Detection And Analysis Of Lung And Gut Microbiota In Experimental Silicosis Mice

Objectives:To detect and analyze the changes in the structure and diversity of the lung microbiota of silicosis mice at different stages of the progression of pulmonary fibrosis.To explore the role of pulmonary microecological imbalance in the development of silicosis and to analyze the structure and diversity of the gut microbiota changes,a preliminary understanding of the relationship between the gut and lung microbiota provide a scientific reference for in-depth study of the mechanism of silicosis progression and related etiology or to find new prevention and treatment methods.Methods:1.Research objects:60 SPF C57BL/6N male mice aged 6 weeks.2.Grouping:Divide mice into experimental group and control group,30 in each group.Using inhalation tracheal drip（once exposure）method,each mouse in the experimental group was given 50μL of 50 mg/mL SiO₂suspension,and the control group was given 50μL of sterile saline.The mice were sacrificed at three time points on the 7,28 and 56 days,with 10 mice per group each time.3.Detection of related indicators of mouse silicosis model.1)Comparison of mouse body weight and organ coefficient.2)HE staining,Masson staining and determination of hydroxyproline content were performed on lung tissue.3)Related indicators:fluorescence quantitative PCR was used to determine the inflammatory indicators IL-6,IL-1β,TNF-α,IFN-γand fibrosis-related indicatorsα-SMA,COL1A1 and TGF-βat the gene level in the lung tissue of each group of mice Relative expression.Western Blot was used to determine the fibrosis-associated proteinα-SMA in the lung tissues of 6 groups of mice.4.Detection of mouse lung and gut microbiota.Samples of mouse alveolar lavage fluid on days 7,28,and 56 were collected,feces samples of mice on day 56 were collected,bacterial genomic DNA was extracted,and 16SrDNA sequencing was used to detect microbial diversity.Use QIIME2 software to perform species composition analysis,Alpha diversity analysis,Beta diversity analysis,species difference and symbol species analysis,and functional potential prediction.5.Statistical AnalysisGeneral statistical analysis uses SPSS21.0,Excel and Graph Pad Prism 8.0.1software,and the results make measurement data expressed as mean±standard deviation.The statistical analysis method between two independent samples uses Student’s t test.Three groups and above are independent One-way analysis of variance（ANOVA）was used for statistical analysis between samples,and Pearson correlation analysis was used for correlation.The inspection level isα=0.05.Results:1.Successfully constructed an experimental silicosis mouse model.On the 56th day,mice in the silica group lost weight and increased their lung coefficient.The expression level of hydroxyproline in the lung tissue of mice in the silica group increased on the 28th and 56th days.The results of HE staining showed that the lung tissue of the silica group mice had alveolar structure destruction and inflammatory cell infiltration on the 7th day,the fibrous tissue increased on the 28th day,and the alveoli were further destroyed.A large number of fibers were formed on the 56th day,and the alveoli the structure disappeared.The results of Masson staining showed that with the development of silicosis,the deposition of collagen fibers gradually increased.The expression levels of pro-inflammatory indexes IL-6,IL-1β,and TNF-αin the silica group mice increased on the 7,28,and 56 days,and the pro-fibrosis indexesα-SMA,COL1A1 and TGF-βwere on the first day.The expression level increased on 28 and 56 days,and the expression level of fibrosis proteinα-SMA increased on 28 and 56 days.Compared with the control group,the above differences were statistically significant（P<0.05）.2.The pulmonary microbiota of mice is dysregulated,and the metabolic function abundance of the lung microbiota is different between the control group and the silica group.As time progresses,the pulmonary microbiota of silicosis mice has changed dynamically.Compared with the control group,Siene＿vulgaris,Acinetobacter lwoffii,Sphingobacterium＿multivorum and other bacteria continue to increase.Lactobacillus brevis,Pediococcus＿acidilactici and other bacteria continue to decrease.These bacteria may be involved in the progress of silicosis.Nucleoside and nucleotide biosynthesis,carbohydrate biosynthesis,TCA cycle,cell wall/membrane/Envelope biogenesis,signal transduction mechanism and other metabolic function abundances increase with time,carbohydrate metabolism,terpenoid and polyketide metabolism,aromatic compound degradation,carboxylate degradation and other metabolic function abundance decrease as time progresses.3.On the 56th day,the gut microbiota of the mice was dysbiosis,and the metabolic function abundance level of the gut microbiota was different between the mice in the control group and the SiO₂ group.On the 56th day,the dominant microbiota in the intestine of mice was similar,but the abundance was different.On the 56th day,the Beta diversity of the mice in the control group and the silica group was different,indicating that the composition of the microbial community between the different groups was different.Compared with the control group,the intestinal tract of mice in the silica group increased in Coprococcus,Clostridium,Verrucomicrobia,Akkermansia,Akkermansia＿muciniphila and Staphylococcus＿saprophyticus decreased.The abundance of metabolic functions such as replication and repair,cell growth and death,translation,ribosomal structure and biogenesis,etc.increase,and the abundance of metabolic functions such as degradation of fatty acids and lipids,biodegradation and metabolism of exogenous substances decreases.4.The lung and gut microbiota have the same different microbiota and the changes are opposite.On the 56th day,the intestinal and pulmonary microbiota were compared at each classification level,and it was found that the silica group mice had Verrucomicrobia,Akkermansia,Akkermansia＿muciniphila and Staphylococcus＿Saprophyticus decreased in the gut microbiota and increased in the lung microbiota.It shows that the gut microbiota may affect the lungs and cause the increase of the corresponding microbiota,thereby protecting the lungs.5.Pro-inflammatory factors and pro-fibrosis factors are related to different microbiota.On the 7th day,Acinetobacter lwoffii in the lungs of the experimental group mice was positively correlated with TNF-α（R=0.9989;P=0.0229）,and Thermus was positively correlated with IL-6（R=0.9987;P=0.0324）.On the 28th day,Sphingomonas＿yabuuchiae in the lungs of the experimental group mice was positively correlated with COL1A1（R=0.9997;P=0.0144）.On the 56th day Lactobacillus It is positively correlated with IL-1β（R=0.9980;P=0.0402）,Allobaculum is positively correlated with IL-1β（R=0.9993;P=0.0233）;Cupriavidus is positively correlated with IL-1β（R=0.9971;P=0.0484）and so on.Conclusions:1.The occurrence and development of silicosis is accompanied by the dysbiosis of the lung microbiota,and the differential lung microbiota at each stage may be related to pro-inflammatory factors and pro-fibrotic factors,suggesting that the microbiota may interact with cytokines in the progress of silicosis.2.In the process of silicosis,the microbial structure and diversity of the gut microbiota have also changed.The imbalanced gut microbiota may have a mutual regulatory relationship with the lung microbiota to affect the occurrence and development of silicosis.