| Research background and purpose:A clean and good poultry house environment is an important prerequisite and guarantee for the healthy growth of poultry.With the continuous improvement of intensive and standardized level,the modern poultry house mostly adopts the cascade cage mode of automatic feeding and defecation,automatic control of temperature,humidity and wind.However,due to the high feeding density,dense cages,uneven indoor temperature,humidity and ventilation,the aerosol concentration of various dust,harmful gases,microorganisms and metabolites increases with the increase of age,which poses a great threat to the health of poultry and breeders.The damage of host immune function caused by excessive concentration of PM2.5 may cause bacterial secondary infection,make the body produce stronger inflammatory reaction and aggravate lung injury.The purpose of this study was to investigate the differences of microbial composition and structure of PM2.5 in poultry houses of different breeding periods,and to further study the synergistic effect of PM2.5 and Pseudomonas aeruginosa on inflammatory damage of respiratory system and its related molecular mechanism.Methods:PM2.5 samples were collected from different growth stages(3 days,22 days and 40 days)of broilers.The morphological differences of PM2.5 were observed by SEM,and the diversity and relative abundance of bacteria in the samples were characterized by16S rDNA sequence analysis.Secondly,the synergistic effects of inactivated PM2.5,inactivated PM2.5(PM2.5-)and Pseudomonas aeruginosa on inflammation were studied in vitro and in vivo.(1)The effect of PM2.5 on the proliferation of RAW264.7 cells stimulated by different concentrations of PM2.5 and PM2.5~—(the final concentration is 1.00,0.50 and 0.25mg/ml respectively)for 24 hours.The influence of samples on the proliferation of RAW264.7cells was analyzed by detecting the change of absorbance of each group.(2)The effect of PM2.5 on the expression of NO in RAW264.7 cells stimulated by different concentrations of PM2.5 and PM2.5~—(the final concentration is 1.00,0.50 and0.25mg/ml respectively)for 6 hours,and the production of no in each group was detected by Griess method.(3)The effects of PM2.5 and Pseudomonas aeruginosa on the expression of inflammatory factors(IL-6,IL-8 and TNF-α)in RAW264.7 cells:The experiment was divided into six groups:normal control group(PBS),PM2.5 group(PM2.5),PM2.5inactivated group(PM2.5~—),PAO1 group(PA),PM2.5 and PA group(PM2.5+PA),PM2.5 inactivated and PA group(PM2.5~—+PA).The final concentrations of PM2.5 and PM2.5~—group samples were 1mg/ml,and the infection ratio(MOI)of PA to cells was10:1.After six hours of stimulation,the expression of IL-6,IL-8 and TNF-αwere detected by ELISA.(4)The effects of PM2.5 and Pseudomonas aeruginosa on the expression of NF-κB signaling pathway related proteins in RAW264.7 cells were grouped as follows:(3).After30 minutes of stimulation,the expression of NF-κB p65 protein and phosphorylated NF- κB p65 protein were detected by Western blot.(5)The effect of PM2.5 and Pseudomonas aeruginosa on inflammatory response in mice 60 male SPF 6-week-old C57BL/6 mice were randomly divided into 6 groups with10 mice in each group.The final concentration of PM2.5 and PM2.5 group is 1mg/ml,and the concentration of PA is 1×10~8cfu/ml.The mice were treated twice a day with 20μl each time.The time interval between the two drops was 4 hours.The mice were weighed and their activity state was observed before,7 days and 14 days.The mice were killed after 14 days.He staining and Masson staining were used to observe the pathological changes of lung tissue;total RNA of lung tissue was extracted and real-time fluorescence quantitative PCR was used to detect the expression of IL-6,IL-8 and TNF- α;ELISA was used to detect the expression of IL-6,IL-8 and TNF-αin serum of mice;Western blot was used to detect the expression of NF-κB p65 protein and phospo NF-κB p65 protein in lung tissue of mice in each group.The results are as follows:(1)Scanning electron microscopy showed that the morphology of PM2.5 aerosol particles is complex and diverse,mainly in the form of filaments,spheroids,clusters and irregularities,and the particle morphology is complex.(2)The trend of PM2.5 concentration was positively correlated with the age of broilers.The results of 16S rDNA sequencing showed that a variety of potential pathogenic bacteria were detected in PM2.5,including Acinetobacter(21.21%),Brevundimonas(6.88%),Cryptococcus(6.56%),Pseudomonas(2.94%)and Faecalibacterium(2.91%).(3)Inhibition of cell proliferation and NO expression:compared with the control group(0.1718±0.02342),PM2.5 group(0.9337±0.03651)significantly inhibited the proliferation of RAW264.7 cells(P<0.001),and its inhibition effect was significantly higher than pm2.5-group(0.7788±0.02933,P<0.05);when the sample concentration was 1mg/ml,PM2.5 could increase the NO expression level in RAW264.7 cells(PM2.5group:55.21±1.678;control group:1.97±1.034,P<0.001),and the NO expression level in PM2.5 group was significantly higher than that in pm2.5-group(21.97±0.8702,P<0.001).(4)The expression of cytokines IL-6,IL-8,TNF-α,NF-κB p65 and phospho-nf-κB p65 in RAW264.7 cells:The expression of pro-inflammatory factors IL-6,IL-8 and TNF-αin PM2.5+PA group(8.022±0.6328,308.7±20.27,1287±56.46)was significantly higher than that in the control group(32.14±3.269,P<0.01;62.43±8.848,P<0.01;795.7±28.64,P<0.01);the level of phospo NF-κB p65/NF-κB p65 in PM2.5+PA group(0.03769±0.007571)was significantly higher than that in the control group(0.0918±0.002178,P<0.001).(5)Pathological changes of mice:the control group mice were active,smooth and glossy coat,good appetite,quick action,even breath,and sensitive to external stimulation;the PM2.5+PA group mice were in the worst state,slow action,dry coat,dark yellow,rapid respiratory frequency,and slow response to external stimulation(drive,light,etc.).There was no significant change in the body weight of mice in the control group(22.68±0.1916,22.11±0.3318,22.3±0.2741),but the body weight of mice in the PM2.5+PA group decreased continuously(22.63±0.1027,21.4±0.1692,19.03±0.7065,P<0.01).Pathological sections of lung tissue showed that:The pathological sections of mouse lung tissue showed that in the control group,the alveolar wall was thin,the bronchial structure was intact,there was no cell detachment in the airway epithelium,and there was occasional infiltration of alveolar macrophages and a few neutrophils.These results indicated that the degree of lung injury in the PM2.5+PA group was the most severe.In these group,the lung tissues exhibited a phenomenon that was similar to that of pulmonary edema.The alveolar walls already exhibited abnormal morphology,and the degree of thickening of the alveolar walls was the most severe,which was accompanied by the hallmark infiltration of pulmonary macrophages and neutrophils into the alveoli.Moreover,the cilia growth on the inner wall of the bronchi was abnormal,showing airway epithelial cell shedding.(6)The expression of IL-6,IL-8,TNF-αmRNA and protein in mice serum:The expression levels of IL-6,IL-8,TNF-αmRNA(9.988±1.877,2.376±0.2811,14.78±5.371)and protein(780.4±82.12,185.3±27.01,968.8±135)in PM2.5+PA group were significantly higher than those in the control group(1.863±0.4443,P<0.01;0.6437±0.1565,P<0.001;0.5504±0.1052,P<0.01;66.29±7.006,P<0.01;45.33±3.243,P<0.001;31.74±4.212,P<0.001).Conclusion:(1)PM2.5 in the poultry house environment is rich in bacteria species and diversity,which has potential hazards.(2)PM2.5 in poultry house can cause respiratory tract inflammation,and the effect of bioactive components in PM2.5 is more significant.(3)Pseudomonas aeruginosa can cooperate with PM2.5 to aggravate the inflammatory reaction and cause more serious respiratory system damage,which is closely related to the activation of NF-κB pathway. |
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