Mechanism Of LPS On The Inflammatory Injury Of Ruminant Respiratory System And The Regulation Of Sodium Butyrate

Bovine respiratory disease syndrome(BRDC)is soften occurs in stressed herds such as weaning,long-distance transportation,and hunger,and occurs extensively in China and even in countries around the world.Stress and multiple pathogens of mixed infections may invade the natural defenses of the bovine respiratory tract.The immune response of host will further damage the body’s defense system.when the immune level of host is reduced to a certain extent,The normal flora of the cow’s nasopharyngeal such as Pasteurella and tracheal parasites or the flora present in the environment can descend into the bronchi and lungs,and colonize the bronchi and lungs,and then proliferate in the bronchus and lungs,causing mucosal damage of upper respiratory tract,thereby causing more serious bacterial infection,eventually leading to the occurrence of respiratory inflammation.The disintegration of Gram-negative bacteria in respiratory infections produces large amounts of LPS,which is the major virulence factors that cause these inflammatory responses.Excessive LPS activates the TLR4/NF-κB inflammatory signaling pathway,which initiates inflammation in the lungs.The occurrence of respiratory inflammation seriously affects the quality of beef cattle and lactating cows,and brings huge economic losses to cattle breeding in China and the world.Sodium butyrate,as a green,safe and efficient feed additive,has significant anti-inflammatory activity.It inhibits the release of pro-inflammatory cytokines and promotes the release of anti-inflammatory by down-regulating the activity of NF-κB,and ultimately,the inflammatory response is alleviated.Our current experiment consists of three parts.The first part is to analyze the collected samples of nasal mucus by high-throughput sequencing.It is found that Gram-negative bacteria are the main pathogens of respiratory inflammation in dairy cows;The second part is aimed to establish acute lung injury by bronchial instillation of LPS and reveals the regulation of sodium butyrate on LPS-induced inflammation;In the third part,bovine embryonic tracheal cells were used as a model to verify the regulating effects of sodium butyrate on LPS-stimulated cells in vitro.1.Detection of microbial community structure and diversity in dairy cows by 16S rRNA high-throughput sequencingTaking the microbes in the collected nasal mucus as the research subject,to analyse the bacterial community structure in the nasal mucus of the herd showing the symptoms of respiratory diseases.Twenty nasal mucus samples from the pasture were treated to determine the 16S rRNA V3-V4 hypervariable region sequence in the bacteria by the Illumina MiSeq high-throughput sequencing platform using 16S rRNA high-throughput sequencing technology.Which is aim to analyze the bacterial community composition in different nasal mucus samples.The results showed that the number of OTU in the nasal mucus samples at the similar level of 97%was 9684.The analysis of the bacterial phylum level showed that the Proteobacteria was the dominant flora,followed by the Firmicutes,Analysis of the bacterial genus level showd that Acinetobacter,Pseudomonas,and Moraxella,those are under Proteobacteria,were the dominant bacteria.Through the analysis of microbial diversity in the sample and the statistics of pathogens with high abundance of bacteria,it is expected to provide a guidance for disease prevention and control and medication in the pasture.This study analyzed the characteristics of microbial diversity and abundance in nasal mucus samples of sick cows,which provided some guidance for the prevention and control of respiratory diseases in cattle farms.2.Protective effect of sodium butyrate on LPS-induced acute lung injury model in mice by TLR4/NF-κB signaling pathwayBacterial pneumonia in cattle is a common disease that brings huge economic losses to the farm.Sodium butyrate is histone inhibitor,which plays an important role in controlling inflammation.This study was to investigate the protective effect of sodium butyrate on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice and to explore the potential mechanism of sodium butyrate in controlling inflammation.Thirty ICR female mice were randomly divided into control group(PBS group),LPS(7.5 mg/kg)group,LPS with pretreatment of sodium butyrate(25 mg/kg)group(LSB group).LPS-induced ALI mice model were established by tracheal instillation of LPS.The protective effect of sodium butyrate on LPS-induced acute lung injury in mice was studied by intragastric administration of sodium butyrate 1 hour before LPS instillation.After 12 h,mice blood and lung tissue samples were collected for experimental analysis.The results showed that the cytokines concentrations of IL-1β and TNF-α,the activity of myeloperoxidase(MPO)in the lung and blood,protein expression level of TLR4,NF-κB p65,p-NF-κB p65,IκB,and p-IκB in LPS group were significantly improved compared with those in PBS group.Expression level of inflammation-related genes such as TLR4,NF-κB,IL-1 β,IL-6 and TNF-α in lung tissues were also significantly increased;however,Compared with the LPS group,the addition of sodium butyrate significantly reduced the concentration of cytokines,MPO activity,and the expression of in flammation-related genes and proteins.In addition,the addition of sodium butyrate can alleviate the damage of lung tissue caused by LPS.The results of immunohistochemistry showed that the positive immune responses of TLR4,NF-κB and TNF-α were enhanced in the LPS group,but the addition of sodium butyrate could alleviate this phenomenon.The results showed that the model of acute lung injury in mice was successfully established by tracheal instillation of LPS and sodium butyrate was effective in protecting LPS-induced acute lung injury in mice.3.Sodium butyrate pretreatment mitigates lipopolysaccharide-induced inflammation through the TLR4/NF-κB signaling pathway in bovine embryo trachea cellsBovine respiratory disease(BRD)is the most important cause of livestock mortality in feedlots in the United States and even worldwide,leading to great losses to dairy farm.Sodium butyrate(SB)has been shown to inhibit the inflammatory response in vivo and in vitro.The present study was conducted to investigate the anti-inflammatory effects and potential mechanisms of SB in bovine embryo tracheal cells(EBTr)stimulated with lipopolysaccharide(LPS).Gene expression of IL-6 and IL-8,as well as cytokine secretion of IL-6,IL-10,and TNF-α,were assessed to optimize the concentration of LPS and SB.Cells were exposed to 1 mmol/L SB for a period of 18 h as pretreatment(SB)and 0.4μg/mL LPS for 6 h as the treatment group(LPS).EBTr were pretreated with SB supplementation for 18 h followed by 6 h of LPS stimulation(LSB).The results showed that with LPS stimulation,the gene expression of TLR4,NF-κB,pro-inflammatory cytokine IL-6 and chemokine IL-8,as well as IL-6 and TNF-α cytokine production,were significantly increased compared with the control group(CON).In contrast,both the gene and cytokine expression of the anti-inflammatory cytokine IL-10 were decreased.However,these inflammatory effects induced by LPS were reversed by SB supplementation.Compared with CON,the protein expression of TLR4,phospho-p65,phospho-IκBα,and IL1α(the end-product of the inflammatory response)were increased in the LPS group and those were decreased in EBTr with SB supplementation.Similarly,increased nuclear translocation of phospho-p65 in the LPS group was suppressed with SB pretreatment.In conclusion,SB can reduce inflammation induced by LPS in bovine embryo tracheal cells,and this positive effect is mediated through the TLR4 and NF-κB signaling pathway.