARDS.Despite the use of lung-protected mechanical ventilation,ARDS still has a mortality rate of over 40%.By now,activation of Cholinergic Anti-inflammatory Pathway(CAP)has been demonstrated to reduce lung injury and decrease the mortality,in some experimental animal models.?7 nictinic acetylcholinergic receptor(a7nAChR)plays the key role in CAP that could be activated by a7nAChR agonist.GTS-21 is one of selective a7nAChR agonist that could down-regulates imflammation and improve cell function in sepsis.ObjectiveThe mechanism of a7nAChR agonist attenuating lung injury remains unclear and there is rare study on the effect of a7nAChR activation by GTS-21 on lung injury caused by Pseudomonas pneumonia.In this study,we examined the protective role of GTS-21 on the lung injury using in vivo and in vitro models inducd by Pseudomonas and inflammatory mdeidiator(TNF-a)in mice and A549 cells.The investigation of degree of lung epithelial cell damage and related molecular mechanisms was performed in order to explore therapeutic targets.MethodI.Pneumonia model.Wild type mice(C57BL/6)(n=30)were divided into 3 groups randomly as follows:i)Control group(n=10),mice were fed normally without any treatment;ii)sham(n=10),received normal saline(NS)intratracheal;iii)lung injury group(n=10),received Pseudomonas Aeruginosa Xen-5 intratracheal;We monitored the mice and identified the bacteria in lung using Living Image System.The mice were sacrificed after 24 hours,and BALF,lung tissue were obtained.Lung was fixed with 10%formalin at least 24 hours and embedded in paraffin,5 um sections were cut and stained with hematoxylin and eosin(H&E).Histopathological photos were taken with a light microscope and lung injury score are analysized.Neutrophil and monocyte cells in BALF were counted.Lung W/D weight was calculated.HMGB1 was measured with Elisa kits.Protein concentration of HMGB1 was measured by Western Blot.?.The effect of GTS-21 on lung injury in mouse pneumonia model1.Study on survival in WT mice treated with GTS-21 or NS after pneumonia.The mice(C57BL/6)(32 mice)were divided into 4 groups randomly as flowing:i)Sham group(n=8),received normal saline(NS);ii)lung injury group(n=8),received Pseudomonas Aeruginosa Xen-5 intratracheally;iii)lung injury+GTS-21 group(n=8),received Pseudomonas Aeruginosa Xen-5 intratracheally and GTS-21 intraperitoneally;iiii)GTS-21 group(n=8),received GTS-21,intraperitoneal.After surgical procedure,all mice were monitored for a week2.Study on survival in a7nAChR KO and WT mice after pneumonia model.Wild type mice(C57BL/6)(n=16)and a7nAChR KO mice(n=16)were divided into 4 groups:i)WT mice sham(n=8),received normal saline(NS);ii)WT mice lung injury group(n=8),received Pseudomonas Aeruginosa Xen-5 intratracheal;iii)KO mice sham(n=8),received normal saline(NS);iiii)KO mice lung injury group(n=8),received Pseudomonas Aeruginosa Xen-5 intratracheal.After made model,After surgical procedure.all mice were monitored for a week3.Wide type mice(C57BL/6)(n=40)and a7nAChR KO mice(n=40)were divided into 4 groups separately:i)sham(n=8),received normal saline(NS);ii)lung injury group(n=8),received Pseudomonas Aeruginosa Xen-5 intratracheal;iii)lung injury+GTS-21 group(n=8),received Pseudomonas Aeruginosa Xen-5 intratracheal and GTS-21 intraperitoneal;iiii)GTS-21 group(n=8),received GTS-21,intraperitoneala.BALF samples were obtained from mice at 24 h time point.After centrifugation,supernatant was used to measure HMGB1 level and sediment was used to count neutrophils after H&E staining.Lung sample was fixed with formalin(10%),embedded in paraffin,sections was sliced and stained with H&E.Lung injury score were analysized.b.Lung tissue were frozen and protein was isolated.Western Blot was used to detect protein expression of HMGB1,p65 NF-?b,p-?b-a,MAPK p-p38,caspase-3,bcl-2(normalized to GAPDH).III.A549 cells were cultured in RPMI medium 1640 containing 10%fetal bovine serum at 37? and 5%C02 saturation.The cells were divided into 4 groups:?)Control group,TNF-a and GTS-21 free;?)injury group,stimulated with TNF-a(10ng/ml);iii)injury group+GTS-21 group,GTS-21(75uM)was given after 4 hours of TNF-a;iiii)GTS-21 group,GTS-21 only.The cells in all groups were incubated for 12 hours totally.Cell viability was detected by MTT assay.Elisa was used to measure HMGB-1 and IL-6 in media.Western Blot was used to measure relative abundance of NF-?B P65,p-iKB-aand total/phosphorylated MAPK p38(t-p38/p-p38)proteins(normalized to?-actin/GAPDH).TUNEL was used to detect epithelial cell apoptosis.IV.Statistical analysis:Results were expressed as the means standard deviation and analyzed using a one-way ANOVA.A Newman-Keuls post hoc analysis was performed for pairwise multiple comparisons if significance was indicated by the ANOVA.The difference was considered to be statistically significant when P<0.05.ResultsI.The appetite in mice with pneumonia decreased obviously compared with Sham group.But there was no significant difference between control group and sham group.24h after pneumonia model,bacteria were noted using living image system in pneumonia model.There were no difference between Control and Sham group in lung injury score,W/D and neutrophils in BALF.Compared with sham group,the number of neutrophils in BALF were much more in pneumonia group(P<0.05).Compared with sham group,HMGB1 level in BALF and expression in lung were both higher in pneumonia group(P<0.05).Compared with sham group,lung injury score was higher in pneumonia group(P<0.05).?.GTS-21 improved survival(P<0.05)in WT mice with lung injury compared with the mice without GTS-21.Survival in a7nAChR KO mice was reduced compared with wild type mice with lung injury induced by pneumonia(P<0.05)In WT mice,compared with sham group,the number of neutrophils in BAL and lung injury score were much higher in lung injury group(P<0.05);HMGB1 level in BALF and HMGB1 expression in lung homogenate were both higher than sham group(P<0.05).GTS-21 attenuated HMGB1 levels in BALF and lung homogenate(P<0.05).GTS-21 also attenuated neutrophils in BALF and lung injury score(P<0.05).Compared with sham group,protein levels of p65 NF-Kb,p-iKb-a,MAPK p-p38,caspase-3 expressed stronger in injury group,however,BCL-2 expressed less compared to control group(P<0.05);GTS-21 attenuated the expression of p65 NF-Kb,p-i?b-?,MAPK p-p38,caspase-3 and BCL-2(P<0.05).The number of the neutrophils in BALF,lung injury score,protein levels of p65 NF-Kb,p-i?b-?,MAPK p-p38,caspase-3 were higher in both a7nAChR KO WT mice(P<0.05),however,even higher of the levels of above indicators was observed in a7nAChR KO mice compared with WT mice(P<0.05).GTS-21 attenuated all indicators induced by lung injury in WT animals(P<0.05),but not in a7nAChR KO mice.?.Compared with control group,MTT showedTNF-? decreased lung epithelia cell viability,however,GTS-21 improved cell viability deduced by TNF-a(P<0.05).Elisa results showed GTS-21 decreased HMGB-1 and IL-6 levels in TNF-a stimu ated cells media(P<0.05).Western Blot results showed protein of p65 NF-?b,p-i?b-?,MAPK p-p38 expressed stronger in TNF-a group and GTS-21 attenuates NF-?B P65 and p38 MAPK signaling in TNF-a treated alveolar epithelial cells(P<0.05).TUNEL result showed TNF-a induced apoptosis with dose of lOng/ml or 20ng/ml and GTS-21 reversed the results(P<0.05).ConclusionsGTS-21 attenuates pneumonia-induced lung injury and HMGB1 levels after lung injury in mice.GTS-21 improves animal survival and cell viability.GTS-21 plays protective role in pneumonia-induced lung injury by attenuating NF-?B P65 and p38 MAPK signaling and reducing cell apoptosis. |
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