Effect Of Guanylate Binding Protein 2 On Rat Alveolar Macrophage Injury Induced By PM_2.5 In Cowshed

Fine particulate matter(PM_2.5)is one of the important components of environmental pollutants.More and more evidence shows that PM_2.5has a potential threat to health.The threat of PM_2.5to respiratory system health has attracted more and more attention,and is an important incentive for many respiratory diseases such as allergic airway diseases,acute airway inflammation,asthma and chronic obstructive pulmonary disease（COPD）.The PM_2.5concentration in the breeding house is higher,and it has complex composition structure,especially high-level microbial components.The research on PM_2.5in breeding environment is still relatively small.The study of PM_2.5in the breeding environment is helpful to analyze the pathogenic mechanism of PM_2.5and the transmission mechanism of some diseases,and improve the prevention and control measures of livestock and poultry breeding.In addition,it is of great significance to ensure the health of long-term staff in breeding environment.Therefore,this study collected PM_2.5samples from cowshed in winter,measured their concentration changes and endotoxin levels,and observed their appearance characteristics.Rat alveolar macrophages were stimulated with different concentrations of PM_2.5,and the effects of PM_2.5on cell damage,oxidative stress and inflammatory factor secretion were evaluated.Transcriptome sequencing technology was used to explore the possible trigger mechanism of PM_2.5on cell damage.Finally,si RNA silencing,RT-q PCR and Western blot were used to explore the effects of PM_2.5stimulation on the expression levels of GBP2 and NLRP3/caspase-1 pathways in cells and the possible reasons.To explore whether GBP2 is associated with the activation of NLRP3/caspase-1 pathway and their effects on cellular immune function.Through the above studies,the specific mechanism of cell damage caused by PM_2.5at the molecular level was explored,so as to provide theoretical reference for the prevention and treatment of respiratory diseases caused by PM_2.5in cowshed.The main research results are as follows:1.Collection and characteristics analysis of PM_2.5in cowshedThe concentration range of PM_2.5in cowshed was 174～291μg/m³,and the concentration was relatively high at 9:00 and 17:00.There were a large number of irregular particle-like substances,crystal particles and filamentous fiber substances in PM_2.5in cowshed,indicating that the concentration of PM_2.5in cowshed was high and the composition was relatively complex.The LPS content in the air of cowshed was 972.453～1512.893 EU/m³.Suggesting that the LPS level in cowshed PM2.5 was high and had the characteristics of high microbial load.2.Toxic effect of PM_2.5on primary rat alveolar macrophagesThe cell viability of primary rat alveolar macrophages was significantly decreased after being stimulated by PM_2.5.The levels of ACP,AKP and LDH in the cell culture supernatant were significantly increased;cells produced significant oxidative stress,including significantly increased intracellular ROS levels,significantly decreased SOD and GSH-PX activities,and significantly increased MDA production and NO release.In addition,it promotes the secretion of inflammatory factors.According to the results of transcriptome sequencing analysis,it was shown that PM_2.5in cowshed had significant damage to cells,and had potential threats to animal health in terms of microorganism infection,inflammation and metabolic abnormalities.3.Upregulation of GBP2 in NR8383 cells induced by cowshed PM_2.5further affects the activation of NLRP3/caspase-1 pathway.Under the induction of cowshed PM_2.5,the expression levels of GBP2,Caspase-11 and NLRP3/caspase-1 pathways were up-regulated.After heat treatment and inactivation of barn PM_2.5,their expression levels,cell viability damage levels and inflammatory factor release levels were reduced,indicating that the microbial components in cowshed PM_2.5were an important reason for up-regulating their expression and cell inflammatory response.After si RNA interfered with the expression of GBP2,the expression of Caspase-11 and NLRP3/caspase-1pathway was inhibited to a certain extent.The secretion and cytotoxicity of LDH,IL-1βand IL-18 were relatively weakened,and the number of intracellular bacteria was significantly increased.The above results showed that after GBP2 was silenced,the toxicity of PM_2.5on cells was weakened,and the autonomous immunity of cells was affected.According to the results of the study concluded:Cowshed PM_2.5concentration is relatively high,the composition is more complex,with a high level of LPS,has obvious toxic effect on cells.When alveolar macrophages devour cowshed PM_2.5,it increases the possibility of microbes entering the cytoplasm.GBP2 identify intracellular bacteria or vesicles containing bacteria,and targeted the structure of the binding membrane to make it lysed.This process increased the content of LPS.Further,LPS binds to the intracellular LPS receptor Caspase-11 and activates NLRP3/caspase-1 pathway in the form of atypical inflammasome to promote the maturation and secretion of downstream IL-1β,IL-18 and LDH,and then play the role of inhibition of intracellular bacteria proliferation.