Lincomycin hydrochloride (LH) was purchased from Anhui Shuanghe Pharmaceutical Co., Ltd. (Anhui, China). ELISA kits were purchased from Shanghai MLBIO Biotechnology Co., Ltd. QIAamp DNA stool mini kit was manufactured by TIANGEN (Germany). Other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

According to our previous methods, CSP-1 was isolated from Cereus sinensis (19, 21). After being soaked in NaCl solution (2%) to remove impurities, fresh Cereus sinensis was crushed and mixed with an equal volume of acetone for 12 h. The solution was filtered and freeze-dried to obtain degreased Cereus sinensis powder. The powder was mixed with distilled water at 72°C for 3 h and then precipitated with 3 times the volume of ethanol for 2 days. The above solution was centrifuged to obtain the precipitate, which was the crude polysaccharides. 50 mg/mL crude polysaccharide solution was mixed with 3% trichloroacetic acid for 12 h to remove protein. The solution was centrifuged, concentrated, dialyzed (3500D MWCO), and lyophilized. 5 mL of 20 mg/mL polysaccharide solution were further purified by column chromatography, involving DEAE-52 ion-exchange column and Sephadex G-100 column. 0.3 mol/L NaCl solution was used as eluate. The solution containing polysaccharides was collected, concentrated, dialyzed, and lyophilized to obtain CSP-1.

A UV-752 spectrophotometer (Qingdao Lubo Jianye Environmental Protection Technology Co., Ltd., Qingdao, China) was used to record the UV spectra of CSP-1 (scanning range 200–400 nm). FT-IR spectrometer (Tianjin Gangdong Technology Co., Ltd., Tianjin, China) was used to measure the FT-IR spectra of CSP-1 (scanning range 4,000–400 cm-1). The polysaccharide sample was mixed with dried KBr and then pressed into a thick pellet for FT-IR determination. The FT-IR and UV spectra of CSP-1 were shown in Supplementary Figure S1.

All experimental procedures were approved by the Committee for Animal Research of Shanghai Ocean University, China (SCXK (HU) 2007-0003). At the same time, animal experiments were carried out strictly following the Guidelines for the Care and Use of Laboratory Animals. Efforts were made to maximize the well-being of mice and minimize their suffering.

C57BL/6 male mice, 6–8 weeks, were obtained from the Shanghai Jiesijie Experimental Animal Co., Ltd and raised in a standard environment (12 h light-dark cycle, 22 ± 0.5°C, RH: 50 ± 5%) for 1 week. And then mice were randomly distributed into 6 groups with 3 mice per group. It was reported that short-term high-dose of LH will cause diarrhea in mice, which is mainly due to the disturbance of intestinal flora and inflammation of the intestinal tissues caused by LH (22). Therefore, LH is often used in the establishment of AAD models. Mice in the antibiotic-associated diarrhea group (MAAD) were gavaged with LH for only 3 days. Mice in the natural recovery group (MNR) were gavaged with LH for 3 days and then gavaged with physiological saline for the next 9 days. Mice in the Low- (MCL), medium- (MCM), and high- (MCH) dosage CSP-1 groups were gavaged with LH for 3 days and then gavaged with CSP-1 (MCL, MCM and MCH groups were 75, 150 and 300 mg/kg, respectively) for the next 9 days. Mice in the normal control group (MNC) were gavaged with physiological saline for 12 days. The dosage of LH in this experiment was 3 g/kg. Mice were gavaged twice a day during the experimental period. The experimental design was shown in Figure 1A.

The state of all mice was recorded daily, such as body weight, water intake, and diarrhea status. Diarrhea status was evaluated as previously reported (Table 1) (20). At the end of the experiment, mice were euthanized. The caecal specimens were temporarily immersed in formalin solution (10%) for histological observation. The caecal contents were collected and next stored at −80°C.

After overnight fixation, the samples of each group were embedded, sectioned and stained by Shanghai Viao Biotechnology Co., Ltd. Briefly, caecal specimens were prepared by ethanol dehydrating and paraffin embedding (23). The sliced (thickness of 5 μm) specimens were stained with hematoxylin and eosin. The sections were observed under a light microscope (40 ×) and images were obtained.

The blood was collected and then centrifuged at 2,000 rpm for 10 min. The serum was obtained by collecting the supernatants. The secretion of TNF-α, IL-1β, and IL-2 was measured using ELISA kits according to the corresponding instructions.

Pretreatment of caecal content was carried out as previously reported (24). 100 mg cecal contents were mixed with 0.4 mL of distilled water and centrifuged at 5,000 rpm for 20 min. The supernatant was mixed with 50% H₂SO₄ (0.2 mL) and 50 μg mL⁻¹ (1 mL) solution containing diethyl ether and 2-methylvaleric acid. The above solution was centrifuged at 12,000 rpm for 10 min and then placed at 4°C for 30 min. The upper liquid was used for the determination of SCFAs. Furthermore, standard solutions of SCFAs (acetate, propionate, isobutyrate, butyrate, 2-methylbutyrate, valerate and hexanoate) at different concentrations (5 μg mL⁻¹, 10 μg mL⁻¹, 50 μg mL⁻¹,100 μg mL⁻¹, 200 μg mL⁻¹, 400 μg mL⁻¹, 1,000 μg mL⁻¹) were prepared in ether. 2-methylpentanoic acid was mixed with diethyl ether as an internal standard solution. The SCFAs were detected using Agilent 7890A-5975C GC-MS System (Agilent, USA). The conditions were as follows: injection volume: 1 μL; carrier gas helium at a flow rate of 1 mL/min; the split ratio was 5: 1; injector temperature: 250°C. column temperature program: 100°C (maintained for 5 min) to 160 °C at 5°C/min; next increased to 240°C (maintained for 10 min) at 40°C/min.

DNA from the intestinal flora was obtained from the cecal contents using the DNA stool kit. PCR amplification of the V3-V4 region of bacterial 16S rRNA gene was carried out using forward primer (5′-ACTCCTACGGGAGGCAGCA-3′) and reverse primer (5′-GGACTACHVGGGTWTCTAAT-3′). Data can be obtained at NCBI with accession no. SUB9203775. The next procedures were the collection, purification, fluorescence quantification of amplification products and preparation of sequencing library. Sequencing of PCR products was performed by the Illumina MiSeq (Illumina, United States) sequencing platform at Personal Biotechnology Co., Ltd (Shanghai, China). The paired-end sequence was filtered using the sliding window method, then paired and connected using the FLASH software (v1.2.7), identified and further assigned to the corresponding sample to obtain the valid sequence for each sample (25). USEARCH (v5.2.236) was applied to filter the sequences obtained above and to cluster the superior reads into operational taxonomic units with a 97% similarity threshold. Using QIIME (v1.8.0) (26). Gut microbiota was analyzed by Illumina MiSeq sequencing based on QIIME analysis (27).

The data was presented as mean ± standard deviation (SD). A significant difference between groups was performed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests using SPSS statistics 17.0 (IBM, USA). Differences were considered significant at P < 0.05.

During LH intragastric administration, the mice showed a gradual increase in diarrhea status score and drinking water (Figures 1B,C). After 3 days, these mice exhibited 100% diarrhea. From the 4th day, the diarrhea status scores and water consumption of the MCL, MCM and MCH groups began to drop. As for the MNR group, the diarrhea status scores began to drop from the 4th day, while water intake began to decrease from the 5th day. On the 12th day, in terms of diarrhea status score and water consumption, the MNR group was obviously higher than the MNC group, while the MCL, MCM and MCH groups were similar to the MNC group. The bodyweight of the mice treated with the LH significantly decreased (Table 2). During the recovery period, the bodyweight of each group was gradually increasing. At the end of the experiments, the bodyweight of the mice in the MCL, MCM and MCH groups was higher than their starting weight, while the bodyweight of the mice in the MNR group was still lower than their starting weight.

The cecal tissue in MAAD group exhibited serious histopathological changes, including edema, massive inflammatory cell infiltration, as well as villus shortening, sparse and irregular arrangement (Figure 2). Compared with the MNR group, CSP-1 significantly reduced the inflammatory cell infiltration and edema of the cecal tissue. Moreover, the cecal villi of the MCL, MCM and MCH groups were longer, thinner and relatively regular arrangement. There were no obvious differences among the MCL, MCM and MCH groups.

LH treatment significantly stimulated the secretion of IL-2, IL-1β, and TNF-α in the serum of mice (Figure 3). Low, medium and high doses of CSP-1 significantly down-regulated levels of IL-2, TNF-α and high dosage of CSP-1 significantly decreased the level of IL-1β compared with the MNR group. No significant differences were observed among the MCL, MCM MCH, and MNC groups in the secretion of IL-2, IL-1β and TNF-α.

Mice treated with low, medium and high dosage CSP-1 showed significant enhancements in acetate and total SCFAs production, compared with mice of the MNR group (Figure 4). Furthermore, mice treated with medium and high dosage CSP-1 also showed significant enhancements in propionate and butyrate production. CSP-1 treatment and MNC groups had no significant difference in terms of the production of acetate, propionate, butyrate and total SCFAs.

Shannon and Chao 1 indexes were used to evaluate the diversity and richness of intestinal flora, respectively (Figures 5A,B). In terms of Shannon and Chao 1 indices, MNR, MCL, MCM and MCH groups were significantly higher than the MAAD group, while MCL, MCM and MCH groups were closer to the MNC group compared to the MNR group. In terms of Chao 1 index, MNC group was significantly higher than MNR group, while there was no significant difference among MCL, MCM and MNC group.

β diversity analysis (Figures 5C,D) showed that the MCL and MCM groups were closer to the MNC group than the MNR group. Meanwhile, the MNR group is closer to the MAAD group than the MCL, MCM, and MCH groups. PLS-DA analysis also demonstrated the structural changes of intestinal flora among the groups (Figure 5E). The significant separation between the MAAD and MNC groups indicated the successful establishment of the AAD model. Figure 5E also revealed that the MCL, MCM and MCH groups were closer to the MNC group than the MNR group.

The main components of each group included Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria at the phylum level (Figure 6A). However, their abundance was different. The MAAD group had significantly higher Firmicutes and lower Bacteroidetes and Proteobacteria than the MNC group. Compared with the MNR group, Bacteroides was enriched, while Proteobacteria and Firmicutes were down-regulated in the MCL, MCM and MCH groups. MNC, MCL, MCM and MCH groups in Firmicutes had no significant difference. Figure 6B showed the genera with the highest abundance in the MAAD group were Coprococcus. Compared to the MAAD group, CSP-1 significantly reduced Coprococcus and increased other bacterial communities in the MCL, MCM and MCH groups. Compared with MNR group, MCL, MCM and MCH groups had lower abundance of Bacteroides, Parabacteroides, Sutterella, Coprobacillus and higher abundance of Phasecolarctobacterium and Bifidobacterium (Figures 6C–H).

LH disrupted normal microbial metabolic pathways in the gut community of mice, like mice in the AAD group (Figure 7). Compared to the MNR group, amino acid metabolism, nervous system, translation, metabolic diseases were obviously enhanced and transcription, membrane transport was significantly weakened in three CSP-1 treated groups. At the same time, mice treated with a medium and high dosage of CSP-1 showed significant enhancements in transport and catabolism, glycan biosynthesis and metabolism. In addition, mice treated with a medium dosage of CSP-1 also showed significant enhancement in digestive system. Moreover, three CSP-1 treated groups and the MNC group did not show significant differences in transport and catabolism, transcription, membrane transport, digestive system, glycan biosynthesis and metabolism. Furthermore, CSP-1 also had good effects on regulating amino acid metabolism, cell motility and translation.