The APS was extracted from Astragalus membranaceus by boiling water extraction, and then the supernatant was deproteinized using Sevag reagent (Sevag et al., 1938). After removing Sevag reagent, the liquid was mixed with 95% ethanol to a final concentration of 80% (v/v). Twenty-four hours later, the precipitation was dried to get the crude APS. The crude polysaccharides were eluted by water using DEAE-cellulose column to obtain the purified APS.

The monosaccharides composition of APS was analyzed with High-Performance Liquid Chromatography (HPLC; Dionex U3000, ThermoFisher) equipped with ZORBAX EclipseXDB-C18 (Agilent) column. The mobile phase was acetonitrile and phosphate-buffered saline (12 g/L of KH₂PO4, 2 M NaOH, and adjust the pH to 6.8) in a proportion of 17:83, the flow rate was 0.8 ml/min and the wavelength was 250 nm. Five point 2 mg of the purified APS was dissolved in trifluoroacetic acid (TFA) solution at 121°C for 2 h and dried with termovap sample concentrator. Then methanol was used to wash the sample till the TFA was completely removed. The sample and standard monosaccharides were reacted with PMP reagent (1-phenyl-3-methyl-5-pyrazolone) before HPLC analysis.

Male C57BL/6 mice aged 5 weeks were purchased from Laboratory Animal Center of Huazhong Agricultural University. Mice were housed under even temperature (22 ± 2°C) and 12 h light/12 h dark cycle. After a week of acclimatization, mice were randomly divided into four groups, control group, LPS group, APS + LPS group, and APS group, respectively. Mice in control group and LPS group were administered with water by intragastric gavage for consecutive 14 days while mice in APS group and APS + LPS group were administered 200 mg/kg of APS (dissolved in water) in the same way. At the 15th day, all the mice were anesthetized with 1% of pentobarbital sodium and intratracheally instilled with PBS (for control group and APS group) or LPS (2 mg/kg, dissolved in PBS, for LPS group and APS + LPS group, Sigma, St Louis, MO, United States).

Lung tissues were separated from thoracic cavity, and the trachea and esophagus were removed. The lungs were numbered, and the wet weights were measured. Subsequently, the tissues were placed into oven at 60°C for 48 h to desiccate followed by weighting the dry lungs. The wet/dry (W/D) ratio of each lung tissue was calculated.

The lung tissues were fixed overnight and embedded in paraffin. Then, the slides were dehydrated using ethanol and stained with hematoxylin and eosin (H&E). The slides were observed under optical microscope (Olympus Shinjuku-ku, Tokyo, Japan).

Bronchoalveolar lavage fluid (BALF) was obtained by inserting 24-gage catheter into trachea and flushing with 1 ml of PBS for three times. The BALF was centrifuged at 400 × g for 10 min to remove liquid, and the cells pellets were collected for cell counting or cell culture. The cells attached to the cell plate were alveolar macrophages. Total protein concentration in cell-free BALF were measured by BCA protein assay kit (Biosharp, cat. no. BL521A).

Total RNA from lung tissues were isolated using RNA isolater Total RNA Extraction Reagent (Vazyme Biotech, cat. no. R401-01) and then were reversely transcribed into complementary DNA using ABScript III RT Master Mix (ABclonal Technology, cat. no. RK20429). Universal SYBR Green Fast qPCR Mix (ABclonal Technology, cat. no. RK21203) and specific primers were mixed with cDNA for qPCR assay. Two delta delta Ct method was used to analyze the relative expression levels of certain genes in lung homogenates. The primer sequences were shown in Table 1. The qPCR values were processed by TBtools software (version 1.0), in which the log scale to base 2 and column scale were selected and heatmap was drawn.

The cytokine concentrations in lung tissue and serum were measured by ELISA assay. The lung tissues were homogenized in cold PBS and centrifuged to collect supernatant, in which the total protein levels were quantified by BCA assay. The lung homogenates and serum were then subjected to ELISA kit (Enzyme-linked Biotechnology) to detect the concentration of IL-1β (cat. no. ml301814), IL6 (cat. no. ml063159), and TNF-α (cat. no. ml002095).

Flow cytometry was performed to identify alveolar cell subpopulations. The cells collected in BALF was stained with CD11b-PerCP Cy5.5 (BD Pharmingen, 561114), Ly-6G-FITC (Thermo Fisher Scientific, 11–9668-82), F4/80-eFlour 450 (Thermo Fisher Scientific, 48–4801-80), CD206-APC (Thermo Fisher Scientific, 17–2061-80), or CD86-PE (Thermo Fisher Scientific, 12–0862-81; all diluted 1:200) according to the manual. Samples were submitted to Cytoflex-LX Flow Cytometer (Beckman), and data were analyzed by using FlowJo (BD Bioscience).

The protein samples from lung tissues were prepared, and BCA method was used to detect the protein concentration in each sample. Twenty micrograms of total protein were loaded into wells of 10% SDS-PAGE gel. After being separated by electrophoresis, proteins were transferred to PVDF membrane (Millipore) and then blocked using 5% milk. Then, the membrane was incubated with NF-kB p65/RelA Rabbit pAb (ABclonal Technology, A2547, diluted 1:1,000), Phospho-NF-kB p65/RelA-S536 Rabbit pAb (ABclonal Technology, AP0475, diluted 1:1,000), and β-Actin Rabbit pAb (ABclonal Technology, AC006, diluted 1:2,000) overnight at 4°C. Appropriated secondary antibody was incubated with membrane at room temperature for 2 h, and then protein bands were visualized using Image-Pro Plus 6.0 software (Media Cybernetics), and grey values were measured by ImageJ software.

The colon samples were immediately frozen and stored at −80°C, and the microbiota compositions were analyzed via 16S rRNA analysis. The total DNA was extracted and then PCR amplification (ABI 2720, United States) of the 16S rRNA genes V3-V4 region were performed using specific primers (338F: 5′-ACTCCTACGGGAGGCAGCA-3′; 806R: 5′-GGACTACHVGGGTWTCTAAT-3′). The products of PCR were then sequenced by Novaseq-PE250 platform at Personal Biotechnology Co., Ltd. (Shanghai, China). The sequence data were analyzed using QIIME2 and R package.

In data analysis, alpha-diversity indices were used to evaluate the intestinal microbiota diversity and abundance. Principal coordinate analysis (PCoA) based on Bray-Curtis distance was used to compare the microbiota composition of each group. The relative species abundances were shown at the phylum and genus levels among groups.

The serum concentrations of SCFAs determined by Gas Chromatography–Mass Spectrometry (GC–MS). Standard SCFAs (Sigma-Aldrich, United States) were diluted in methyl tert-butyl ether (MTBE). 100 μl of serum samples were mixed with 0.5 ml of MTBE (with internal standard) solution followed by incubating for 3 min. The mixtures were then ultrasonicated for 5 min, and centrifuged at 12,000 r/min for 10 min. The supernatant was collected and used for GC–MS/MS analysis. Gas chromatograph (Agilent 7890B) coupled to a mass spectrometer (7000D) with a DB-5MS column (30 m length × 0.25 mm i.d. × 0.25 μm film thickness, J&W Scientific, United States) was used for GC–MS/MS analysis. Helium was used as the carrier gas at a flow rate of 1.2 ml/min. Injections in splitless mode were made with an intection volume of 2 μl. The oven temperature was kept at 90°C for 1 min, raised to 100°C at a rate of 25°C/min, raised to 150°C at a rate of 20°C/min, hold for 0.6 min, raised to 200°C at a rate of 25°C/min, and held for 0.5 min after running for 3 min. All samples were analyzed in multiple reaction monitoring mode. The temperatures of inlet and transfer line were 200 and 230°C, respectively.

All data were presented as the mean ± SD. Significant differences among groups were analyzed by one-way analysis of variance (ANOVA) and t-tests using GraphPad Prism 8 software. The heatmap was drawn by TBtools software following data normalization by using log scale and column scale.

High-Performance Liquid Chromatography was used to detect the monosaccharides composition of APS according to the standard monosaccharide samples. Results showed that the APS used in this study was composed of mannose (Man, 8.922%), rhamnose (Rha, 10.255%), glucuronic acid (GlcA, 12.552%), glucose (Glc, 39.283%), galactose (Gal, 5.418%), and arabinose (Ara, 23.570%) (Figure 1).

To confirm the protective role of APS on mice lung tissue, the mice were treated with APS solution for consecutive 2 weeks and then inflammatory lung injury mice models were established by LPS (Figure 2A). Data showed that APS treatment did not affect the body weight of mice (Figure 2B). Intratracheal instillation of LPS resulted in notably pathologic changes including destroyed alveolar structure, thickened alveolar wall and infiltration of inflammatory cells and red blood cells. However, in APS pre-treatment group, the pathologic changes induced by LPS were less severe, as more alveoli and less inflammatory cells were observed (Figure 2C). We calculated the W/D ratio of the lungs to evaluate their edema. Results showed that LPS instillation markedly increased the W/D ratio, suggesting the development of lung edema. As expected, APS pre-treatment prevented the increase of W/D ratio and mitigated the lung edema (Figure 2D).

We also collected the BALF and measured the total cell count and total protein concentration. The results showed that LPS treatment increased the quantity of total cells as well as the concentration of total protein in BALF, while these two indices were significantly reduced in APS pre-treatment (Figures 2E,F). This further proved that APS pre-treatment alleviated the inflammatory lung injury induced by LPS.

Lipopolysaccharides activates immune cells to overproduce inflammatory cytokines chemokines and lead to severe inflammation in tissues. Neutrophils are of importance in the onset of tissue inflammatory injury. Thus, we firstly evaluated the role of APS on the neutrophils infiltration. Flow cytometry analysis showed that abundant neutrophils were detected in lungs with inflammatory injury, and APS pre-treatment significantly reduced the number of neutrophils (Figure 3A). The concentration of myeloperoxidase (MPO) in lung homogenates and serum were measured by ELISA. Results showed that LPS induced remarkable increase of MPO concentration in both lung homogenates and serum. Consistent with flow cytometry results, APS pre-treatment downregulated the MPO concentration (Figure 3B). Moreover, we also detected the relative mRNA levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which were responsible for the adhesion of neutrophils to vascular endothelium (Wiesolek et al., 2020). Results showed that LPS treatment significantly increased the expression of Icam-1 in mice lung but had no effect on the expression of Vcam-1. APS pre-treatment led to less Icam-1 expression but did not change the expression of Vcam-1 either (Figure 3C). These results further confirmed that APS prevented the neutrophils infiltration in lung inflammatory response.

We then detected the expression of various pro-inflammatory markers. Twenty-four hours post LPS exposure, qPCR was used to analyze the relative mRNA levels. The results showed that LPS treatment greatly increased the relative expressions of Il-1β, Il-6, Tnf-α, Ccl2, and Cxcl1, which were significantly reduced in APS pre-treatment group (Figure 3D). Same trends were also observed by ELISA analysis, the concentrations of IL-1β, IL-6, and TNF-α in both lung homogenates and serum remarkably increased in LPS treatment group yet decreased when mice were pretreated with APS (Figures 3E,F).

We also observed the phenotype of alveolar macrophages (AMs), which is another type of immune cells responsible for the secretion of cytokines in lung. Pro-inflammatory macrophages express high CD86 while anti-inflammatory macrophages express high mannose receptor (MRC1, also known as CD206). After 24 h of LPS stimulation, AMs were collected from BALF and qPCR was conducted to detect the mRNA expression of Cd86 and Mrc1. We observed that LPS treatment significantly increased the expression of Cd86, and APS pre-treatment reduced its expression (Figure 3G). LPS treatment also induced the increase of Mrc1, this suggested adaptive anti-inflammatory response of AMs was elicited by LPS. APS combined with LPS further increased the Mrc1 expression (Figure 3G). This indicated that APS pre-treatment instructed the transition of macrophages from pro-inflammatory phenotype to anti-inflammatory phenotype.

Lipopolysaccharides can be recognized by toll like receptor 4 (TLR4) that located on the surface of several immune cells, and then the downstream NFκB signaling pathway was activated to produce inflammatory cytokines (Lu et al., 2008). The activation of NFκB is a crucial step to produce inflammatory cytokines. We observed the increase of the phosphorylation of p65 protein in lung following LPS treatment, while the phosphorylated p65 expression level in APS pre-treatment group was significantly decreased, suggesting the inhibition of NFκB signaling pathway (Figure 3H).

To explore the role of APS on mice intestinal microenvironment, the microbiota of colon mucosa was analyzed by 16S rRNA sequencing. Alpha-diversity based on Chao1 index and Shannon index was used to evaluate the community richness and diversity (Figure 4A). Results showed that compared to control group, Chao1 index and Shannon index values were increased in all other three groups. Principal coordinates analysis (PCoA) was used to evaluate the overall difference in microbiota classification. Samples in same group clustered together and were separated from other groups, suggesting the microbiota composition was affected by LPS and APS treatment (Figure 4B). The unweighted pair-group method with arithmetic mean (UPGMA) analysis showed that samples within same group were clustered together and distinguished with each other, confirmed that those treatment significantly altered the microbiota composition (Figure 4C). The Venn diagram illustrated the overlaps of Operational taxonomic units (OTUs) profiling and showed that a total of 545 OTUs among four groups (Figure 4D). Besides, the total number of microorganisms was increased in LPS treatment groups and decreased with APS pre-treatment. What is more, APS alone treatment also increased the number of microorganisms (Figure 4D).

Then, we analyzed the relative abundance of dominant taxa. At phylum level, Firmicutes and Bacteroidetes, constituted the principal part of microbiome community. Lung injury mice had higher level of Bacteroidetes but lower level of Firmicutes. On the contrary, mice pretreated with APS had higher Firmicutes but lower Bacteroidetes (Figure 4E). At genus level, LPS treatment increased the proportion of Prevotella. Compared with control group and lung injury group, APS pretreatment significantly reduced the Prevotella proportion and increased the Oscillospira proportion. LPS treatment also reduced the proportion of Allobaculum, whereas APS pre-treatment did not alter this reduction (Figure 4F). Besides, APS pre-treatment also increased the proportion of Akkermansia and Coprococcus, inspite of their relative low level in the whole genus (Figures 4F,G).

Short-chain fatty acids are important metabolite of intestinal microbiome that have been demonstrated to have anti-inflammation effects. To confirm if the anti-inflammation effects of APS was associated with SCFAs, the SCFAs concentration in serum were measured by GC–MS. The results showed that acetic acid, propanoic acid and butyric acid were significantly increased by APS treatment (Figures 5A–C). Next, we isolated mice primary AMs to further observe the anti-inflammation effects of butyrate and propionate. Cells were pretreated with butyrate or propionate and then were exposed to LPS. The qPCR results showed that butyrate and propionate pre-treatment significantly reduced the mRNA levels of pro-inflammatory cytokines, and reduced the expression level of Cd86, the marker of M1 phenotype of macrophages (Figure 5D). These proved the anti-inflammation effects of butyrate and propionate. We further measured the activation of NFκB signaling. Western blot results showed that the ratio of p-p65/p65 was significantly increased following LPS treatment, while butyrate and propionate pre-treatment inhibited the increase of p-p65 induced by LPS, suggesting the inhibition of NFκB signaling (Figure 5E).