The mice on the high-fat diet show significant increase in body weights and dyslipidemia, which was aborogated by saturated hydrogen.
As shown in Figure 1A, the average weight of mice in the HF+H2 group on the 42nd day was 34.05 ± 2.15 g, 20% higher than that of mice in the Control group, which was 27.45 ± 1.15 g, meaning that the average weight of mice in the HF+H2 group meet the weight standard of obese mice. The average weights of mice in the H2 and HF+H2 groups decreased after treatment with saturated hydrogen when compared with those in the corresponding Control and HF groups, as shown in Figure 1B, indicating that saturated hydrogen inhibited the increase in body weights of mice.
On day 28, total cholesterol (TC) and low-density lipoprotein (LDL) in the HF group increased significantly compared with those in the Control group, and LDL in the HF+H2 group decreased considerably compared with the HF group (Figure 1 C, E). On day 42, TG, TC, and LDL in the HF group increased markedly compared with those in the Control group, which decreased substantially after saturated hydrogen treatment (Figure 1C, D, and E). However, high-density lipoprotein (HDL) in the HF group decreased remarkably compared with the Control group, and it increased dramatically in the HF+H2 group compared with the HF group (Figure 1F), suggesting that saturated hydrogen reduces TC, TG, and LDL levels and increases the content of HDL in the peripheral blood of obese mice.
In the high-fat diet group, the infiltration of inflammatory cells in the lung tissues increased and EMT increased.
It can be seen from the HE staining results of the lung histopathological sections (Figure 2A) that after day 42 on high-fat diet, the infiltration degree of inflammatory cells around the bronchus in HF group increased compared with that of the Control group, with a small amount of inflammatory cells infiltrating in the lumens and a little shedding of bronchial cilia. Compared with the HF group, the pathological changes were decreased in the HF+H2 group. The results of qPCR showed that the expression of E-cadherin was significantly decreased and twist was significantly increased in the HF group compared with the Control group. Compared with the HF group, the expression of E-cadherin gene in the HF+H2 group increased, and twist decreased, but there was no significant statistical difference (Figure 2B). The results of immunofluorescence showed that the expression of E-cadherin protein decreased significantly at day 42, while the expression of twist protein increased significantly in the HF group compared with the Control group, which was in consistent with the results of qPCR (Figure 2 C, D). These results have shown that high-fat diet can lead to inflammation of lung tissue and accelerate the process of EMT of respiratory tract.
The normal diet group and the high-fat diet group show significant differences in the number and composition of microbes in the lungs.
After the question sequence is eliminated, the total sequencing quantity is 559453. The length distribution of the sequence contained in all samples was statistically analyzed in R software, and the sequence length was mainly distributed between 250-450bp (Supplementary figure 1). Operational taxonomic unit (OTU) whose abundance value was less than 0.001% (1/100,000) of the total sequencing amount of all samples was removed, and the distribution of the remaining OTU in each group is shown as (Figure 3A, Supplementary figure 2). The results showed that the number of OTU on the HF group was significantly higher than that of the Control group at the level of phylum, class, order, genus and species. The analysis of the same OTU between the groups showed that the number of unique OTU in the HF group increased significantly compared with that of the Control group, and the number of unique OTU in the Control and HF groups all decreased after hydrogen administration (Figure 3B). According to the OTU classification and classification status, the specific composition of each sample at the level of phylum, class, order, family, genus and species classification was obtained. The results showed that the number of bacteria in the lung of mice in the HF group was significantly higher than those in the Control group, and there was no significant difference between the hydrogen group and the non-hydrogen group (Figure 3C, D).
The diversity and difference of microflora in the lung of each group.
The rarefaction sparse curve (Chao1), speccaccum species accumulation curve and abundance level curve in alpha diversity analysis tend to be gentle, indicating that the sequencing depth is enough to reflect the diversity and richness of the microbial community contained in the community sample, and the total number of OTUs in the community will no longer increase by significant. With the addition of new samples, and the abundance difference among OTUs in the community, the difference is small and the community composition is high (Figure 4A). According to the alpha diversity index (Chao1, ACE, Shannon, Simpson) (Figure 4B), the diversity of bacteria in the lung of mice in the high fat diet group was significantly higher than that of the normal diet group, and the diversity of mice in the normal hydrogen feeding group was lower than that of the normal control group.
Discriminant analysis by PLS-DA partial least squares and beta diversity based on weighted UniFrac NMDS UPGMA cluster analysis chart of UniFrac distance matrix (Figure 4C) shows that there are obvious differences in the structure of microbial community in the lung of the HF group compared with the Control group, and also some differences in the hydrogen feeding group compared with the non-hydrogen feeding group, and the difference of each mouse within the same group is small. The results show that the animal classification model is effective.
Identification of key microbes with significant difference in pulmonary microflora of mice in each group.
Using R software, cluster analysis and heat map (Figure 5A) were made for the first 50 genera of abundance, which showed that there were significant differences between the high-fat diet group and the normal diet group, and there were also some differences between the hydrogen group and the non-hydrogen group.
Compared with the Control group, the abundance of the common opportunistic pathogens Acinetobacter, Pseudomonas, Corynebacterium, Strepcoccus, Clostridium, Haemophilus and Porphyromonas increased significantly in the HF group. After hydrogen administration, the abundance of Acinetobacter, Clostridium and Porphyromonas decreased significantly. There was a significant decrease in Bifidobacterium in the HF group compared with that of the Control group (Figure 5B, C). These results showed that high-fat diet significantly alters the diversity of microbes in the lung of mice, opportunistic pathogens increased while probiotic microbes decreased, thus increased susceptibility of respiratory inflammation.
Functional prediction of bacterial metabolism
The 16S rRNA gene sequence was predicted in KEGG, COG and rfam3 functional spectrum databases by PICRUSt functional prediction analysis. The predicted functional spectrum data are clustered according to the abundance distribution of functional groups or the similarity between samples, and the functional groups and samples are sorted according to the clustering results. Using R software, the functional groups in the top 50 of the abundance were clustered and analyzed, and a heat map (Figure 6A) was drawn, showing that there were significant differences between the high-fat diet group and the normal diet group. R software was used to calculate the number of common functional groups of each sample, and the proportion of common and unique functional groups of each sample was visualized through Venn diagram. The results showed that the number of functional groups in the HF group was significantly increased compared with the Control group, and that in the hydrogen group was decreased compared with the non-hydrogen group (Figure 6B).
Violin diagram was drawn to show the abundance distribution of the predicted functional groups in each sample (Figure 6C). The results showed that the flora abundance of the HF group involved in lipid metabolism, energy metabolism and amino acid metabolism was significantly increased compared with the Control group, while the flora abundance of the HF group involved in glycan biosynthesis, metabolism and glucose metabolism was decreased. The abundance of bacteria involved in cell communication, cell migration, cell growth and death increased significantly. The number of bacteria involved in the transcription of genetic information increased and the abundance of bacteria involved in translation decreased significantly. The abundance of bacteria involved in neurodegenerative diseases and cardiovascular diseases increased significantly. The abundance of bacteria involved in the body's immune system decreased significantly and the abundance of bacteria involved in the endocrine system and circulatory system increased significantly.
The expression of ICL gene in P. aeruginosa, S. aureus, A. baumannii, K.pneumoniae, C. albicans and X. maltophilia
To investigate whether changes in microbes in the lungs of mice on a high-fat diet were due to changes in glyoxylic acid circulation in bacteria caused by a high-fat environment. We designed primers using the ICL sequence, a key enzyme in the glyoxylic acid cycle, to detect the expression of ICL genes in several clinical pathogens significantly associated with the development of asthma. qPCR results (Figure 7) showed that the ICL gene expression in P. aeruginosa, S. aureus and A. baumandii in the HF group was significantly increased compared with that of the Control group, and P. aeruginosa was significantly decreased after hydrogen supplementation. However, K. pneumoniae, C. albicans and X. maltophilia showed no significant changes.