Research progress of transcription activating factor 3 in regulating the alveolar macrophages in acute respiratory distress syndrome / 中华危重病急救医学

Acute respiratory distress syndrome (ARDS) refers to acute diffuse lung injury caused by a variety of intrapulmonary and/or extrapulmonary factors such as infection and trauma. Uncontrolled inflammatory response is the main pathological feature. Different functional states of alveolar macrophages have different effects on inflammatory response. Transcription activating factor 3 (ATF3) is a fast response gene in the early stage of stress. In recent years, it has been found that ATF3 plays an important role in regulating the inflammatory response of ARDS by regulating the function of macrophages. This paper reviews the regulatory effects of ATF3 on alveolar macrophage polarization, autophagy and endoplasmic reticulum stress and its effects on the inflammatory process of ARDS, aiming to provide a new research direction for the prevention and treatment of ARDS.

Pulmonary delivery of siRNA against acute lung injury/acute respiratory distress syndrome

The use of small interfering RNAs (siRNAs) has been under investigation for the treatment of several unmet medical needs, including acute lung injury/acute respiratory distress syndrome (ALI/ARDS) wherein siRNA may be implemented to modify the expression of pro-inflammatory cytokines and chemokines at the mRNA level. The properties such as clear anatomy, accessibility, and relatively low enzyme activity make the lung a good target for local siRNA therapy. However, the translation of siRNA is restricted by the inefficient delivery of siRNA therapeutics to the target cells due to the properties of naked siRNA. Thus, this review will focus on the various delivery systems that can be used and the different barriers that need to be surmounted for the development of stable inhalable siRNA formulations for human use before siRNA therapeutics for ALI/ARDS become available in the clinic.

Increased leishmanicidal activity of alveolar macrophages from mature horses with mild equine asthma / Aumento da atividade leishmanicida de macrófagos alveolares de equinos adultos com asma equina leve

Alveolar macrophages (AMs) are an essential part of defense mechanisms within the lungs and their phagocytic activity is important for organ homeostasis. The phagocytic ability of AMs obtained from bronchoalveolar lavage from 17 mature mixed-breed pleasure horses (8 healthy and 9 diagnosed with mild equine asthma) was studied through assays with Leishmania (Viannia) braziliensis promastigotes, which enabled the calculation of a phagocytic index (PI) and a survival index (SI). Results indicate that phagocytic activity of AMs in asthma affected horses is similar to healthy horses, while leishmanicidal activity is significantly increased in horses with asthma.(AU)

Os macrófagos alveolares (MAs) são uma parte essencial dos mecanismos de defesa dentro dos pulmões e sua atividade fagocítica é importante para a homeostase desse órgão. A capacidade fagocitária dos MAs obtidos do lavado broncoalveolar de 17 equinos adultos, sem raça definida (oito saudáveis e nove com diagnóstico de asma equina leve), foi estudada por meio de ensaios com promastigotas de Leishmania (Viannia) braziliensis. Foi calculado o índice fagocítico e o índice de sobrevivência. Os resultados indicam que a atividade fagocítica de MAs em cavalos com asma é semelhante a cavalos saudáveis, enquanto a atividade leishmanicida está significativamente aumentada em cavalos com essa enfermidade.(AU)

Transcriptome sequencing and analysis of bovine alveolar macrophages infected with BCG / 吉林大学学报(医学版)

Objective:To reveal the differentially expressed genes and long non-coding RNAs (lncRNAs) by sequencing the transcriptome of bovine alveolar macrophages (BAM) infected with Bacillus Calmette-Guérin (BCG) and analyzing their bioinformations, and to provide theoretical foundation for the research on immune regulation mechanism of anti-infection of Mycobacteriumtuberculosisof macrophages.Methods:The BAM were collected by pulmonary lavage and centrifugation and cultured and divided into infected group and uninfected group.After infection for 12hin infected group, the expression profiles of mRNA and lncRNA in infected group and uninfected group were detected by RNA-Seq, and bioinformatics analysis was carried out.Results:compared with uninfected group, there were 119differentially expressed lncRNAs and 1111differentially expressed mRNA in infected group (P＜0.05) .Gene Ontology functional enrichment analysis showed that the most significant enrichment was immune response (GO:0006955, P＜0.05) , including 125genes, in which 63 were up-regulated and 62 were downregulated, and the expressions of proinflammatory factors interleukin-1 (IL-6) , interleukin-7 (IL-7) , and interleukin-23A (IL-23A) were up-regulated.Cis target gene prediction and KEGG pathway analysis showed that the differentially expressed lncRNAs were involved in the regulation of transforming growth factor-β (TGF-β) signaling pathway and ATP-binding cassette transporter (ABC transporter) signaling pathway (P＜0.05) .Conclusion:The host cells are stimulated to produce a strong immune response after the BAM are infected by BCG and results in the changes of lncRNA and mRNA expression profiles.

Transcriptome sequencing and analysis of bovine alveolar macrophages infected with bcg / 吉林大学学报(医学版)

Objective: To reveal the differentially expressed genes and long non-coding RNAs (IncRNAs) by sequencing the transcriptome of bovine alveolar macrophages (RAM) infected with Bacillus Calmette-Guerin (RCG) and analyzing their bioinformations, and to provide theoretical foundation for the research on immune regulation mechanism of anti-infection of Mycobacterium tuberculosis of macrophages. Methods: The RAM were collected by pulmonary lavage and centrifligation and cultured and divided into infected group and uninfected group. After infection for 12 h in infected group, the expression profiles of mRNA and IncRNA in infected group and uninfected group were detected by RNA-Seq, and bioinformatics analysis was carried out. Results: Compared with uninfected group, there were 119 differentially expressed IncRNAs and 1111 differentially expressed mRNA in infected group (P

MicroRNA-155 reduces inflammatory response induced by lipopolysaccharide in alveolar macrophages / 中华危重病急救医学

Objective To observe the effect of microRNA-155 (miR-155) on the inflammatory response of rat alveolar macrophages induced by lipopolysaccharide (LPS). Methods The alveolar macrophages NR8383 of rat were cultured in vitro, the macrophages in logarithmic growth phase were harvested to conduct experiment. ① The 1 mg/L LPS was used to stimulate the rat alveolar macrophages for 3, 6, 12, and 24 hours, a phosphate buffer solution (PBS) control group was also set up. Enzyme linked immunosorbent assay (ELISA) was used to detect the dynamic changes of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the supernatant, and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the dynamics expression of miR-155 in the cells, which confirmed the optimal time for LPS stimulation was 12 hours. ② Carboxyfluorescein (FAM) labeled mimic (FAM mimic) and inhibitor (FAM inhibitor) were used to transfect the alveolar macrophage, and the transfection effect was observed under inverted fluorescence microscope 6 hours later to confirm the optimal transfection concentration of mimic was 20 nmol/L, and the optimal transfection concentration of inhibitor was 100 nmol/L. miR-155 mimic and miR-155 inhibitor were transfected to alveolar macrophages respectively at the optimal transfection concentration for 24 hours, and 1 mg/L LPS was used to stimulate the cells for 12 hours. A mimic negative control + LPS group and an inhibitor negative control + LPS group were set up. The expressions of IL-1β and TNF-α in the supernatant were determined by ELISA to observe the regulation of miR-155 on inflammatory response of alveolar macrophages. Results ① After stimulation of 1 mg/L LPS on alveolar macrophages, the contents of IL-1β and TNF-α in the supernatant and the expression of miR-155 in the cells were increased gradually with time prolongation, IL-1β and TNF-α contents peaked at 12 hours, and the expression of miR-155 peaked at 24 hours [as compared with PBS control group, IL-1β (ng/L): 910.43±36.09 vs. 22.66±7.84, TNF-α (ng/L): 3 138.39±394.10 vs. 233.92±8.84, miR-155 (2-ΔΔCt): 7.82±0.30 vs. 1, all P < 0.05]. ② Under inverted fluorescence microscope, after 20 nmol/L FAM mimic or 100 nmol/L FAM inhibitor transfected alveolar macrophages for 6 hours, a large number of cells showed green fluorescence, indicating that the transfection was successful. The expression of miR-155 in the cells transfected with 20 nmol/L miR-155 mimic was up-regulated by (236.73±46.49) times as much as that in the negative control group (P < 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly lower than those in the negative control group [IL-1β (ng/L): 324.37±36.59 vs. 799.31±39.44, TNF-α (ng/L): 1 554.01±342.48 vs. 3 020.49±418.30, both P < 0.05]. The miR-155 activity was significantly inhibited in the cells transfected with 100 nmol/L miR-155 inhibitor, and the expression of miR-155 was decreased by (4.00±3.26)% as compared with the negative control group, but the difference was not statistically significant (P > 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly higher than those in the negative control group [IL-1β (ng/L): 1 358.98±212.04 vs. 878.68±53.42, TNF-α (ng/L): 4 225.57±281.11 vs. 2 881.32±286.08, both P < 0.05]. Conclusion In LPS induced inflammatory response of alveolar macrophages, miR-155 plays an obvious inhibitory role.

Effects of Ghrelin on Inflammatory Signaling Akt,NF-κB and iNOS in Alveolar Macrophages from Septic Rats / 中山大学学报(医学科学版)

[Objective]To investigate the effects of ghrelin on inflammatory signaling protein kinase B(Akt),nuclear factor-κB(NF-κB)and inducible nitric oxide synthase(iNOS)in alveolar macrophage(AM).[Methods]24 Male SD rats were randomly divided into Sham,CLP,CLP+ghrelin,and Sham+ghrelin groups. Cecal ligation and puncture(CLP)was used to induce sepsis. Ghrelin(20 nmol/kg)was administered by intraperitoneal injection at 3 h and 15 h post-operation. Histopathological changes of lungs were observed and scored.AM were extracted from bronchoalveolar lavage fluid(BALF). Interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in BALF were detected by ELISA. IL-1β,TNF-α,and IL-6 mRNA in AM were detected by qPCR.NF-κB p65,IκBα,p-IκBα,Akt,p-Akt and iNOS in AM were detected by immunofluorescence(IF)and Western blotting.[Results]The histologic score(6.7±0.8),BALF IL-1β[(146±12)pg/mL]and IL-6[(182±10)pg/mL]from CLP+ghrelin group were respectively 35.4%,44.5% and 46.42% lower than those from CLP group[(10.3±0.7),(263±17)pg/mL,and(273±5)pg/mL],P<0.05.No significant difference was found in BALF TNF-α between CLP group and CLP+ghrelin group.The IL-1β,TNF-α and IL-6 mRNA in AM from CLP+ghrelin group were respectively 54.38%,53.6% and 46.42% lower than those from CLP group,P<0.05. The nuclear NF-κB p65 and cytoplasmic p-IκBα,p-Akt and iNOS from CLP+ghrelin group were respectively 32.58%,45.42%,27.6% and 48.33% lower than those from CLP group,P<0.05. There was no significant difference in all data between Sham group and Sham+ghrelin group.[Conclusion]Ghrelin can decrease the activity of inflammatory signaling proteins Akt,NF-κB and iNOS in AM,therefore restricts AM expressing pro-inflammatory cytokines IL-1β,TNF-α,and IL-6,thus alleviates sep-sis-induced acute lung injury(ALI).

Effect of caveolin-1 scaffolding domain peptides on heme oxygenase-1 activity increasing and M1/M2 phenotype polarization in rat alveolar macrophages induced by lipopolysaccharide / 中华危重病急救医学

Objective To investigate the effect of caveolin-1 scaffolding domain (CSD) peptides on heme oxygenase-1 (HO-1) activity increasing and M1/M2 phenotype polarization in rat alveolar macrophages (AMs) induced by lipopolysaccharide (LPS).Methods Bioinformatics was used to analyze the binding of full-length wild-type CSD polypeptide and 101 amino acid deleted truncated mutant CSD polypeptide (Δ101CSD) to HO-1. Primary AMs were isolated from rats, when cell fusion reached 80％, they were synchronized with serum-free medium and divided into five groups: no treatment was given to the blank control group; LPS group was treated with 100μg/L LPS for 16 hours;LPS+ hemin group was treated with 100μg/L LPS and 20μmol/L hemin for 16 hours; wild-type CSD polypeptide+ LPS+hemin group was pretreated with 10μmol/L wild-type CSD polypeptide 6 hours before LPS treatment; Δ101CSD+ LPS+hemin group was pretreated with 10μmol/L Δ101CSD polypeptide 6 hours before LPS treatment. After treatment for 16 hours, the co-localization between caveolin-1 (Cav-1) and HO-1 was displayed by confocal microscope; the mRNA expressions of inflammatory cytokines interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) and M1/M2 polarization cytokines tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), leukocyte differentiation antigen 206 (CD206) and IL-10 were determined by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-qPCR); the HO-1 activity and nitric oxide (NO) production were determined by spectrophotometry.Results Bioinformatics analysis showed: both wild-type CSD and Δ101CSD peptides could bind to HO-1, and there was no significant difference in the binding ability between the two peptides, but the deletion of 101 Arg resulted in the disappearance of part of the binding region between Δ101CSD and HO-1. The results of laser confocal microscopy showed: the expressions of Cav-1 and HO-1 were lowed in the blank control group, and Cav-1 was bound to HO-1 in LPS group and LPS+ hemin group. Both wild-type CSD and Δ101CSD peptides pretreatment could significantly reduce the binding of HO-1 to Cav-1 induced by LPS. HO-1 activity analysis showed: after LPS stimulation, the activity of HO-1 was significantly higher than that of the blank control group; the activity of HO-1 induced by LPS was increased by hemin; after pretreatment with two kinds of CSD peptides, the activity of HO-1 was further increased, and the effect of wild-type CSD peptide was more significant, which showed a statistically significant difference as compared with that of LPS+ hemin group (pmol·mg-1·h-1: 3683±266 vs. 2408±132,P < 0.05). RT-qPCR results showed: LPS could induce elevation of cytokines and M1 markers and decrease of M2 markers, while hemin could inhibit LPS-induced inflammatory response and M1/M2 phenotypic polarization. Compared with LPS+ hemin group, after pretreatment with wild-type CSD peptide, the levels of inflammatory factors in AMs were decreased, and the mRNA expression levels of TNF-α and iNOS, M1 markers, were decreased [TNF-α mRNA (2-??Ct): 6.82±0.05 vs. 8.70±0.24, iNOS mRNA (2-??Ct): 331.50±32.05 vs. 506.70±0.10, bothP < 0.05], and IL-10 mRNA expression level was increased (2-??Ct: 269.09±6.54 vs. 119.05±3.30,P < 0.05). The deletion of 101 site partially weakened the inhibitory effect of CSD peptides on inflammatory factors and only reduced the expression of iNOS mRNA (2-??Ct: 429.11±8.92 vs. 506.70±0.10,P < 0.05), indicating that its ability to transform AMs from M1 phenotype to M2 phenotype was poor. The two peptides had no effect on the expression of CD206.Conclusion Wild-type CSD had beneficial effects of anti-inflammation by reducing Cav-1 binding to HO-1 induced by LPS, restoring the HO-1 activity and driving M2 phenotype in alveolar macrophages.

Pyroptosis mediated by mitochondrial DNA amplifies the inflammatory response of alveolar macrophage / 中华危重病急救医学

Objective To observe the amplification effect of mitochondrial DNA (mtNDA) on the inflammatory response of rat alveolar macrophage induced by lipopolysaccharide (LPS), and to preliminarily explore its mechanism. Methods mtDNA of Sprague-Dawley (SD) rat liver tissue was harvested, ultra-micro spectrophotometer and 1% agarose gel electrophoresis were used to detect the concentration and quality of mtDNA. The alveolar macrophages of SD rat were isolated and cultured, the macrophages in logarithmic growth phase were divided into phosphatic buffer solution (PBS) group, LPS group, mtDNA group and LPS+mtDNA group. The first three groups were added equal volumes of PBS, LPS 1 mg/L or mtDNA 10 mg/L to the alveolar macrophages medium for 6 hours and 12 hours, respectively; the alveolar macrophage medium of LPS+mtDNA group was stimulated with 1 mg/L LPS for 6 hours and then stimulated with 10 mg/L mtDNA for 6 hours. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell supernatant were detected by enzyme linked immunosorbent assay (ELISA);the expression of the key protein of Pyroptosis-Gasdermin D (GSDMD) was detected by Western Blot. Results ① The mtDNA A260/280 ratios were between 1.8-2.0, and agarose gel electrophoresis showed a single band, with a size of about 16 kb. ② After alveolar macrophages stimulated by LPS or mtDNA for 6 hours or 12 hours, respectively, the levels of IL-1β and TNF-α were higher than those in PBS group. When cells were treated with mtDNA for 6 hours after LPS stimulation 6 hours, the levels of IL-1β were higher than those in LPS 12 hours group (ng/L: 366.27±23.35 vs. 154.70±23.32, 1 < 0.01), but the levels of TNF-α had no significant difference compared with LPS 12 hours group (ng/L: 836.13±25.01 vs. 802.67±30.48, 1 > 0.05). ③ The protein expressions of GSDMD in LPS group, mtDNA group and LPS+mtDNA group were significantly higher than those in PBS group (GSDMD/β-actin: 1.77±0.05, 1.65±0.04,2.40±0.05, 1.00±0.02, all 1 < 0.01), and the protein expression of GSDMD in LPS+mtDNA group was higher than that in LPS group (1 < 0.01). Conclusion mtDNA amplifies LPS-induced alveolar macrophage inflammatory responses,which mechanism may be related to the increase in pyroptosis mediated by mtDNA.

Assessment of the oxidative metabolism of calves' bronchoalveolar cells: comparison between NBT and fluorimetric techniques / Mensuração do metabolismo oxidativo de células broncoalveolares de bezerros: comparação entre as técnicas de NBT e fluorimétrica

Evaluation of alveolar macrophage functions of cattle is an important tool in order to assess whether measures taken during the cattle husbandry can decrease the respiratory tract defense. The aim of this study was to determine whether dexamethasone used at therapeutic dose can affect the oxidative metabolism of alveolar macrophages of cattle. This was evaluated by two tests, the fluorometric and colorimetric. The similarity of the results was studied, using alveolar macrophages of six healthy cattle, obtained from bronchoalveolar lavage on a basal and an immunosuppressant moment after the application of dexamethasone. For the fluorometric test, alveolar macrophages were incubated with Staphylococcus aureus and 2'-7'dichlorohidroflurescein, and analyzed by flow cytometer. For the colorimetric test, alveolar macrophages were incubated with Phorbol 12- miristate-13 acetate and nitroblue tetrazolium, dissolved and analyzed in a spectrophotometer. It was noted that dexamethasone therapeutic dose (0.05 mg/kg) reduced the functions of alveolar macrophages from healthy bovine. This result was observed by both tests with the difference that the flow cytometry assay was more informative for identifying which specific cellular function has been compromised.(AU)

A avaliação das funções de macrófagos alveolares de bovinos é uma ferramenta importante no intuito de analisar se medidas adotadas durante a criação desses animais diminuem a defesa do trato respiratório, aumentando a susceptibilidade à pneumonia. O objetivo deste trabalho foi verificar se a dexametasona em dose terapêutica afeta o metabolismo oxidativo de macrófagos alveolares de bovinos por meio de duas provas, a fluorimétrica e a colorimétrica, avaliando a similaridade dos resultados. Para tanto, utilizaram-se macrófagos alveolares de seis bovinos sadios, obtidos por meio de lavados broncoalveolares em um momento basal e em um momento imunossupressor, após a aplicação da dexametasona. Na prova fluorimétrica, os macrófagos alveolares foram incubados com Staphylococcus aureus e o fluorófilo 2'-7' diclorohidrofluresceína e analisados por citometria de fluxo. Na prova colorimétrica, os macrófagos alveolares foram incubados com Phorbol 12- miristate-13 acetate e nitroazul de tetrazolium, dissolvidos e analisados em espectofotômetro. Observou-se que a dexametasona em dose terapêutica (0,05 mg/kg) reduziu as funções dos macrófagos alveolares de bovinos sadios, sendo este resultado obtido em ambas as provas, com a diferença de que o teste de citometria de fluxo foi mais esclarecedor por identificar qual função celular específica foi comprometida.(AU)

Free SiO2 promotes the foaming of alveolar macrophages / 医学研究生学报

Objective Lipid metabolism disorders caused by cell foam plays an important role in atherosclerosis,but wheth-er it is involved in the development and progression of silicosis has not yet been elucidated. This study aimed to investigate the effect of free silica(SiO2) in inducing foam cell formation of NR8383 alveolar macrophages in rats. Methods NR8383 cells were cultured in vitro by the routine method (the control group) or in 50 μg/mL SiO2 (the SiO2group), 50 μg/mL ox-LDL (the ox-LDL group), or 50 μg/ml SiO2and ox-LDL (the model group), all for 36 hours. The survival rate of the cells was calculated with the cell proliferation and cytotoxicity assay (MTS),the lipid deposition observed by oil red O staining,the levels of total cholesterol (TC), free cholesterol (FC) and cholesterol esters(CE) measured by ELISA,and the mRNA and protein expressions of PPARγ and CD36 in the cells determined by RT-PCR and Western blot, respectively. Results Compared with the control group,the cells treated with ox-LDL showed a significantly increased survival rate, which reached the peak at 50 μg/mL ([1.501±0.201]%) (P<0.05). Foam cells were observed in the SiO2,ox-LDL and model groups,but most significantly in the model group. In comparison with the ox-LDL group,the model group exhibited remarkable increases in TC([14.195±2.260] vs[35.764±4. 226] μg/mg,P<0.05),FC([7.722±0.690] vs[10.049±0.698] μg/mg,P<0.05),CE([6.473±1.707] vs[25.715±4.243] μg/mg,P<0.05),and CE/TC (45.057% vs 71.642%, P<0.05). Conclusion Free SiO2promotes the lipid metabolism disorder in macrophages and enhances the foaming of the cells,in which PPARγ and CD36 may play an important role of regulation.

Isolation of mouse alveolar macrophages by flow cytometry and identification / 安徽医科大学学报

Objective To establish a method for isolating alveolar macrophage (AM) of mouse based on flow cy tometry.Methods The lungs were digested by collagen ⅣV in vitro to prepare single-cell suspension that was stained by CD11 b and CD1 1 c antibody.CD1 1 b1owCD1 1 c + cell population were AM and isolated by flow cytometry.After that,the cell viability was measured via the Typan blue staining,and the identification of AM was through flow cytometry and real-time PCR.Results CD1 1 b1owCD1 1 c + cell population was isolated by flow cytometry,the purity was (93 ± 2)％ and the cell viability was (80 ±5)％.The real-time PCR results showed that peroxisome proliferator-activated receptor γ (PPARγ) mRNA was highly expressed in AM isolated by flow cytometry (P ＜ 0.001).In addition,the functional assay showed that the isolated AM possess high phagocytic activity.Thus,the results described above demonstrate that the isolated cells were AM.Conclusion A method for obtaining AM based on flow cytometry was established.The method has high cell purity and good cell activity which can be used for functional experiments.

The TNF-α/TNFR/NF-κB pathway in regulating alveolar macrophage apoptosis in coal workers' pneumoconiosis / 中国职业医学

OBJECTIVE: To study the mechanism of tumor necrosis factor( TNF)-α and its receptor( TNFR) signal transduction pathways in regulating cell apoptosis of alveolar macrophage( AM) in coal workers' pneumoconiosis( CWP).METHODS: Twenty-four coal workers with pneumoconiosis at stage Ⅰ were selected as CWP group and four observation subjects exposed to coal were chosen as observation group by using simple random sampling method. The bronchoalveolar lavage fluids of whole-lung lavage of two groups were collected. AMs were separated and purified. Then they were divided into 6 groups: a control group,a superoxide dismutase( SOD) group,a TNF/TNFR group,an anti-TNF-α antibody group,a Caspase-8 suppression group and a nuclear factor-κB( NF-κB) suppression group. The AMs of 6 groups with corresponding treatment were cultivated. After 24 hours,the cells were harvested and proteins extracted. The relative expression of TNF-α,TNFR1,TNFR2,Caspase-8,Caspase-3,NF-κB P50 and NF-κB P65 protein was detected by Western blotting. RESULTS: The protein relative expression of TNF-α,TNFR2,Caspase-8,Caspase-3,NF-κB P50 and NF-κB P65 in CWP group was significantly higher than those in the observation group( P < 0. 05). The protein relative expression of TNF-α,TNFR1,Caspase-8,Caspase-3 and NF-κB P50 in the TNF/TNFR group and the anti-TNF-αantibody group was lower than that of the control group( P < 0. 05). The above indexes in the anti-TNF-α antibody group were lower than that of the NF-κB suppression group( P < 0. 05). The protein relative expression of TNFR1,Caspase-8and Caspase-3 in the TNF/TNFR group was higher than that of the SOD group and the Caspase-8 suppression group( P <0. 05). The protein relative expression of TNFR1,Caspase-8 and NF-κB P50 in the TNF/TNFR group was lower than that of the NF-κB suppression group( P < 0. 05). Among the CWP patients,the relative expression of TNFR2 and NF-κB P65 in the TNF/TNFR group was lower than that of the control group( P < 0. 05),and higher than that of the SOD group( P <0. 05). CONCLUSION: AM apoptosis mediated by TNF-α/TNFR/NF-κB signal transduction pathway plays an important role in the occurrence and development of CWP. The TNF-α/TNFR/NF-κB signal transduction pathways inhibited or blocked at different stages can affect the expression of proteins related to AM apoptosis.

Mechanism of epithelial-mesenchymal transition induced by free silicon dioxide in type Ⅱ alveolar epithelial cells / 中国职业医学

OBJECTIVE: To investigate the epithelial-mesenchymal transition(EMT) induced by direct or indirect exposure to free silicon dioxide(SiO_2) and the expression of surface protein marker in rat typeⅡalveolar epithelial cell RLE-6TN.METHODS: i) The alveolar macrophages(AM) were isolated from specific pathogen-free SD rat by bronchoalveolar lavage.AM and RLE-6TN were treated with 0-140 mg/L(final concentration) of SiO_2 suspension and were cultured conventionally for 24,48 and 72 hours. The cell viability was detected by CCK-8 assay. The result of CCK-8 essay was used to choose the SiO_2 concentration for the following study. ii) To establish models of RLE-6TN co-cultured with AM that were seeded in Transwell. The cells were divided into 4 groups: the direct control group(RLE-6TN,no SiO_2 exposed),the direct exposure group(RLE-6TN,treated with 100 mg/L SiO_2),the indirect control group(RLE-6TN and AM were cocultured,no SiO_2 exposed) and the indirect exposure group(RLE-6TN and AM were co-cultured,AM was treated with 100 mg/L SiO_2 directly). Western blotting was used to detect the expression of E-cadherin(E-cad) and α-smooth muscle protein(α-SMA) after cells were cultured for 0,24,48 and 72 hours. RESULTS: i) According to the CCK-8 assay,the final concentration of 100 mg/L SiO_2 was chosen for the following study. ii) The difference of relative expression of E-cad andα-SMA in RLE-6TN was statistically significant in different treatment combination and time(P < 0. 01). The E-cad expression of RLE-6TN at 48 and 72 hours in the direct exposure group and the indirect exposure group was lower than that in direct control group at the same time point(P < 0. 05). The E-cad expression in RLE-6TN at 72 hours in the direct exposure group was lower than that in the 0 and 24 hours(P < 0. 05). The E-cad expression in RLE-6TN at 48 and 72 hours in the indirect exposure group was lower than that in the 0 hour(P < 0. 05). At 48 and 72 hours,the α-SMA expression in the indirect exposure group and the direct exposure group was higher than that in their control groups at the same time point(P < 0. 05). The expression of α-SMA in the indirect exposure group was higher than that in the direct exposure group(P < 0. 05). The expression of α-SMA in both exposure groups increased in a time-effect relationship(P <0. 05). CONCLUSION: Direct or indirect exposure to free SiO_2 can induce EMT in RLE-6TN,and decrease the expression of E-cad and increase the expression of α-SMA in a time-effect relationship. Indirect exposure group is more susceptible to EMT.

Porcine circovirus type 2 increases interleukin-1beta and interleukin-10 production via the MyD88–NF-kappa B signaling pathway in porcine alveolar macrophages in vitro

Porcine alveolar macrophages (PAMs) represent the first line of defense in the porcine lung after infection with porcine circovirus type 2 (PCV2) via the respiratory tract. However, PCV2 infection impairs the microbicidal capability of PAMs and alters cytokine production and/or secretion. At present, the reason for the imbalance of cytokines has not been fully elucidated, and the regulatory mechanisms involved are unclear. In this study, we investigated the expression levels and regulation of interleukin-1beta (IL-1β) and IL-10 in PAMs following incubation with PCV2 in vitro. Levels of IL-1β and IL-10 increased in PAM supernatants, and the distribution of nuclear factor kappa B (NF-κB) p65 staining in nucleus, expression of MyD88 and p-IκB in cytoplasm, and DNA-binding activity of NF-κB increased after incubation with PCV2, while p65 expression in PAM cytoplasm decreased. However, when PAMs were co-incubated with PCV2 and small interfering RNA targeting MyD88, those effects were reversed. Additionally, mRNA expression levels of Toll-like receptors (TLR)-2, -3, -4, -7, -8, and -9 increased when PAMs were incubated with PCV2. These results show that PCV2 induces increased IL-1β and IL-10 production in PAMs, and these changes in expression are related to the TLR–MyD88–NF-κB signaling pathway.

Relationship between the expression of Cofilin in alveolar and peripheral blood mononuclear cells in COPD patientsand the function of phagocytic aspergillus / 实用医学杂志

Objective To investigate the expression of Cofilin in alveolar and peripheral blood mononuclear cells in COPD patients and the correlation with the function of phagocytic aspergillus. Methods 20 COPD pa-tients were selected from July 2015 to May 2016 in the outpatient department of respiratory medicine of Zhujiang Hospital of Southern Medical University ,then divided into the experimental group 1(group Aand B in 2011 version of GOLD),group 2(test group C and group D in 2011 version of GOLD),and the healthy control group in 10 people. The AM and MDM in the peripheral bloodwere extracted respectively in the 3 groups by bronchoalveolar lavage and,and the ability of AM and MDM in each group were detected. The expression of Cofilin protein was measured by real-time fluorescence quantitative(qRT-PCR) and Western blotting ,and the differenceswere compared among the three groups. Results The colony numbers of MDM/AM in the 3 groups were 17 ± 3,16 ± 2, 42 ± 3(F = 73.446 ,P < 0.001),and the colony numbers of MDM/AM in the two test groupshad significantly different from the healthy group ,while no significant difference was found in the two test groups. The results of fluorescence quantitative reverse transcription polymerase chain reaction and Western blot analysis showed that the expression of Cofilin in the two test groups was significantly higher than that in the healthy group. Conclusion The decreased function of phagocytic aspergillus in the alveolar and peripheral blood mononuclear cells in COPD patients may relates to the increase of cofilin.

Isolation, culture and identification of goat alveolar macrophages / 中国比较医学杂志

Objective In order to study the biological characteristics of macrophages and provide the materials to study the survival mechanism of intracellular parasites, we conducted this study to establish a high-purity alveolar macrophage isolation and culture method.Methods Goat lungs were lavaged with normal saline in sterile environment several times, and cells were collected and then goat alveolar macrophages were purified by density gradient centrifugation using peripheral blood mononuclear cells (PBMC) solution.The isolated goat alveolar macrophages were cultured in cell culture medium containing 10% fetal bovine serum and cell morphology was observed under an inverted microscope every day,and the phagocytic activity of the cells was detected by chicken red blood cell phagocytosis test.Flow cytometry was used to detect CD14, a characteristic monocyte-macrophage surface marker.Results The adherent cells were characterized by typical macrophage morphology, pseudopodia and protrusions, showing round and irregular shape, rich cytoplasm, and large cell body.Of the cultured macrophages, 54.5% could phagocytize chicken erythrocytes and showed good phagocytic activity.After one month of in vitro culture, 93.7% of the cells were able to express CD14 antigen, which had a macrophage-specific immunophenotype.Conclusions The alveolar macrophages obtained in this study have high purity and good bioactivity, thus provide a cell model for studying the immune mechanism of intracellular parasites.

A study on the effect of indomethacin on SiO2 induced lysozyme over-expression in rat alveolar macrophages / 预防医学

Objective To learn the effect of indomethacin (IND)of different concentrations on the expression of lysozyme protein induced by SiO2 in rat alveolar macrophages (AM).Methods Pure AM was prepared with the method of bronchoalveolar lavage in rats.In the model group,the silica dust poisoning model was replicated by adding SiO2 dust suspension (50 μg /ml).In the intervention group,following the adding of SiO2 dust suspension,the IND of 1 0 -5 ,1 0 -4 , and 1 0 -3 g/ml were added respectively.In the control group,the same volume of PBS was given.After 8 h,1 6 h,the cell morphology was observed.Results Compared with the model group,the AM cells in the intervention group were relatively complete,and that there was a concentration dependent trend.The expression of LSZ protein in AMof the model group was significantly higher than that of the control group (P<0. 05 ),while the expression of LSZ protein in the intervention group decreased compared with that of the model group.After incubation with IND and SiO2 ,the expression of LSZ protein in the intervention group decreased compared with that of the model group.Conclusion IND can inhibit the increased expression of LSZ protein in AMcaused by the stimulation of SiO2dust,and can reduce the damage of SiO2on the membrane of alveolar macrophage and thus has the protective effect on the AM cell membrane.

Effects of PI3K / Akt signaling pathway on the autophagy of rat alveolar macrophages exposed to dust / 中国职业医学

OBJECTIVE: To determine the regulating role of phosphatidylinositol 3-kinase( PI3K) / protein kinase B( Akt)signaling pathway in the autophagy activity of rat NR8383 cells exposed to silicon dioxide( SiO_2). LY294002 was used to block PI3 K pathway. METHODS: i) The normal NR8383 cells were used and divided into blank group and silica exposure group( final concentrations of SiO_2 suspension were 0 and 50 mg / L respectively). They were cultured for 3,6,12,20 and24 hours. The enzyme linked immunosorbent assay( ELISA) was used to assess the amount of tumor necrosis factor-α( TNF-α) and transforming growth factor-β1( TGF-β1) in supernatants of cultured cells,and then the optimal time of cells exposed to dust was determined. ii) NR8383 cells were divided into control group( treated with a same volume of F-12 K medium without serum),silica group( treated with SiO_2 suspension,final concentration 50 mg / L) and intervention group( treated with SiO_2 suspension and PI3 K inhibitor LY294002,final concentration 50 mg / L and 20 μmol / L,respectively).Cells were harvested following incubation. ELISA was used to detect the levels of TNF-α and TGF-β1 at the time point of20 hours after incubation. To reveal the autophagy status of cells,Western blotting was used to detect Akt and microtubuleassociated proteins 1 light chain 3( LC3) protein at time point of 20 hours; laser scanning confocal microscope( LSCM)was used to observe the immunofluorescence expression of autophagy at time points of 3,6,12 and 20 hours. The cells were also treated with the lysosomal inhibitor chloroquine diphosphate( CDP) at the same time of SiO_2 treatment. RESULTS: i) The time point of 20 hours was confirmed to be the best dust exposure time for in vitro cell model of NR8383 cells.ii) The levels of TNF-α and TGF-β1 of supernatant in the silica group were higher than those of the control group( P <0. 05). The levels of TNF-α and TGF-β1 of supernatant in the intervention group were higher than those of the control group and silica group( P < 0. 05). The Akt protein expression of the intervention group was lower than those in the control group and the silica group,respectively. The LC3 Ⅱ / Ⅰ protein level of the silica group was higher than those of the control group and intervention group( P < 0. 05),but no statistical significance was found between the control group and intervention group( P > 0. 05). LSCM results indicated that autophagy expression at time points of 3 and 6 hours were stronger than those of 12 and 20 hours in control group; autophagy expression at time point of 12 hours was stronger than those of 3 and 6 hours in the silica group,while the autophagy expression at time point of 20 hours was slightly weaker than that of 12 hours,but still stronger than those of 3 and 6 hours. Compared with the same time point in control group,autophagy expression at 3 and 6 hours were weaker in the silica group,while the expressions increased obviously at time points of 12 and 20 hours. Autophagy expression at all time points decreased in the intervention group compared with silica group,especially at the time point of 20 hours. The autophagy expression in each group increased in varying degrees after added with CDP blocking. CONCLUSION: Silica dust exposure can induce autophagy in rat NR8383 cells. PI3 K inhibitor LY294002 can reduce the autophagy expression indicating that the PI3 K / Akt signaling pathway might participate in the autophagy process of silica dust inducing autophagy in alveolar macrophages.

Role of AMP-activated protein kinase in regulating LPS-induced secretion of inflammatory cytokines in mouse alveolar macrophages / 中华微生物学和免疫学杂志

Objective To investigate the mechanism of 5′adenosine monophosphate-activated pro-tein kinase ( AMPK) in the regulation of LPS-induced secretion of inflammatory cytokines in mouse alveolar macrophages.Methods Alveolar macrophages were isolated from wild type and AMPKα1-/-C57BL/6J mice.ELISA was used to measure the concentrations of IL-1βand TNF-αin the culture supernatants of LPS treated alveolar macrophages with or without 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide ( AICAR ) stimulation.Western blot assay was performed to analyze the expression of AMPKα1 and AMPKα2 in alveolar macrophages isolated from wild type and AMPKα1-/-mice as well as the LPS-induced changes of p-AMPKαactivity in wild type mice.Results The LPS-induced secretion of inflammatory cyto-kines in alveolar macrophages isolated from AMPKα1-/-mice were significantly higher than that of wild type mice.The activities of p-AMPKαin wild type mice were suppressed by LPS treatment.Treatment of AICAR inhibited the LPS-induced secretion of TNF-αand IL-1β.Conclusion The LPS-induced secretion of in-flammatory cytokines was enhanced in AMPKα1-deficient mice, but could be suppressed upon the treatment of AICAR, an agonist of AMPK.AMPK was involved in the regulation of LPS-induced secretion of inflamma-tory cytokines by alveolar macrophages in mice.