| High-density and intensive pig production leads to serious air pollution inside the pig houses.A large amount of particulate matter(PM)generating from the pig house can cause respiratory diseases frequently,which seriously damages the pig health and production,and affects the economic benefits of the pig farm.Because the fine particulate matter(PM2.5)has very small particle size and large specific surface area,it is more likely to carry harmful substances and can directly enter the alveoli.The component of PM2.5 inside the pig house is complex and toxic,which can easily induce respiratory inflammation for pigs.As an important vector for disease transmission,the harmful effect of PM2.5 can not be ignored.Alveolar macrophages,as the first line defensing against the foreign substances in the airway and lung,play important roles in maintaining pulmonary healthy and homeostasis.At present,the distribution and characteristics of PM2.5 inside the pig house,and the mechanism of inducing the pulmonary inflammatory response are still not clear.Hence,firstly,the environmental factors inside the nursery pig house were monitored in spring,summer,autumn and winter,respectively.The temporal and spatial distribution of PM concentrations were detected.In addition,the correlation between PM concentrations and other environmental indicators was analyzed.Secondly,the microbial assemblages contained in PM2.5 were identified by microbial second generation sequencing technology.Thirdly,we collected the PM2.5 samples in the nursery pig house in winter.The morphology and chemical composition of PM2.5 were detected.Then combining the in vitro and in vivo experiments to investigate the mechanism of PM2.5 inducing pulmonary inflammatory response.In vitro,the porcine alveolar macrophage 3D4/21 was chosen as cell model.In vivo,the mouse was chosen as animal model.This research is helpful for further studying the source and microbial assemblages of PM2.5 from the nursery pig house,and providing the theoretical basis for preventing the pulmonary inflammation induced by PM2.5.The main research findings are as follows:1.The distribution of airborne particulate matter from a nursery pig house and the correlation with environmental factorsA nursery pig house from an intensive pig farm in Jiangsu province was chosen to study the distribution of PM and its influencing factors.The environment inside the nursery pig house was monitored during spring,summer,autumn and winter,respectively,seven days for each season.There are total nine monitoring time(7:00,9:00,11:00,13:00,15:00,17:00,19:00,23:00 and 3:00)and five monitoring locations(the forepart,middle,south,north and rear part),and the height was 0.5 m above the floor(the breathing height of nursery pigs).The environmental indicators included temperature,relative humidity,wind speed,illumination,different sized PM(TSP,PM10,PM2.5,PM1),NH3 and CO2.Also,the correlation analysis was performed between the PM concentrations and other environmental factors.The results showed that the temperature was significantly different among seasons(p<0.05),and it was the highest in winter and the lowest in summer.The relative humidity in winter was significantly lower than that in other seasons(p<0.05).In terms of gas,the NH3 concentration in spring was the highest and the CO2 concentration was the lowest in summer.Overall,the PM concentrations were the highest in winter,followed by spring and autumn,then the summer was the lowest.The results of temporal distribution for PM concentrations indicated that the PM concentrations have the same trend in spring,autumn and winter.During the daytime(7:00-19:00),the PM concentrations peaked at 7:00,11:00 and 17:00,while during the night time(19:00-3:00),the PM concentrations increased gradually.However the variation trend of PM concentrations in summer was different from those in other seasons,they peaked at 13:00,19:00 and 3:00.The results of spatial distribution for PM concentrations indicated that except the PM10 concentration in summer,and TSP and PM10 concentrations in winter have significant difference,there is no difference among others.According to the correlation analysis between PM concentrations and other environmental factors among different seasons,the concentrations of TSP and PM10 in spring and summer was negatively correlated with wind speed and positively correlated with illumination(p<0.05);the PM2.5 concentration in autumn was positively correlated with NH3(p<0.05);and the PM2.5 concentration in winter was positively correlated with temperature and NH3,and negatively correlated with wind speed(p<0.05).2.Microbial assemblage contained in airborne PM2.5 from a nursery pig houseTo study the microbial assemblages contained in PM2.5,the PM2.5 samples were collected in the middle of the nursery pig house across four seasons.The 16 S rDNA and ITS 1 gene sequencing was employed to analyze the bacterial and fungal assemblage contained in PM2.5,respectively.And the correlation between microbial assemblages and environmental indicators was analyzed.The results showed that,for bacterial assemblage,the samples of summer and autumn clustered together,while they were distinct from those in spring and winter.In contrast,for fungal assemblage,the samples clustered by season.The alpha diversity was higher in summer and autumn compared to the spring and winter.Bacterial OTU number and Chao 1 index in summer and autumn were significantly higher than those in spring and winter(p<0.05).Fungal OUT number and Chao 1 index in autumn were significantly higher than those in spring and winter(p<0.05).At the phylum level,the dominant bacteria and fungi were Firmicutes and Basidiomycota,respectively,across four seasons.At the genus level,a total of four potential pathogen bacterial and 20 potential allergen fungal genera were identified across the samples.The relative abundance of the total pathogen bacterial genera was the highest in summer,followed by winter and autumn,and the spring was the least;the relative abundant of the total allergen fungal genera was the highest in autumn(p<0.05),up to 49.4%,followed by summer and spring,and the winter is the least.In addition,the correlation results showed that the relative abundance of the total potential pathogen bacterial and allergen fungal genera did not have a significant correlation with PM2.5 concentration(p>0.05).Moreover,the OTU number of bacteria and fungi and the relative abundance of total allergen fungal genera were positively correlated with temperature and relative humidity(p>0.05).The above results showed that the alpha diversity was higher than that in summer and autumn compared to the spring and winter.The bacterial assemblage in autumn was similar to that in summer and the fungal assemblage separated from each season.Some potential pathogenic bacterial and allergenic fungal genera were found in PM2.5 samples,indicating that the PM2.5 from a nursery pig house had potential risk.3.The effect of airborne PM2.5 from a nursery pig house on inflammatory response in porcine alveolar macrophagesThis chapter was aimed to investigate the effect of PM2.5 on alveolar macrophages.The airborne PM2.5 samples were collected in winter.The morphology and chemical elements were measured by SEM-EDX.The endotoxin concentration of PM2.5 was measured by a specific chromogenic end-point tachypleus amebocyte lysate kit.The porcine alveolar macrophage 3D4/21 was chosen as cell model.Different concentrations of PM2.5(0,3.125,6.25,12.5,25.0,50.0,100 and 200 ?g/mL)were used to treated 3D4/21 cells for 12 h.The effect of PM2.5 on cell viability was detected by MTT and LDH release trial,and according to the result,appropriate concentrations were chosen for further studying.Then,the cell apoptosis and Ca2+level were measured by flow cytometry.The intracellular ROS level was detected by immunofluorescence method.The mRNA expression levels of IL-1?,IL-18,TNF-?and COX-2 were measured by qRT-PCR.The results showed that the PM2.5 samples contained various morphologies and abundant chemical elements.The endotoxin concentration was 681.80±19.47 EU/mg in PM2.5 on average,equally to 91.86±2.62 EU/m3 in the air.The cell experiment results showed that 25-200 ?g/mL PM2.5 significantly decreased cell viability in a dose dependent manner(p<0.01),then the concentrations of 12.5,25.0 and 50.0 ?g/mL were chosen for further experiment.Compared to the control group,25 and 50 ?g/mL PM2.5 significantly increased the ratio of apoptotic cells(p<0.01),promoted the release of intracellular ROS and Ca2+(p<0.05),and increased the mRNA expression levels of IL-1?,IL-18,TNF-? and COX-2 in a dose dependent manner.The above results showed that the PM2.5 samples from a nursery pig house contained various various morphologies,abundant chemical elements and high concentration of endotoxin,and it can induce inflammatory response in alveolar macrophages.4.The mechanism of airborne PM2.5 from a nursery pig house inducing inflammatory response in porcine alveolar macrophagesThe results of the previous chapter have confirmed that the PM2.5 can induce inflammatory response in porcine alveolar macrophage 3D4/21,but the specific mechanism needs to be further studied.This chapter focused on the inflammatory response induced by PM2.5 based on TLR4/MAPK/NF-?B pathway and NLRP3 inflammasome.12.5,25.0 and 50.0 ?g/mL PM2.5 were used to treated 3D4/21 cells for 12 h.The mRNA expression levels of IL-1?,IL-18,TNF-? and COX-2 were detected by qRT-PCR.The protein expression levels of MAPK(ERK1/2,p38,JNK),NF-?B p65 and NLRP3 inflammasome(NLRP3,ASC,caspase-1)were detected by Western blot.The results showed that 25 and 50 ?g/mL PM2.5 significantly increased the mRNA expression levels of TLR4 and MyD88,the protein expression levels of p-p38,p-JNK,p-p65,NLRP3,ASC and caspase-1(p<0.05),while the protein expression level of p-ERK1/2 was increased at 50.0 ?g/mL PM2.5(p<0.05).To confirm whether the MAPK and NF-?B p65 signaling pathways were involved in NLRP3 inflammasome activation,some inhibitors were used to suppress the protein expression levels of p-p38,p-ERK1/2,p-JNK and p-p65,respectively.We found that the protein expression levels of NLRP3 and caspase-1 were significantly decreased(p<0.05).Next,to investigate the role of intracellular ROS and endotoxin present in PM2.5,N-acetylcysteine(NAC)and polymyxin B(PMB)were used to inhibit the ROS production and neutralize the endotoxin activity,respectively.The results showed that compared to PM2.5 treatment,pretreated with NAC and PMB,to different degree,could increase cell viability,reduce the LDH release,decrease the mRNA expression levels of proinflammatory cytokines,and the combined effect was stronger the separate one.In terms of protein expression level,except no significant effect of NAC alone on p-p38 and p-JNK,and PMB alone on p-p38(p>0.05),pretreated with NAC alone,PMB alone and combined NAC and PMB significantly increased other protein expression levels(p<0.05).Furthermore,the combined effect of the both on these proteins was superior to the alone one,and the effect of PMB was slightly stronger than that of NAC.The above results suggested that PM2.5 from pig house could induce inflammatory response in porcine alveolar macrophage 3D4/21 via TLR4/MAPK/NF-?B pathway and NLRP3 inflammasome activation,and the TLR4/MAPK/NF-?B pathway participated in NLRP3 inflammasome activation.In addition,endotoxin and ROS both participated in PM2.5 from a nursery pig house inducing inflammatory response in porcine alveolar macrophage 3D4/21,and the effect of PMB was stronger than NAC,which indicated that endotoxin played a vital role in PM2.5 from a nursery pig house inducing inflammatory response.5.The effects of airborne PM2.5 from a nursery pig house on mouse pulmonary inflammatory responseTo study the effect of PM2.5 on pulmonary inflammatory response in vivo,32 male mice aged six weeks old were chosen.The mice were divided into two groups,the control group and the PM2.5 treatment.The mice were exposed to PM2.5 by the method of intratracheal instillation,twice a week,and the dose was 1 mg for each time.The control group was instilled with the same volume of saline.The experiment period was four weeks.The mice was weighted once a week,as well as the feed intake.When the experiment finished,the mice were sacrificed 24 h after the last instillation.Each organ was isolated and weighted,and the blood,lung and the bronchoalveolar lavage fluid(BALF)were sampled.The results showed that PM2.5 treatment had no significant effect on the average daily feed intake and body weight of the mice(p>0.05),while increased the weight and indexes of lung and spleen(p<0.05).The HE staining results showed that PM2.5 treatment damaged the alveolar structure of mice,causing the alveolar space to thicken and irregular.PM2.5 treatment significantly increased the number of white blood cells,neutrophils,monocytes and platelets in the blood of mice,as well as the number of total cells,macrophages and neutrophils in BALF(p<0.05).Meanwhile,T-SOD activity and MDA content in serum,as well as T-SOD,GSH-PX activity,and MDA content in lung were significantly increased(p<0.05).LDH and MPO content in BALF were significantly increased(p<0.05).qRT-PCR results showed that the mRNA expression levels of IL-1?,IL-6,IL-17,TNF-? and MMP9 in lung were significantly up-regulated(p<0.05).Western blot results showed that PM2.5 treatment could significantly increase the protein expression levels of TLR4,MyD88,p-p65,p-ERK1/2,p-p38,NLRP3 and caspase-1 in lung(p<0.05),but had no significant effect on p-JNK expression level(p>0.05).The above results indicated that PM2.5 from a nursery pig house could induce oxidative stress and inflammatory response in the lung of mice,then further caused oxidative damage and inflammation for the body,and during the process,TLR4/NF-?B/MAPK signaling pathway and NLRP3 inflammasome played important roles.In summary,this study indicates that the airborne PM concentration and the microorganisms contained in PM2.5 inside the nursery pig house show obvious seasonal variation,which can be attributed to the environmental factors inside.PM2.5 can induce pulmonary inflammation through TLR4/MAPK/NF-?B signaling pathway and NLRP3 inflammasome,and during this process oxidative stress and endotoxin contained in PM2.5 play vital roles. |
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