Microbiological Aerosol Monitoring And Study On The Lung Damage Of Mice And Toxicological Mechanism Of BEAS-2B Cells From The Duck House

The microorganisms in the atmosphere combine with the surrounding dry solid particles and liquid droplets to form a stable colloid system,which can diffuse to the surrounding with air flow and form microbial aerosols.Bacteria and fungi are the main components of microbial aerosols.The pathogenic or conditionally pathogenic microorganisms in them pose a certain threat to the health of animals and human beings.PM2.5 is a fine particle with aerodynamic diameter less than or equal to 2.5?M.PM2.5 has small particle size and large surface area,which slows down its settling speed and increases its transmission distance.It can enter into the deep bronchioles of respiratory tract,and even enter the whole body through air-capillary ventilation,which is harmful to the health of the body.The basis of PM2.5 toxicological effect is to induce oxidative stress and inflammatory response.Epidemiological studies show that high concentration of PM2.5 exposure can induce oxidative stress,inflammatory reaction and apoptosis.It cause multiple organs and organs damage,such as respiratory system,immune system and cardiovascular system,which increase incidence rate and mortality rate,and even lower human lifespan.At present,the research of PM2.5 at home and abroad focuses on the analysis and research of its physical and chemical properties as well as its pathogenic mechanism,but there are few researches on its microbial components and their functions,and even fewer researches on PM2.5 in animal houses.Autophagy is an important physiological process of material circulation in cells,which plays an important role in maintaining the homeostasis of intracellular environment.Autophagy is related to the occurrence of many diseases.Many studies have proved that oxidative stress is a common way to induce oxidative damage in cells.ROS is an important signal molecule in the process of cell survival and death,and ROS can activate autophagy through different signal molecular pathways.Based on this,we collected PM2.5 from the duck house environment and detected the microbial components?bacteria and fungi?;In order to reveal the harm of PM2.5 in duck house to animals and human body,we conducted short-term and long-term experiments in mice exposed to PM2.5 in duck house to clarify its effect on pathological damage,oxidative stress,apoptosis and expression of related immune factors in mammalian lung,to make a comprehensive assessment of its damage to lung,and to clarify the role of biological components.This study includes the following five parts:1.Diversity analysis of bacteria and fungi in duck house environment PM2.5In this study,the concentration of PM2.5 in the duck house is 112?g/m³-208?g/m³,which greatly exceeds the PM2.5 standard value of 75?g/m³ in China,which also means that the potential health threat of the particles in the duck house to animals and people is greater.Through 16S high-throughput sequencing technology to analyze the bacterial samples,160 OTUs common to the five duck houses PM2.5,the unique OTUs of A,B,C,D and E are 26,6,4,13 and 17,respectively,with high cross,indicating that the composition of bacterial samples among the duck houses is similar.The top five species in the Phylum classification level of duck house are Actinobacteria,Firmicutes,Proteobacteria,Bacteroidetes,and Patescibacteria.Actinobacteria is dominant species.The top 10 species in genus level are corynebacterium-1,Corynebacterium,Staphylococus,Romboutsia,Turicibacter,Vagococcus,Macrococcus,Jeotgalicoccus,Rothia and Aerococcus.Corynebacterium-1 has the highest relative abundance and is an dominant species.Corynebacterium,Staphylococus and Aerococus with high abundance are pathogenic or conditional pathogens,which have potential harm to human health.Based on ITS high-throughput sequencing technology,96 OTUs common to the five duck houses PM2.5,and the unique OTUs of A,B,C,D and E were 8,4,1,79 and 14,respectively.The OTUs cross of the five samples was very high,which indicated that the composition of fungus samples was similar.Ascomycota and Basidiomycota were detected at the fungi phylum level.The top 10 species in genus level are:Aspergillus,Cladosporium,Trametes,Alternaria,unclassified-p-Ascomycota,Diutina,Schizophyllum,Bjerkandera,Coprinellus,Mycosphaerella.Among them,the highest relative abundance is Aspergillus,which belongs to the dominant species and has potential harm to human health.Among them,Aspergillus and Cladosporium are pathogenic or conditional pathogens,which have potential harm to human health.2.Study on the expression of immune related factors and lung injury in BALB/c mice exposed to PM2.5(PM_2.5^-,PM_2.5⁺)in duck houseIn order to evaluate the effects of short and long-term exposure to PM2.5 on lung pathological damage and immune response,we conducted a mouse PM exposure experiment.The results of lung histopathology showed that PM2.5 exposure could cause different degrees of pathological damage to the lung and pulmonary fibrosis,the pathological changes of chronic group were more obvious.The double immunofluorescence of E-cadherin and vimentin,which are closely related to the epithelial mesenchymal transition?EMT?,showed that with the increase of PM2.5 exposure time,the expression of E.cadherin decreased and the expression of vimentin increased,it is further proved that the level of pulmonary fibrosis is increased.The results showed that TLR2,TLR4 and TLR9 were significantly up-regulated?P<0.05?compared with the control group,and the repeated PM_2.5⁺exposure group were significantly up-regulated?P<0.01?.There was no significant change in TLR3 and TLR7 expression.This shows that TLR2,TLR4 and TLR9 receptors play an important role in the recognition of PM2.5,and the role of biological components in them cannot be ignored.The detection of cytokines showed that the expression of TNF-?,IL-6 and VEGFA were significantly up-regulated compared with the control group?P<0.05?,indicating that they were the main cytokines in the process of PM exposure.In conclusion,this part of the experiment preliminarily confirmed the role of atmospheric particulate matter and its microbial components in mammalian pathological damage and related immune response,and provided theoretical basis for further study of its pathogenesis.3.Study on oxidative stress and apoptosis induced by single and repeated exposure of PM2.5(PM_2.5^-,PM_2.5⁺)to BALB/c miceIn this part,we evaluated the effects of oxidative stress and apoptosis in lung tissue of mice after PM exposure.Firstly,the total antioxidant capacity?T-AOC?,superoxide dismutase?SOD?,glutathione peroxidase?GSH-Px?and malondialdehyde?MDA?were measured.Compared with the control group,with the increase of PM2.5 exposure time,the expression of T-AOC,SOD,GSH-Px decreased,the expression of MDA increased,and within the same exposure time,the effect of PM_2.5⁺exposure group on each index of oxidative stress was biger than that of PM^-_2.5exposure group.This shows that PM exposure can reduce the antioxidant capacity of lung,increase lipid peroxidation,and cause oxidative stress injury.The study on lung apoptosis after PM2.5 exposure showed that PM2.5 exposure could significantly increase the apoptosis rate of mice,and repeated PM_2.5⁺exposure group had the highest apoptosis rate.In summary,this part of the study shows that PM exposure can induce a certain degree of oxidative stress and apoptosis in the body,which is harmful to the health of the body.4.Study on the survival rate of BEAS-2B cells exposed to PM2.5In this part,the cytotoxic effects of PM2.5 on BEAS-2B cells were studied.The results showed that the survival rate of BEAS-2B cells was significantly reduced in a time and dose-dependent manner by PM exposure,and PM2.5 exposure had a certain toxicological effect on BEAS-2B cells.The previous part of the study shows that PM exposure can induce oxidative stress in the body,so we detect ROS produced in the process of PM2.5 exposure.With the increase of exposure time,the amount of ROS produced increases.Next,we used ROS scavenger NAC to pretreat cells and then exposed to PM,and found that NAC pretreatment could alleviate the effect of PM on cell survival.The results of this part of the study are similar to those of animal experiments.PM exposure can also cause oxidative stress and affect the activity of BEAS-2B cells.5.Study on autophagy and related signaling pathway of BEAS-2B cells exposed to PM2.5In this study,autophagy induced by PM2.5 and its signaling pathway were studied.After PM2.5 was exposed to BEAS-2B cells,it was confirmed that PM2.5 could induce autophagy of BEAS-2B cells and the autophagy flow was smooth by electron microscopy and StubRFP-SensGFP-LC3 lentivirus infection experiment.In order to detect whether SRC/STAT3 signaling pathway is involved in the process of PM2.5 induced autophagy in BEAS-2B cells.WB experiments showed that PM exposure could increase the expression of SRC and STAT3 in a concentration dependent manner.The phosphorylation of STAT3 was inhibited by PM exposure after SRC interference.PM2.5 exposure after cells were pretreated with autophagy inhibitor 3-MA,the phosphorylation of SRC and STAT3 was inhibited significantly.These results indicate that PM2.5 can induce the autophagy of BEAS-2B cells through SRC/STAT3pathway.In order to verify the role of ROS in PM exposure,ROS scavenger NAC pretreated cells were exposed to PM2.5,which indicated that NAC could affect the expression of SRC/STAT3 by PM2.5.The above experiments show that PM2.5 exposure can induce autophagy in bronchial epithelial cells through ROS-SRC-STAT3 pathway.In conclusion,this study laid a foundation for further exploring the mechanism of lung pathological damage induced by PM2.5 exposure in duck house,and provided a new idea for the prevention and treatment of respiratory diseases caused by PM2.5.