Objective To investigate the effects of IL-10 on lipopolysaccharide( LPS) -induced MyD88 /NF-κB signaling activation. Methods Ana-1 macrophages were divided into a LPS group and a LPS + IL-10 group. The cells and the culture supernatant were collected at 0, 0. 5, 1, and 2 hours respectively. The expression levels of NF-κB p65 and MyD88 in cytoplasm and nucleus were detected by Western blotting. The concentration of TNF-αin the culture supernatant was determined by ELISA. Results Through 0 to 2 hours, MyD88 expression increased significantly after LPS stimulation. The expression was attenuated by the pretreatment of IL-10, which returned to normal levels at 2 hours( 8. 8 ±0. 3 vs 21. 4 ±1. 8,P lt;0. 05) . IL-10 had no effect on total expression of NF-κB, but decreased nuclei / cytoplasm ratio of NF-κB p65 after LPS stimulation. The ratio was lower in the LPS + IL-10 group compared and the LPS group at 1 hour and 2 hour ( 1. 1 ±0. 1 vs 2. 4 ±0. 4, 0. 6 ±0. 7 vs 3. 1 ±0. 6, P lt; 0. 05) . Consequently, IL-10 pretreatment decreased TNF-α concentration after LPS stimulation at 1 hour and 2 hours [ ( 222. 5 ±33. 5) pg/mL vs ( 365. 2 ±22. 7) pg/mL, ( 212. 7 ±15. 9) pg/mL vs ( 566. 2 ±31. 5) pg/mL, P lt;0. 05] .Conclusion IL-10 attenuates inflammation via MyD88 /NF-κB signal pathway depression.
Objective To evaluate the effects of N-acetylcysteine ( NAC) on bleomycin-induced lung fibrosis in mice and to investigate the therapeutic mechanisms of NAC on lung fibrosis. Methods Forty-five KM female mice were randomly divided into 3 groups. The mice in the control group were administered with saline aerosol intratracheally. The mice in the fibrosis group were administered with bleomycin ( 3 mg/kg) dissolved in normal saline aerosol intratracheally. The mice in the NAC group were gastric perfused with NAC at a dose of 400 mg · kg- 1 · d - 1 after administering bleomycin aerosol intratracheally. All animals were sacrificed 28 days after the treatments. The left lung was fixed in 10% neutral formalin, then stained with hematoxylin eosin and Masson’s trichrome respectively for the pathological examination. The right lung was sampled and the content of hydroxyproline ( HYP) was assayed by alkaline hydrolysis method. The serum was collected and the concentrations of malondialdehyde ( MDA) and totalantioxidant capacity ( T-AOC) were measured by colorimetric method. The RNA and total tissue protein were extracted for the examination of NOX1 /2/4 by RT-PCR and Western blot respectively. Results NAC prevented lung fibrosis induced by bleomycin with significantly reducing lung collagen accumulation and the level of HYP in the NAC group ( P lt;0. 05) . The serum concentration of MDA were reduced and serum TAOC raised by treating NAC after intratracheal administration of bleomycin ( P lt;0. 05) . NOX1 /2/4 gene and protein expression were increased in the fibrosis group compared with the control group. NAC had no effect on the gene expression of NOX1/2 /4( P gt;0. 05) , but inhibitted the NOX4 protein expression in lung tissue significantly ( P lt; 0. 05) . Conclusion NAC inhibits the expression of NOX4 and prevents bleomycin-induced lung fibrosis in mice.
Objective To investigate the effects of glutathione S-transferase M5 (GSTM5) on the inflammation in human bronchial epithelial 16HBE cells and its possible molecular mechanisms. Methods Acute lung injury cell model was constructed with 16HBE cells induced by tumour necrosis factorα (TNF-α, 10 ng/mL). The cells were devided into a control group, a TNF-α group (TNF-α), a GSTM5 group (GSTM5+TNF-α), a negative control group (negative control plasmid+TNF-α). GSTM5-GFP plasmid and negative control plasmid were respectively transfected to the cells of the GSTM5 group and the negative control group using Lipofectamine2000. The contents of interleukin-6(IL-6), IL-8, IL-10 in the cell supernatant were measured by ELISA.The expression of nuclear factor-κB (NF-κB) mRNA was detected by RT-PCR, and the expression of NF-κB, phospho-NF-κB, p38, phospho-p38 protein were detected by Western blot. Results The GSTM5-GFP eukaryotic expression vector was successfully constructed and transfected successfully confirmed by fluorescence microscope. The contents of IL-6, IL-8, IL-10 in the TNF-α-induced cell supernatant were significantly higher than those in the control group(P < 0.05), and the contents of IL-6, IL-8, IL-10 in the GSTM5 group were lower than those in the TNF-α group (P < 0.05)with statistically significant difference. At the same time, the total NF-κB mRNA, phospho -NF-κB and phospho-p38 protein were increased in TNF-α stimulated cells compared with the control group (P < 0.05), while the GSTM5 group was lower than that in the TNF-α group and the negative control group (P < 0.05). Conclusion Overexpression of GSTM5 inhibits the phosphorylation of p38MAPK and NF-κB and down-regulates the inflammation of TNF-α-induced human bronchial epithelial 16HBE cells.
Objective To study the differences in gene expression in A549 cells transfected with Forkhead box protein O1(FOXO1),and provide clues to further exploring the mechanism of FOXO1 in acute lung injury. Methods After using TNF-α to stimulate A549 cells,the eukaryotic expression vector GV230-FOXO1 was transfected into A549 cells by using lipofectamine transfection reagent.The RNA was isolated and differentially expressed genes were screened with high-throughout DNA microarray. Results The eukaryotic expression vector GV230-FOXO1 was successfully constructed and verified.High quality mRNA was isolated and prepared for microarray screening,which passed RNA quality control.The DNA microarray data indicated that 317 genes were up-regulated and 237 genes were down-regulated in A549 cells transfected with FOXO1.The function of these differentially expressed genes involved in many aspects,such as proliferation,apoptosis and differentiation. Conclusions Differentially expressed genes in A549 cells transfected with FOXO1 can be successfully screened by using DNA microarray.FOXO1 may influence the progression of the disease by changing the level of cell proliferation,apoptosis and differentiation in acute lung injury.
Objective To evaluate the effects of two different epidermal growth factor receptor tyrosine kinase inhibitors ( EGFR-TKIs) , Gefitinib and Erlotinib, on lung fibrosis induced by bleomycin.Methods Forty BALB/c female mice were randomly divided into four groups, ie. a control group( saline given orally and intratracheally) , a fibrosis group( saline given orally with bleomycin instillation) , a Gefitnib group( Gefitnib 20 mg/kg given orally with bleomycin instillation) , and an Erlotinib group ( Erlotinib25 mg/kg given orally with bleomycin instillation) . Bleomycin ( 3 mg/kg) was intratracheally instilled on the first day. Gefitinib or Erlotinib was given orally daily and normal saline as control. Then they were sacrificed by abdominal aortic bleeding 14 days after the bleomycin instillation. The left lung was stained with HE and Masson’s trichrome staining respectively for pathological examination. Total EGFR and phosphorylated EGFR were detected by immunohistochemistry. Hydroxyproline ( HYP) assay was performed in the right lung.Results Both Gefitinib and Erlotinib significantly reduced lung collagen accumulation and the content of HYP. Immunohistochemistry revealed that phosphorylation of EGFR in lung mesenchymal cells induced by bleomycin was inhibited. Furthermore, there was no difference between Gefitinib and Erlotinib in inhibiting lung fibrosis. Conclusion Our findings suggest that, in the preclinical setting, EGFR-TKIs may have aprotective effect on lung fibrosis induced by bleomycin.
Objective To establish a cell culture model in vitro of acute lung injury and investigate the effects of NF-κB p65 on the inflammation and oxidative stress in TNF-α-activated type Ⅱ alveolar epithelial cells. Methods A549 cells were treated with TNF-α ( 10 ng/mL, 24 h) in the absence or presence of NF-κB p65 siRNA ( 50 nmol /L) . RT-PCR and Western blot were performed to analyze the silence efficiency of RNAi targeting NF-κB p65. The contents of IL-1β, IL-4, and IL-6 in the culture supernatant were measured by ELISA. The concentration of MDA and SOD were detected by colorimetric method. The survival rate of cell was assessed by the methyl thiazolyl tetrazolium ( MTT) assay. Results P65 RNAi significantly decreased the transcription and translation of NF-κB p65 induced by TNF-α( P lt; 0. 05) . The levels of IL-1β, IL-4, and IL-6 were significantly lower in the supernatants of A549 cells pretransfected with NF-κB p65 siRNA ( P lt;0. 05) , while the concentration of MDA markedly decreased ( P lt; 0. 05) , and the activation of SOD increased dramatically ( P lt; 0. 05) . Consequently, the survival rate of A549 in the p65 siRNA group improved( P lt; 0. 05) . Conclusions NF-κB p65 plays a key role in the oxidative stress induced by TNF-α. NF-κB p65 silencing can down-regulate the inflammation and oxidative stress induced by TNF-αand enhance the proliferation of alveolar epithelial cells.
Objective To compare three approaches of lipopolysaccharides ( LPS) administration for inducing acute lung injury ( ALI) in mice. Methods LPS ( 5 mg/kg) was intratracheally aerosol administered ( ITA group) , intratracheally instilled ( ITI group) , or intraperitoneally injected ( IPI group) to induce ALI in BLAB/ c mice. Evans Blue instead of LPS was intratracheally administered to observe the liquid distribution in the lungs. Two hours after LPS administration, the mice were sacrificed and the lungs were removed to determine wet-to-dry lung weight ratio ( W/D) , and the histological changes were evaluated by HE staining. Phosphorylation level of IκB-αand NF-κB p65 in lung tissue were investigated by Western blot. Transcription intensity of TNF-α and IL-1β mRNA in lung tissue were detected by real-time quantitative PCR. Results Evans Blue distributed more uniformly in the ITA group than the ITI group. The lung W/D ratio and histological changes score in three LPS administration groups were all significantly higher than the normal control group ( P lt;0. 01) , with the ITA group being the highest. The phosphorylation levels of IκB-αand NF-κB p65 were significantly higher in the ITA group than the ITI group ( P lt;0. 05) , and were significantly higher in the ITI group than the IPI group ( P lt; 0. 05) . Transcription intensity of TNF-αand IL-1βmRNA was significantly higher in the ITA group than the ITI group ( P lt;0. 05) , and were significantly higher in the ITI group than the IPI group ( P lt;0. 05) . Conclusion Being non-invasive and convenient,intratracheal LPS aerosol inhalation is an optimal method to induce ALI in mice because it induces more extensive and uniformly distributed injuries in lung.
Objective To investigate the role of alveolar macrophages ( AMs ) in airway inflammation of smoke-induced COPD rat model and its possible regulating mechanism. Methods Twelve Wistar rats were randomly divided into a COPD group and a control group. The rat model of COPD was established with smoke exposure and LPS intrathacheal instillation. Bronchoalveolar lavage fluid ( BALF)was collected for measurement of total and differential cell counts. Then AMs were isolated and identified byimmunofluorescence. Western blot was employed to analyze the cytoplasmic and nuclear NF-κB p65 expression of AMs. The concentrations of TNF-α,macrophage inflammatory protein 2 ( MIP-2) and IL-10 in cell culture supernatantwere assayed by ELISA.Results The scores of bronchitis and mean liner intercepts in the COPD group were significantly higher than those in the control group [ 4. 33 ±1. 16 vs. 1. 33 ±0. 58,P =0. 016; ( 168. 77 ±11. 35) μm vs. ( 93. 61 ±4. 16) μm, P = 0. 000) ] . The total cell count in BALF of the COPD group was significantly higher than that in the control group ( P lt; 0. 05) , and the AMs and neutrophils were predominant [ ( 72. 00 ±2. 22) % and ( 18. 29 ±8. 34) % ] . The cytoplasmic NF-κB p65 expression of AMs in the COPD group was significantly lower , while the nuclear NF-κB p65 expression was significantly higher ( P lt; 0. 05) compared with the control group. The ELISA results showed that the concentrations of TNF-αand MIP-2 in culture supernatant of AMs in the COPD group were significantly higher than those in the control group ( P lt;0. 05) , while the concentration of IL-10 was not significantly different between the two groups ( P gt;0. 05) . Conclusions COPD rat model was established successfully with smoke exposure and LPS intratracheal instillation with a profile of macrophage-based chronic inflammation and increased secretion of TNF-αand MIP-2. The mechanismis closely related to activation of NF-κB.
ObjectiveTo investigate the mechanism of lung tissue apoptosis in LPS-induced mice ARDS via TNF-α neutralization. MethodsThirty-six mice were randomly divided into a control group,a LPS group,and TNF-α neutralization group.LPS(5 mg/kg) was intratracheally nebulized to induce ARDS in the LPS group and the TNF-α neutralization group.Twenty-four hours before LPS treatment,etanercept (0.4 mg/kg) was abdominal injected to the mice in the TNF-α neutralization group.Mice were sacrificed 2 hours after LPS treatment.PCR were used to detected the expression of NF-κB p65,Bax and Bcl-2 in lung tissue.Western blot were used to detected protein level of NF-κB p65,Erk1/2 and their phosphorylation and Bax,Bcl-2.The lung dry-to-wet ratio was measured.The lung histological changes were evaluated by HE staining. ResultsActivation level of NF-κB p65 and Erk1/2 was elevated,the ratio of Bcl-2 and Bax was decreased in the LPS group(P<0.05).After TNF-α neutralization,the activation level of NF-κB p65 and Erk1/2 were reduced,the ratio of Bcl-2 and Bax was increased (P<0.05).Compared with the LPS group,the lung dry-to-wet ratio and lung injury semi-quantitative score were significantly decreased in the TNF-α neutralization group (P<0.05). ConclusionTNF-α neutralization can suppress lung injury in LPS-induced ARDS mice by inhibiting activation of NF-κB p65 and Erk1/2,increasing the ratio of Bcl-2 and Bax ratio,and eventually reducing apoptosis.