A total of 72 6-8-wk-old male C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in a standard specific pathogen-free environment. The mice were isolated and allowed to adapt for one week. All procedures involving animals were approved and supervised by the Animal Experimentation Ethics Committee of Zhejiang Eyong Pharmaceutical Research and Development Center. Mycoplasma free HUC-MSCs (HUM-iCell-e009) were purchased from iCell Bioscience Inc. (Shanghai, China) and cultured in a specialized medium (PriMed-iCELL-012; iCell Bioscience, China) containing supplements at 37 °C in a 5% CO₂ incubator. The purity of HUC-MSCs was assessed by flow cytometry and was typically greater than 90%. Cell identification was performed by iCell Bioscience Inc.

The random number method was used to divide mice into four groups, namely, sham, sham + MSCs, LPS, and LPS + MSCs groups, with 18 mice in each group. The 36 randomly selected mice were intraperitoneally injected with 100 mL of LPS (10 mg/kg) to induce ALI[7], and sham mice were administered 100 mL of 0.9% NaCl as controls. After 6 h, half of the ALI mice and half of the sham mice were given 0.5 mL of phosphate buffered saline (PBS) containing HUC-MSCs (1 × 10⁶ cells/mL) by intraperitoneal injections[24], and the other half of the ALI mice and sham mice were given 0.5 mL of 0.9% NaCl. The mice were reared under standard laboratory conditions. The experiments were carried out by expert technicians who were blinded to the animal grouping.

Three days after HUC-MSC intervention in ALI mice, BALF was obtained after the mice were anesthetized with isoflurane as reported by Wu et al[24]. Briefly, the trachea was flushed five times with PBS via a 20-gauge catheter, and all liquids were collected. Subsequently, all mice were euthanized with CO₂, and the blood, lungs, ileum, and feces were obtained using aseptic techniques. The lungs (one lung was evenly divided into four) from three mice in each group were taken, weighed, and then baked in an oven at 80 °C for 48 h to determine the dry weight to calculate the ratio of wet lung weight to dry lung weight (W/D weight ratio) as reported by Li et al[25]. In addition, a part of the remaining lung and ileum were made into paraffin blocks separately, and the other were stored at 80 °C for further analysis. The BALF was divided into two parts for inflammation cytokine detection and 16S rDNA sequencing, respectively.

The protein concentration in the BALF was measured using the BCA assay (Beyotime, China). Additionally, the cells in BALF were precipitated and resuspended, and mononuclear cells and neutrophils were counted after Wright’s staining. The levels of tumor necrosis factor (TNF)-α (ml002095-1, Enzyme-linked, China), IL-1β (ml063132-1, Enzyme-linked, China), and IL-6 (ml002293-1, Enzyme-linked, China) in the serum, lungs, and ileum were measured by enzyme-linked immuno sorbent assay (ELISA) according to the manufacturer’s instructions.

Hematoxylin-eosin (H&E) staining was performed to observe the histopathology of the lungs and ileum. Briefly, the lungs and ileum were fixed in 10% formalin. The tissues were then placed in paraffin and sectioned, and sections were stained with H&E. The injury of lungs and ileum were scored in a blinded fashion as previously reported[26,27].

Three mice were randomly selected from each group for the Evans blue dye leakage assay to explore the function of the lung tissue endothelial barrier as previously reported[28]. This assay was performed using 25 mg/kg of 0.5% blue dye (Sigma, United States) injected into mice via the tail vein 2 h before isoflurane anesthesia. After the heart of the anesthetized mice was exposed, the left ventricle was intubated and flushed with 4% normal saline. Subsequently, the mice were euthanized using CO₂. Lungs were homogenized in 2 mL PBS and treated with formamide (Sigma, United States) at 60 °C for 24 h. Finally, the concentration of Evans blue dye in the lung tissue was determined at 620 nm using a microplate reader (CMaxPlus, Molecular Devices, United States).

Immunohistochemistry (IHC) was performed as described by Peng et al[29]. Paraffin sections of lung tissue were deparaffinized, permeabilized, and blocked. The sections were then incubated at 4 °C overnight with anti-TLR4 antibody (1:100 dilution; Affinity, United States). After washing with PBS, the slides were incubated with goat anti-rabbit IgG HRP (Abcam, United States) at 37 °C for 30 min. The slides were washed again, mounted with DAPI (Vector Laboratories, Burlingame, CA), and examined using an E100 fluorescence microscope (Nikon, Japan).

Fluorescence in situ hybridization (FISH) was performed on slides of ileum and lung tissues as previously described[30], using a pan-bacteria FITC-labeled probe (EUB338) and an RNA FISH kit (Genepharma, China). After DAPI staining, the slides were visualized under an inverted fluorescence microscope (Ts2-FL; Nikon, Japan). Images were acquired using Micro-Manager and analyzed using ImageJ/FIJI software (National Institutes of Health, United States, version 1.53c).

The antibodies used for western blot (WB) are shown in Table 1. The lung and ileum tissues were homogenized in a lysis solution for the extraction of proteins. After centrifugation and supernatant collection, the samples were uniformly concentrated and denatured. Briefly, sodium dodecyl sulfate-gel electrophoresis and membrane transfer were performed for 20 μg protein per group as reported by Li et al[3]. The membranes were incubated with primary antibodies overnight and then with secondary antibodies for 1 h. Finally, the membranes were subjected to chemiluminescence reactions and protein levels were measured by enhanced chemiluminescence. ImageJ/FIJI software was used for semiquantitative analysis.

High-throughput 16S rDNA sequencing was performed to analyze the gut and pulmonary microflora by BioDeep Co., Ltd (Suzhou, China). A sequencing library was prepared using the TruSeq Nano DNA LT Library Prep Kit of Illumina. A NovaSeq6000 system (PE250, Illumina, United States) was used for sequencing as reported by Yi et al[31]. Then the QIIME2 DADA2 and OmicStudio platforms[32] were used to analyze the data.

The non-targeted metabolomics approach was performed by PANOMIX Biomedical Tech Co., Ltd. (Suzhou, China). The samples were treated with 75% methanol-chloroform (9:1) and 25% water for sonication and centrifugation to extract the metabolites. The samples (2 μL) were analyzed by liquid chromatography-mass spectrum (MS) detection with the Vanquish UHPLC System (Thermo, United States). Additionally, an Orbitrap Exploris mass spectrometer (Thermo, United States) was used for mass spectrometry analysis. After data acquisition, the Proteowizard package (v3.0.8789) and the R xcms package were used to preprocess the data. Metabolites were identified on the public databases such as the Human Metabolome Database[33], MassBank (https://massbank.eu/MassBank/), Lipid Maps (https://Lipidmaps.org/), mzCloud (https://www.mzcloud.org/), Kyoto Encyclopedia of Genes and Genomes (KEGG)[34], and self-building repository, and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) analysis was performed to screen the differential metabolites.

Statistical analyses were performed using SPSS (version 16.0; IBM, Armonk, NY, United States). Data from multiple in vivo experiments were analyzed using one-way analysis of variance (ANOVA) with a post-hoc Tukey test. The independent-samples Student’s t-test was used for comparison between the two groups. The Wilcoxon rank-sum test was conducted to explore the microflora with a significant difference in abundance. Pearson’s correlation analysis was used to explore the intrinsic associations. Additionally, the Variable Importance for the Projection (VIP) of OPLS-DA was used to screen for metabolites with biomarker potential (VIP > 1). Two-way Orthogonal Partial Least Squares (O2PLS) analysis was used to explore the links between BALF microbiota and lung metabolism. The threshold for significance was P < 0.05 for all tests.

This study used LPS to induce ALI in mice and then collected their lungs and BALF to explore the ameliorating effect of HUC-MSCs on ALI by H&E staining and ELISA. ALI mice had a higher lung W/D weight ratio and more mononuclear cells and neutrophils than sham mice (P < 0.01), whereas HUC-MSC treatment on ALI mice decreased the lung W/D weight ratio (P < 0.05), mononuclear cell and neutrophil counts, and protein concentration (P < 0.01) (Figures 1A and B). Additionally, compared to sham mice, the lung of ALI mice had markedly thickened alveolar septa with significant inflammatory cell infiltration; however, HUC-MSC treatment alleviated the degree of alveolar septal thickening and inflammatory cell infiltration in ALI mice (Figure 1C). Likewise, the score of lung injury in the LPS group was higher than those in the sham (P < 0.01) and LPS + MSC groups (P < 0.05) (Figure 1D). Moreover, ALI mice had significantly increased levels of TNF-α, IL-1β, and IL-6 in their serum and lung tissues (P < 0.01) (Figures 1E and F). In particular, the above-mentioned inflammatory factor levels were decreased in the ALI mice treated with HUC-MSCs (P < 0.01) (Figures 1E and F).

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Figure 1 Human umbilical cord mesenchymal stem cells attenuate lipopolysaccharide-induced lung injury and inflammation in acute lung injury mice. A: In acute lung injury (ALI) mice, the wet-to-dry (W/D) weight ratio of the lung was higher than that of human umbilical cord mesenchymal stem cell (HUC-MSC)-treated ALI mice (n = 3); B: ALI mice had more mononuclear cells and neutrophils, and higher protein concentration in bronchoalveolar lavage fluid (BALF) than sham mice, while HUC-MSC treatment decreased these parameters (n = 12); C: Representative images of hematoxylin-eosin (H&E)-stained lung tissues (× 400, scale = 50 μm); D: Lung histopathological damage in mice (H&E staining; n = 3); E: Serum tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and lipopolysaccharide levels were higher in ALI mice and HUC-MSC treatment decreased their levels (n = 12); F: TNF-α, IL-1β, and IL-6 levels in the lungs of ALI mice were hugely increased than those in sham mice, while HUC-MSC treatment in ALI mice decreased them (n = 12). ^aP < 0.05, ^bP < 0.01. LPS: Lipopolysaccharide; MSC: Mesenchymal stem cell; BALF: Bronchoalveolar lavage fluid; H&E: Hematoxylin-eosin; IL: Interleukin; TNF: Tumor necrosis factor.

In addition, this study observed the endothelial barrier function and integrity of the lungs using Evans blue, WB, and IHC assays. As shown in Figure 2A, ALI mice had a higher concentration of Evans blue dye in the lungs than the sham mice, and HUC-MSC treatment reduced Evans blue concentration in the lungs of ALI mice (P < 0.01). In particular, the levels of endothelial barrier-associated proteins, such as vascular endothelial (VE)-cadherin, zonula occludens-1 (ZO-1), and occludin, were markedly decreased in ALI mice (P < 0.05 or P < 0.01); however, HUC-MSC treatment reversed the expression levels of these proteins (P < 0.01) (Figures 2B-D). In addition, this study explored the signal intensity of the TLR4/myeloid differentiation factor 88 (Myd88)/NF-κB signaling pathway. The TLR4, Myd88, p-NF-κB/NF-κB, and p-inhibitor α of NF-κB (p-IκBα)/IκBα expression levels in the lung were all increased in ALI mice compared to the sham mice (P < 0.01); however, in ALI mice treated with HUC-MSCs, the expression levels of these proteins were decreased (P < 0.05 or P < 0.01) (Figures 2E-I). Similarly, TLR4 detected by IHC had a strong positive expression in the lungs of ALI mice, whereas HUC-MSC treatment attenuated the positive expression of TLR4 (P < 0.05 or P < 0.01) (Figures 2J and K).

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Figure 2 Human umbilical cord mesenchymal stem cells improve endothelial barrier function in vivo and regulate related protein expression. A: The Evans blue assay was carried out to assess lung endothelial barrier function in acute lung injury (ALI) mice (n = 3). The ALI mice [lipopolysaccharide (LPS) group] had the largest amount of Evans blue contents; human umbilical cord mesenchymal stem cell (HUC-MSC) treatment in ALI mice (LPS + MSC group) decreased the contents in the lung; B-H: LPS treatment in mice inhibited the expression of vascular endothelial-cadherin (B), zonula occludens-1 (C), and occludin (D) and raised the expression of Toll-like receptor 4 (TLR4) (E), myeloid differentiation factor 88 (F), p-nuclear factor kappa-B (NF-κB)/NF-κB (G), and p-inhibitor α of NF-κB (IκBα)/IκBα (H) (n = 3), while HUC-MSC treatment antagonized the effects of LPS; I: Representative western blot bands of the above proteins; J and K: Statistical results of immunohistochemistry and its representative images. It was showed that TLR4 was highly expressed in the lung tissue of ALI mice and HUC-MSC treatment could decrease its expression (n = 3). ^aP < 0.05, ^bP < 0.01. LPS: Lipopolysaccharide; MSC: Mesenchymal stem cell; LPS: Lipopolysaccharide; VE: Vascular endothelial; ZO-1: Zonula occludens-1; TLR4: Toll-like receptor 4; Myd88: Myeloid differentiation factor 88; NF-κB: Nuclear factor kappa-B; IκBα: Inhibitor α of nuclear factor kappa-B.

Studies have reported not only lung function injury but also intestinal dysfunction in ALI[17]. Therefore, the ileum was examined using H&E staining, ELISA, WB, and FISH. The ileal tissue of ALI mice had shorter and ruptured villi with significant inflammatory cell infiltration compared to sham mice, whereas these ileal injuries in ALI mice treated with HUC-MSCs were improved (P < 0.05 or P < 0.01) (Figure 3A). In addition, LPS treatment on mice markedly increased the ileal TNF-α, IL-1β, and IL-6 levels (P < 0.01), whereas HUC-MSC treatment inhibited them (P < 0.01) (Figure 3B). The suppressed expression of endothelial barrier-associated proteins, including claudin, ZO-1, and occludin by LPS was enhanced by HUC-MSC treatment in ALI mice (P < 0.05) (Figures 3C and D). Furthermore, the EUB338 counts of the ileum epithelium and lungs in ALI mice were measured to observe the lung translocation of gut bacteria, and it was found that they were increased in ALI mice, whereas HUC-MSC treatment reduced them (P < 0.01) (Figures 3E-H).

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Figure 3 Human umbilical cord mesenchymal stem cells improve histopathology, inflammation, and endothelial barrier integrity of the ileum in acute lung injury mice. A: Ileum tissue injury in acute lung injury (ALI) mice was observed after hematoxylin-eosin staining. ALI mice had severe ileum tissue injury while human umbilical cord mesenchymal stem cell (HUC-MSC) treatment improved such injury (n = 3); B: Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and lipopolysaccharide (LPS) levels in the ileum were measured by ELISA (n = 12), and ALI mice had higher levels of TNF-α, IL-1β, IL-6, and LPS compared to sham mice, while HUC-MSC treatment decreased the levels of these factors; C: Zonula occludens-1 (ZO-1), claudin-1, and occludin levels were measured to observe the integrity of the ileum barrier (n = 3); D: Representative western blot bands of ZO-1, claudin-1, and occludin; E-H: Bacterial translocation was determined by fluorescence in situ hybridization, and the EUB338 counts in the ileum epithelium and lung per field were quantified (n = 3). ^aP < 0.05, ^bP < 0.01. MSC: Mesenchymal stem cell; LPS: Lipopolysaccharide; ZO-1: Zonula occludens-1; HE: Hematoxylin-eosin; IL: Interleukin; TNF: Tumor necrosis factor.

16S rDNA sequencing was used to explore the effect of the lung and gut microbiota on the HUC-MSC-mediated amelioration of ALI. BALF and fecal samples were collected for 16S rDNA sequencing. After homogenizing the sequencing depth of each group, 48 BALF and fecal samples from the four groups were identified at the genus level, with an average of 48.25 units in each group. Figures 4A and B shows the top 20 flora with the highest average abundance at the genus level. The Shannon and Simpson indices of alpha diversity reflect richness and community evenness, respectively. According to the Kruskal-Wallis rank-sum test, the Shannon index showed no significant differences among the groups (P = 0.056) (Figure 4C). Additionally, the Bray-Curtis distance of beta diversity reflects microbial diversity between groups and was analyzed by principal coordinates analysis, which revealed that the projection distance of the LPS + MSC group on the coordinate axis was closer to that of the negative control group than that of the LPS group (Figure 4C). Similarly, the Simpson index showed no significant differences among the groups (P = 0.058), and the projection distance of the gut microflora was closer to that of the negative control group than to that of the LPS group (Figure 4D).

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Figure 4 Microflora homeostasis in bronchoalveolar lavage fluid and feces of acute lung injury mice (n = 6). A and B: Relative abundance of top 20 genera of microflora in bronchoalveolar lavage fluid (BALF) and feces; C and D: The within-sample richness and evenness (alpha diversity) were statistically analyzed by Shannon index, Simpson index, and the bray-Curtis based principal coordinates analysis of similarity coefficients in the BALF and feces of different groups (beta diversity); E and F: The Wilcoxon rank sum test was used to explore microflorae with a significant difference in abundance on OmicShare Tools. The number of differential microflorae in lipopolysaccharide (LPS) + mesenchymal stem cells (MSCs) group vs sham + MSC group, sham group vs LPS group, sham group vs sham + MSC group, and LPS group vs LPS + MSCs group is presented. The length of the red bar indicates the number of up-regulated microflorae and the length of the blue-green bar indicates the number of down-regulated microflorae. LPS: Lipopolysaccharide; MSC: Mesenchymal stem cell; BALF: Bronchoalveolar lavage fluid.

Subsequently, the Wilcoxon rank-sum test was performed to explore the microflora with a significant difference in abundance (marked microflorae) (Figures 4E and F). In particular, there were 21 microflorae with upregulated abundance and 12 microflorae with downregulated abundance in the BALF of mice in the LPS + MSC group compared to the LPS group (P < 0.05) (Figure 4E), and 17 microflorae with upregulated abundance and 3 microflorae with downregulated abundance in feces (P < 0.05) (Figure 4F). The 33 marked microflorae with the largest upregulation or downregulation of operational taxonomic units in the BALF of ALI mice with/without HUC-MSC treatment are shown in Figure 5A, and 20 marked microflorae in feces are shown in Figure 5B. Rhizobiales had the largest log2 fold change (FC) in the BALF of mice in the LPS + MSC group compared to the LPS group [log2(FC) = 9.3264, P = 0.0284], and Elizabethkingia had the lowest log2FC in the BALF of mice in the LPS + MSC group compared to that of the LPS group [log2(FC) = -5.1799, P = 0.028] (Figure 5A). In fecal samples, the log2FC of unclassified_Bacteroidales was the highest in the marked microflorae of the LPS + MSC group compared to that of the LPS group [log2(FC) = 4.7549, P = 0.027], and that of the unidentified_F16 was the lowest [log2(FC) = -4.6328, P = 0.012] (Figure 5B).

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Figure 5 Differences in genus abundance of different microflorae in bronchoalveolar lavage fluid and feces of acute lung injury mice treated with human umbilical cord mesenchymal stem cells (n = 6). A and B: Heatmaps showing the differences in the genus abundance of different microflorae in bronchoalveolar lavage fluid and feces. Red represents the genus that is more highly abundant; blue/purple represents the genus that is less abundant; white/yellow indicates no difference in expression between groups. Abundance is homogenized by Z-score. The value displayed in the middle of the grid is log2 (fold change). LPS: Lipopolysaccharide; MSC: Mesenchymal stem cell; BALF: Bronchoalveolar lavage fluid.

Furthermore, the Pearson’s correlation analysis was used to analyze the correlation between marked microflorae of the gut and lungs, and most of the bacteria in the BALF and feces had a strong or extremely strong correlation (Figure 6). The Desulfovibrio genus in feces was positively correlated with Stenotrophomonas in BALF (P < 0.05) (Supplementary Table 1).

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Figure 6 Correlation analysis between gut and lung microflorae. Person correlation analysis was used to analyze the correlation between marked microflorae of the gut and lung with OmicsStudio Tools. Red indicates positive correlation and blue indicates negative correlation. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001 vs lipopolysaccharide group. BALF: Bronchoalveolar lavage fluid.

The base peak chromatograms in the positive and negative modes of the four groups showed similar trends, suggesting good repeatability and reliable results (Figure 7A). Additionally, Partial Least Squares-Discriminant Analysis (PLS-DA) was used to distinguish metabolite differences between groups in the positive/negative mode. In particular, all blue Q2 positions in the permutation test chart were lower than the original points on the far right, suggesting that the PLS-DA models were valid (Figures 7B and C). In the positive mode, MS analysis identified a total of 1206 common biomarkers between the sham and LPS + MSC groups, 362 between the LPS and sham + MSC groups, 347 between the LPS and sham groups, 343 between the LPS + MSC and LPS groups, and 181 between the sham + MSC and sham groups (Figure 7D). In the negative mode, this study identified 905 common biomarkers between the sham and LPS + MSC groups, 476 between the LPS and sham + MSC groups, 225 between the LPS and sham groups, 112 between the LPS + MSC and LPS groups, and 139 between the sham + MSC and sham groups (Figure 7E).

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Figure 7 Metabolomics for the lung (n = 6). A: Base peak chromatogram in positive/negative mode; B and C: The Partial Least Squares-Discriminant Analysis was used to distinguish metabolite differences between groups in positive/negative mode; D and E: Upset Venn diagrams were used to count the number of common and unique differential metabolites in different groups. The bar chart at the bottom left represents the total number of differential metabolites between two groups. The bar chart above shows common metabolites. The results are based on mass spectrum analysis data. LPS: Lipopolysaccharide; MSC: Mesenchymal stem cell.

Subsequently, precise screening of the metabolic profiles by MS/MS was performed to eliminate false positives. This study identified four upregulated metabolites (3-succinoylpyridine, nicotinamide ribotide, aldosterone, and phenylacetic acid) and one downregulated metabolite (lithocholic acid) in the sham + MSC group compared to the sham group. In addition, KEGG enrichment analysis suggested that these differential metabolites might participate in nicotinate and nicotinamide metabolism, aldosterone-regulated sodium reabsorption, and aldosterone synthesis and secretion pathways (Figures 8A-C). Additionally, 12 markedly upregulated metabolites (hydroxyindole, xanthine, and N-acetyl-L-aspartic acid, etc.) and 20 markedly downregulated metabolites (prostaglandin E2, argininosuccinic acid, and guanine, etc.) were identified in the LPS group compared to the sham group, and these metabolites were predicted to be potentially involved in alanine, aspartate, and glutamate metabolism, oxidative phosphorylation, the cAMP signaling pathway, and so on (Figures 8A, D and E). In particular, five upregulated metabolites (anabasine, IMP, lidocaine, salicylic acid, and propionylcarnitine) and 11 downregulated metabolites (N-acetylleucine, guanosine, guanine, etc.) were identified in the LPS + MSC group compared to the LPS group (Figures 8A and F). They were related to purine metabolism and the taste signaling transduction pathways (P < 0.001) (Figure 8G).

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Figure 8 Screening of differently expressed metabolites in acute lung injury mice treated with human umbilical cord mesenchymal stem cells (n = 6). A: Statistic of differently expressed metabolites under liquid chromatography-tandem mass spectrometry mode; B: Heatmaps of sham + mesenchymal stem cells (MSCs) group vs sham group; C: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched by differently expressed metabolites of sham + MSC group vs sham group; D: The lipopolysaccharide (LPS) group vs sham group were used to analyze and display differently expressed metabolites; E: KEGG pathway enriched by differently expressed metabolites of LPS group vs sham group; F: Heatmaps of LPS group vs LPS + MSC group; G: KEGG pathways enriched by differently expressed metabolites of sham + MSC group vs sham group. LPS: Lipopolysaccharide; MSC: Mesenchymal stem cell.

To explore the role of HUC-MSCs in regulating the microflora of the lung-gut axis to improve ALI, we performed an O2PLS correlation analysis on the expression of microflora in BALF and the expression of metabolites in the lungs to determine the microflora involved in the improvement of ALI by HUC-MSCs and their impact on metabolism. In Figure 9A, the top 25 bacteria and top 25 metabolites are shown with large absolute joint loading values, suggesting that they have a large weight in the improvement of ALI following HUC-MSC treatment. Subsequently, these flora and metabolites were analyzed using correlation analysis (Supplementary Table 1). Significantly related bacteria and metabolites were screened based on P < 0.05. The number of metabolites that significantly correlated with BALF microbes is shown in Figure 9B. Additionally, metabolites with a large role in the improvement of ALI by HUC-MSC treatment were subjected to KEGG enrichment analysis and were found to be mainly involved in the signaling pathways of drug metabolism-other enzymes, tyrosine metabolism, autophagy-animal, and endocytosis (Figure 9C).

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Figure 9 Extent of correlation between lung metabolites and bronchoalveolar lavage fluid microflorae. A: Top 25 lung metabolites and top 25 bronchoalveolar lavage fluid (BALF) microflorae were analyzed by the two-way Orthogonal Partial Least Squares analysis. A larger absolute value in a coordinate indicates a greater degree of association. Circles represent metabolites in lung tissues and squares represent microflora in BALF; B: The number of lung metabolites which were related with BALF microflorae; C: Kyoto Encyclopedia of Genes and Genomes enrichment analysis of top 25 BALF microflorae with strong association. KEGG: Kyoto Encyclopedia of Genes and Genomes; BALF: Bronchoalveolar lavage fluid.

Acute lung injury (ALI) has high morbidity and mortality rates and needs effective treatment. Research has found that the gut microbiota improves lung injury through the lung-gut axis. Human umbilical cord mesenchymal cells (HUC-MSCs) can improve ALI.

Although HUC-MSCs can improve ALI, their biological mechanism of action is not yet clear.

To explore changes in the microbiota in the lung-gut axis and the relationship with HUC-MSC treatment.

C57BL/6 mice were used to establish an ALI animal model by intraperitoneal injections of lipopolysaccharide. Wright’s staining, ELISA, hematoxylin-eosin staining, Evans blue dye leakage assay, immunohistochemistry, fluorescence in situ hybridization, and western blot were used to observe the improvement of ALI mice by HUC-MSCs. High-throughput 16S rDNA sequencing was used to observe the microbiota homeostases in the lung-gut axis. The non-targeted metabolomics was used to explore changes in lung tissue metabolites.

HUC-MSCs ameliorated histopathological damage in the lung and ileum of ALI mice. HUC-MSC treatment improved inflammation, endothelial barrier integrity, and bacterial translocation in the lungs and ileum of ALI mice. HUC-MSCs regulated lung-gut microbiota homeostasis. HUC-MSC treatment regulated the metabolic profile in the lung and ileum of ALI mice.

This study shows the improvement of changes in the lung and ileum of ALI mice by HUC-MSCs, and suggests a correlation between HUM-MSC-improved ALI and gut and lung microbiota homeostases.

This study explores the biological mechanism of HUC-MSCs in improving ALI from the perspective of the correlation between the microbiota in the lung-gut axis and lung tissue metabolites, providing a research basis for HUC-MSC treatment.