| Objectives In this study,we used a combination of 16 Sr DNA gene sequencing technology and UHPLC-Q-TOF-MS metabolomics to detect and analyze the changes of respiratory tract flora and metabolomics of lung lavage fluid in a rat model of pneumoconiosis,to explore their roles in the development of pneumoconiosis,and to provide new ideas for the study of pneumoconiosis.Methods The experimental subjects were SPF grade healthy SD rats,all males,randomly divided into three groups,namely,the Si O2 group(using silica dust),the model group(using coal mine dust),and the normal saline group(using sterilized normal saline).The screened coal mine dust and pure Si O2 dust were prepared into dust suspension with a final concentration of 50 mg/m L.After autoclaving at 121℃,rats were given one-time dust exposure(the dosage was 1 m L of dust suspension or 1 m L of normal saline).The method of dust exposure was non-tracheal exposure,and the day of dust exposure was recorded as the first day.Lung tissues,pharyngeal swabs,and lung lavage fluid samples were collected24 w after dusting.Lung tissues were used to make lung pathology sections and stained using HE to observe the occurrence of disease in the rats.Respiratory tract flora variation between groups of rats was analyzed using 16 Sr DNA gene sequencing,and differential metabolites of lung lavage fluid were analyzed using UHPLC-Q-TOF-MS untargeted metabolomics,and Spearman’s statistical method was used to correlate differential flora and metabolites.Results 1 After 24 weeks of dust exposure,the lung tissue structure of rats in the Si O2 group was severely damaged,and there were obvious silica nodules;rats in the model group showed a large number of cellular nodules;while the alveolar structure of rats in the normal saline group was normal.2 Upper respiratory tract flora(pharyngeal swabs)detection: 1)Alpha diversity analysis: The rat pharyngeal swab microbial communities Chao1,Ace,Shannon,and Simpson indices were not significantly different between the three groups.2)Beta diversity analysis: Both the Si O2 group and the model group had differential microbial communities in the upper respiratory tract compared to the normal saline group.3)Differential flora: The abundance of Pasteurella＿sp.＿V6,Bacteria,Rodentibacter and uncultured＿bacterium＿Rodentibacter in the upper respiratory tract flora of rats in the model group was lower than that in the normal saline group,and the abundance of Muribacter,Muribacter＿muris,uncultured＿bacterium＿Stenotrophomonas were significantly higher than the normal saline group;The abundance of Pasteurella＿sp.＿V6 and Streptococcus＿agalactiae in the upper respiratory tract flora of rats in the Si O2 group was significantly lower than that in the normal saline group.3 Lower respiratory tract flora(lung lavage fluid)detection: 1)Alpha diversity analysis: Compared with the control group,the microbial Chao1 index in the lung lavage fluid of rats in the Si O2 group was significantly increased(P < 0.05);There was no significant difference in the Ace,Shannon and Simpson indices of the rat lung lavage fluid microbial community between the three groups.2)Beta diversity analysis: There were differences in the lower respiratory tract microbial communities between the model group and the normal saline group and between the Si O2 group and the normal saline group.3)Differential flora: The abundance of Mycoplasma,Gammaproteobacteria,Betaproteobacteriales and Burkholderiaceae in the lower respiratory tract flora of rats in the model group was lower than that in the normal saline group,and the abundance of Bacteria was significantly higher than that in the normal saline group;The abundance of Bifidobacteriales,Bifidobacteriaceae and Bacteria in the lower respiratory tract flora of rats in Si O2 group was higher than that in the normal saline group,and the abundance of Betaproteobacteriales and Burkholderiaceae was significantly lower than that in the normal saline group.4 Differential metabolites in lung lavage fluid: Compared to the normal saline group,the contents of 7-Oxocholesterol,Pantothenol,1-Stearoyl-2-hydroxysn-glycero-3-phosphocholine,and Cer(d18:1/18:1(9Z))in the lung lavage fluid of rats in the model group were significantly lower,and the contents of Stearic acid were significantly higher,and the main metabolic pathways involved included Biosynthesis of unsaturated fatty acids and fatty acid biosynthesis;The content of glycerol in the lung lavage fluid of rats in the silicon dioxide group was significantly increased,1-Stearoyl-2-arachidonoyl-sn-glycerol,Acetylcarnitine,Uracil,Cytidine,N-Acetylglutamine,L-Carnitine,Betaine,Thioetheramide-PC,Uridine,(3-Carboxypropyl)trimethylammonium cation,Erucamide,LGlutamate,Cyclohexylamine,Erucic acid,Phytanic acid,N-Palmitoylsphingosine,PC(16:0/16:0)content was significantly reduced,and the metabolic pathways mainly involved included Pyrimidine metabolism,D-Glutamine and D-glutamate metabolism,Nitrogen metabolism,Aminoacyl-t RNA biosynthesis,Histidine metabolism,Pantothenate and Co A biosynthesis,Glycerolipid metabolism,Beta-Alanine metabolism,Butanoate metabolism,Alanine,aspartate and glutamate metabolism,Galactose metabolism,Glutathione metabolism,Porphyrin and chlorophyll metabolism,Glycine,serine and threonine metabolism,Biosynthesis of unsaturated fatty acids,Arginine and proline metabolism.Pyrimidine metabolism,D-glutamine and D-glutamic metabolism were the most obvious metabolic pathways(P < 0.05).5 Spearman’s correlation analysis: 1)Association analysis of differential flora of upper respiratory tract with differential metabolites of lung lavage fluid: Among differential flora and differential metabolites of the model group and normal saline group,Pasteurella＿sp.＿V6 were positively correlated with stearic acid;Rodentibacter were positively correlated with Cer(d18:1/18:1(9Z));uncultured＿bacterium＿Rodentibacter were negatively correlated with Cer(d18:1/18:1(9Z)),1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine;Both Muribacter＿muris and Muribacter were positively correlated with Cer(d18:1/18:1(9Z))and Pantothenol;uncultured＿bacterium＿Stenotrophomonas were negatively correlated with stearic acid.Among the differential flora and differential metabolites in the Si O2 group and normal saline groups,Pasteurella＿sp.＿V6 were positively correlated with glycerol;Streptococcus＿agalactiae were negatively correlated with uracil,phytanic acid,PC(16:0/16:0),L-Carnitine,Erucamide,and(3-carboxypropyl)trimethylammonium cation.2)Analysis of the association between differential flora of the lower respiratory tract and differential metabolites of lung lavage fluid: Among the differential flora and differential metabolites of the model group and the normal saline group,Mycoplasma was positively correlated with stearic acid;Gammaproteobacteria were negatively correlated with Cer(d18:1/18:1(9Z));Betaproteobacteriales were negatively correlated with Pantothenol and Cer(d18:1/18:1(9Z));Burkholderiaceae were negatively correlated with 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine,Cer(d18:1/18:1(9Z)),and 7-Oxazolidinol.Both Bifidobacteriales and Bifidobacteriaceae were positively correlated with uracil in the differential flora and differential metabolites in the Si O2 group and normal saline groups;Betaproteobacteriales was negatively correlated with uridine,thioetheramide-PC,Npalmitoylsphingosine,N-acetylglutamine,L-glutamate,cytidine,cyclohexylamine,acetylcarnitine,and 1-stearoyl-2-arachidonoyl-sn-glycerol;Burkholderiaceae was positively correlated with glycerol and negatively correlated with uridine,thioetheramide-PC,LGlutamate,cyclohexylamine,acetylcarnitine,and 1-stearoyl-2-arachidonoyl-sn-glycerol were negatively correlated.Conclusions 1 Exposure to dust causes disorders in the respiratory tract flora of the body:In the upper respiratory tract,the abundance of Muribacter,Muribacter＿muris,and uncultured＿bacterium＿Stenotrophomonas in the model group were increased,while that of Pasteurella＿sp.＿V6,Bacteria,Rodentibacter,and uncultured＿bacterium＿Rodentibacter was decreased.The abundance of Pasteurella＿sp.＿V6 and Streptococcus＿agalactiae in the Si O2 group was decreased.In the lower respiratory tract,the abundance of Bacteria in rats of the model group was increased,while the abundance of Mycoplasma,Gammaproteobacteria,Betaproteobacteriales,and Burkholderiaceae was decreased.In the Si O2 group,the abundance of Bifidobacteriales,Bifidobacteriaceae and Bacteria was increased,while the abundance of Betaproteobacteriales and Burkholderiaceae was decreased.2 After the dust exposure,the metabolic state of the body was disordered: Five potential metabolic markers including Pantothenol and Stearic acid have been identified in the model group,which are mainly involved in unsaturated fatty acid biosynthesis and fatty acid biosynthesis;In the Si O2 group,18 potential metabolic markers such as L-Glutamic,Uracil,Cytidine,and Uridine were identified,and among the main metabolic pathways involved,pyrimidine metabolism,D-glutamine and D-glutamic metabolism may be the key metabolic pathways in the occurrence and development of pneumoconiosis.3 The imbalance of Betaproteobacteriales,Burkholderiaceae,Bifidobacteriales,Bifidobacteriaceae and Streptococcus＿agalactiae may cause the abnormalities of pyrimidine metabolism,Dglutamine and D-glutamic metabolism,and thus affecting the occurrence and development of pneumoconiosis.Figure 28;Table 13;Reference 117... |
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