| ObjectivesThe main pathogenesis of the acute respiratory distress syndrome (ARDS) was excessive uncontrolled inflammatory response mediated by various inflammatory cy-tokines, but the treatment was limited effective. And a large number of polymorpho-nuclear neutrophil (PMN) accumulated in lung tissue plays an important role in dam-age of pulmonary capillary endothelial cells and alveolar epithelial cells. Previous studies have demonstrated that a large number of polymorphonuclear neutrophil (PMN) accumulated in lung tissue and release inflammatory cytokines, such as Inter-leukin-1β(IL-1β),Il-6,IL-8and tumor necrosis factor-alpha (TNF-a) and so on when body against infection or trauma, which plays an important role in starting and main-taining the inflammatory response. And it could promote the excessive production and expression of inflammatory mediators including cytokines, chemokines, adhesion molecules, myeloperoxidase (MPO) and ROS.As an important nuclear transcription factor, Nuclear factor-κB(NF-κB) is the intersection of multiple signaling pathways. The protein exists in the form of inactive in the cytoplasm by the form of dimer and directly combines with inhibitory protein IκB to form the trimeric complex at usually state. P50/P65heterodimer plays a major physiological function during inflammation, and NF-κB P65was as its main subunit. Lipopolysaccharide (LPS) is the major component of the outer membrane of gram-negative bacteria and is a common trigger of sepsis, which is the important init-iation factor to activate NF-κB. The Rel protein localization signal (NLS) exposed under the stimulation of LPS, then NF-κB bound to specific κB sequences of DNA in the nucleus in order to regulate the transcription and expression of gene. IL-10is a major anti-inflammatory cytokines, and could inhibit activation of NF-κB and reduce the expression of pro-inflammatory cytokines.The expression of CD11b/CD18plays an important role in PMN aggregation and activation. CD11b/CD18is a heterodimer of the aM(CD11b) and β2(CD18) subunits, which is a key factor in inflammatory response to combine with the ICAM-1and me-diate the adhesion between PMN and microvascular endothelial cells, then transmit the intracellular signal. CINC, one of the IL-8family, is the specific chemokines of PMN, which plays an important role in the aggregation process of PMN in lung tissue. CINC-1and CINC-3played important role in PMN recruitment to the lung in LPS-induced ARDS.Another chemokine neutrophil activating peptide (ENA-78) also could chemotaxis PMN. In addition, ENA-78could bind to CD11b/CD18receptor, increase the free Ca2+in endothelial cells and the permeability pulmonary capillary.Pyrrolidine dithiocarbamate(PDTC) is a metal chelating agent and an antioxidant, which is the specific inhibitor of NF-κB. The study has confirmed that PDTC could directly reduce binding capacity of NF-κB to DNA, and increase the synthesis of IκB in order to prevent the phosphorylation of IκB and the subsequent degradation. The process could prevent NF-κB move into the nucleus and NF-κB signaling pathway, thus that would inhibit the function of NF-κB. In conclusion, the response above could reduce the gene expression of inflammatory mediators and inflammation and reduce the PMN adhesion and the expression of molecules and chemokines in endo-thelial cell surface.This study investigated the role of pro-inflammatory-anti-inflammatory cyto-kine imbalance and NF-κB activation in PMN accumulation in the lungs of ARDS mice. At the same time, the expression of CD11b/CD18, its ligand ICAM-1and CINCmRNA and the expression of ENA-78were measured, in order to clarify the relationship between adhesion molecule (CD11b/CD18and its ligand ICAM-1) and chemokine (CINC,ENA-78) and PMN aggregation in lung tissue of ARDS. The present study is aimed at evaluating direct effects of PDTC on PMN activities charac- terized by the protein changes of NF-κBp65, infiltration of PMN and excessive re-lease of inflammatory cytokines.MethodsAnimalsBALB/c mice6-8-weeks of age, weighing20±2g, were maintained at24℃room temperature. To further study the protective effect of PDTC on mice with LPS, mice were randomly divided into3groups:Control group (saline,20ml/kg,i.p.), LPS group (LPS,20mg/kg,i.p.), and PDTC+LPS group (PDTC,120mg/kg,i.p.; LPS,20mg/kg,i.p.). The mice were sacrificed using aortic phlebotomy at2h,6h,12h and24h.Specimen collectionThe blood, lung tissue and BALF samples in each group of mice were collected at the same time after modeling2h,6h,12h and24h. The mice were anesthetized by intraperitoneal injection of10%chloral hydrate (3.5ml/kg). The first part of sampling: mouse orbital blood was collected and collected serum protein was stored at-80℃for cytokines and total protein. Subsequently apical blood was collected for blood gas analysis. Lungs were collected, and take the right lung to prepare for HE staining and immunohistochemistry; and take the left lung calculate lung wet/dry weight ratio (W/D). The second part of sampling:the trachea exposed in another mice, and the BALF was collected. After centrifuged, the supernatant of BALF was collected and the expression of IL-1β, IL-8, TNF-a, IL-10and protein were measured and count the number of PMN was measured. The third part of sampling:Take the mouse lung tis-sue was stored at-80℃liquid nitrogen to prepare for Western blot,Quantitative Real-time RT-PCR and the MPO activity.Detections1. The lung injury and scores were evaluated by histopathological analysis; the lung permeability index (LPI=BALF protein/serum protein),W/D and P(A-a)O2were measured.2. After centrifugation, the cells in BALF sediment were smeared and count the num-ber of PMN by Wright-Giemsa staining. 3. The expression levels of TNF-a, IL-1β, IL-8and IL-10was measured in serum and BALF supernatant by ELISA. The MPO activity was detected by colorimetric.4. Total protein of lung tissue was extracted and the phosphorylated NF-κB (p-NF-KB) was determined by Western blot in order to indicate that NF-κB was activated. The protein in nuclear and cytoplasm were extracted and the production of protein NF-κBp65in lung tissue was measured by Western blot.5. The expression of NF-κB、CDllb/CD18、ICAM-1and ENA-78in lung tissue was measured by immunohistochemistry.6. The expression of CINC mRNA in lung tissue was measured at each time by quan-titative-polymerase chain reaction (Quantitative Real-time, RT-PCR).7. Statistical analysisThe results were expressed as means±standard deviations. Statistical analysis was performed with analysis of variance and t test was used for comparison among groups. P value less than0.05was considered statistically significant. The statistical analysis was conducted by SPSS19.0software.Results1. General informationThe mice in control group breath smooth and the lung appearance was pink. The mice in LPS group were shortness of breath, oral cyanosis, listlessness, and hemorr-hagic secretions could be seen in the nose, less activities and eating. And the lung vo-lume increased when dissected, meanwhile, the reddish liquid was leaked on the sheet section. In the PDTC intervention group, the shortness of breath was reduced, and the lung volume has no obvious increase when dissected.2. Pathology results of lung tissue and scores2.1HE stainingThe structure of lung tissue was integrated and there were no inflammatory cells in control group. But in the LPS groups, we could observe the widened lung interval, highly congested pulmonary interstitial, fracture alveolar wall and a large number of infiltrative inflammatory cells, and inflammatory cell infiltration increased and lung tissue damage aggravated as the time went on. There were similar symptoms with ARDS groups in PDTC intervention group, but the lung tissue injury was milder than that in ARDS group.2.2The pathology scoresThe pathology score was obviously higher LPS group than that in control group (P<0.01), while the score was decreased after PDTC intervention than that in the LPS group (P<0.05), and the score was similar to lung tissue damage.3. The changes of pulmonary vascular permeability3.1lung wet/dry weight ratio (W/D)The lung W/D values were significantly increased in mice of intraperitoneal in-jection LPS compared with the control group at2h,12h(P<0.01), and W/D values gradually increased as time extended. Compared with LPS group, W/D values was significantly decreased in mice of intraperitoneal injection of PDTC (P<0.05) but higher than that in control group.3.2lung permeability index, LPILPI was significantly higher in LPS groups than that in the control group (P<0.01), and PDTC could inhibit LPI increased (P<0.05).4. The total cells and PMN ratio in BALFThe total number of cells were obviously increased in LPS group compared with the control group(P<0.01); after the PDTC intervention the cells were decreased at2h and6h compared with LPS group (P<0.05) and significantly decreased at12h and24h(P<0.01).The cells were mainly PMN in BALF of LPS group and PDTC group, and the number was obviously higher than the control group (P<0.01), but that was lower in PDTC group than LPS group (P<0.05).5. Expression of TNF-a, IL-1β, IL-8and IL-10in serum and BALFThe protective effect of PDTC on the overproduction of proinflammatory cyto-kines induced by LPS was observed in our study. The expression of TNF-a, IL-1β, and IL-8in serum and BALF in LPS group were markedly higher than that in control group (P<0.01). But in the group treated with PDTC(120mg/kg) before induced by LPS, the expression of TNF-α, IL-1β and IL-8were decreased compared with LPS group at2h,6h and12h (P<0.05) and dramatically decreased compared with LPS group at24h (P<0.01). The expression of IL-10in serum and BALF of LPS group was higher than that in control group in the initial6h (P<0.05). But as the time went on, the levels of IL-10was markedly increased (P<0.01). In the group treated with PDTC, the level of IL-10was slightly lower than that in the LPS group at2h, there was no significant difference (P>0.05), but it was lower than the LPS group at6h,12h and24h (P<0.05).6. MPO activity in lung tissueMPO activity is an important index to evaluate the accumulation of neutrophil in lung tissues. The activity of MPO in LPS group was markedly increased compared with control group (P<0.01). However, this change was blocked significantly in the group treated with PDTC before challenged by LPS (P<0.05).7. Changes of p-NF-KB and NF-κB P65protein expression in cytoplasm and nucleus of lung tissueThe p-NF-KB was increased in LPS lung tissue compared with the control group (P<0.01), and after the intervention of PDTC, the p-NF-KB was decreased compared with LPS group (P<0.05).NF-κB P65protein expression was significantly lower cy-toplasm of LPS group than control group, but P65protein expression was significant-ly higher in pulmonary nucleus than control group (P<0.01). However, P65protein in the cytoplasm of lung tissue expression intensity was increased in PDTC+LPS group compared with LPS group (P<0.05), while P65protein expression was decreased in nucleus of PDTC+LPS group compared with LPS group at2h,6h and12h(P<0.05) and significantly decreased at24h(P<0.01).8. The expression of CINCmRNAThe CINCmRNA expression was obviously increased in LPS group compared with control group (P<0.01), while the CINCmRNA expression was significantly re-duced when intervened by PDTC at each phase points compared with LPS group (P<0.01).9. The expression of NF-κB、CD11b/CD18and ICAM-1in the lung tissueNF-κB was mainly expressed in PMN, and the positive cells were stained brown. NF-κB positive cells were increased in LPS group and the positive cells were de- creased in PDTC intervention group compared with the control group. CD11b/CD18was mainly expressed in PMN of lung tissue and these cells were stained brown. CD11b/CD18positive cells were increased in LPS group and the cells were decreased after treated with PDTC compared with the control group. ICAM1was mainly ex-pressed in vascular endothelial cells. ICAM1positive cells were increased in LPS group and the cells were reduced after intervened by PDTC compared with the control group. The results of immunohistochemistry showed that ENA-78was highly ex-pressed in endothelial cells in LPS-induced ARDS mice than that in the control group. The expression of ENA-78in PDTC+LPS group was markedly lower than that in LPS group.Conclusions1. The NF-κB inhibitory protein IκB phosphorylation degradation and NF-κBp65ac-tivated in lung tissue from cytoplasm to nucleus which initiate the inflammation in ARDS. The activation of NF-κB induced by LPS could regulate the expression and release of cytokines such as TNF-a,IL-1β, IL-8and IL-10.2. The pro-inflammatory-anti-inflammatory response was imbalanced in the early stage of ARDS, and the expression of pro-inflammatory cytokines was increased. Therefore, the production of anti-inflammatory cytokines was relative lack, and the high expression time was delayed.3. The interaction between CD11b/CD18on PMN surface and its ligand ICAM-1is involved in ARDS induced by LPS. Meanwhile, CINC and ENA-78were highly ex-pressed in the lung tissue which prompting a large number of PMN aggregation in the lungs.4. The expression of cytokines, adhesion molecules and chemokines and PMN aggre-gation were reduced by the inhibition of PDTC on NF-κB, in order to improve the pro-inflammatory-anti-inflammatory imbalance, reduce the release of MPO from PMN and the lung injury.Innovation and Significance1. In this study, we clarify the relationship among the p-NF-κB, p65protein in cytop-lasmic and nuclear and inflammatory cytokines in lung tissue in order to investigate the role of PMN infiltration in ARDS.2. We study the mechanism between high expression of adhesion molecule and che-mokine in lung tissue of ARDS in order to study the mechanism of PMN aggregation in ARDS.3. We investigate the protective mechanism about PDTC, a specific inhibitor of NF-κB, in ARDS mice induced by LPS. That would provide broad prospects and the-rapeutic approaches for the study of the pathogenesis and development of ARDS. |
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