Antibiotics are widely used in the world, saving countless lives and making great contributions to humanity. However, the side effects caused by nonstandard use of antibiotics have attracted more and more attention, especially diarrhea. Antibiotic-associated diarrhea (AAD) is a common phenomenon in antibiotic treatment [1
], accompanied with changes of composition and diversity of gut microbiota, destruction of the gut structure, and dysbiosis of the gut environment, which might aggravate the ill process and is harmful for the recovery of patients.
Traditional Chinese medicine (TCM) has been widely used to treat many diseases as a supplemental or alternative medicine in Asian countries. Astragalus membranaceus
) root, a plant commonly used in TCM, contains various bioactive compounds, such as flavones, saponins, and polysaccharides, which are always used as TCM to treat diarrhea. Scientific evidence shows that polysaccharides in Astragalus
have multiple biological activities, including anti-cancer [2
], immune-modulatory [3
], anti-inflammatory [4
], renal protective [5
], antioxidant, antidiabetes, and cardioprotective activities [6
]. It has been reported that polysaccharides from Astragalus
(APS, MW, ~3.6 × 104
Da) have α-(1→4)-D-glucan chain that contains one α-D-glucose at the C-6 position for every nine residues, which have shown anti-gastric cancer activity in rats [8
]. Another report about the Astragalus
polysaccharides APS-I and APS-II is composed of α-(1→3) glucose and 1→4, 1→6 glucose in the main chain, and with arabinose and xylose serving as the side chains; both APS-I and APS-II can inhibit tumor growth [9
]. Other studies also reported that polysaccharides and oligosaccharides from TCM can alter the gut microbiota as well as maintain its homeostasis [10
]. However, there are little reports on the effect of Astragalus
polysaccharides in antibiotic-associated diarrhea.
In this study, we established an AAD rat model containing the composition and diversity of the gut microbiota, colon structure, short-chain fatty acid (SCFA) metabolites, and various metabolic processes. This model enables us to investigate the effects of Astragalus polysaccharides on AAD.
2. Materials and Methods
Astragalus membranaceus was collected from Fusong, Jilin, China and characterized by Professor Yinshi Sun. Lincomycin hydrochloride was purchased from CR Double-Crane Pharmaceuticals Co., Ltd. (Jinan, China). The TIANamp Stool DNA Kit (cat. No. DP328) was obtained from Tiangen Biotech Co., Ltd. (Beijing, China). Acetate, propionate, and butyrate were purchased from Sigma-Aldrich Co. (Darmstadt, Germany). All other chemicals and reagents were obtained from Sinopharm Group (Shanghai, China).
2.2. Extraction of Astragalus Polysaccharide
The dried roots of Astragalus (500 g) were suspended in 6 L of distilled water and heated at 100 °C for 3 h. The solution was cooled, filtrated, and the heating step was repeated. The solution was collected, concentrated to 1 L at 60 °C and centrifuged (4500 rpm, 10 min). Afterward, the solution was mixed with four volumes of anhydrous ethanol and centrifuged (4500 rpm, 10 min). The precipitates were collected and dissolved in 800 mL of distilled water, followed by an additional 3.2 L of anhydrous ethanol. The precipitates were dissolved in 800 mL distilled water, Sevag reagent (Chloroform: n-butyl alcohol = 4:1, v:v) was used three times to remove the protein layer, and then the precipitates were freeze-dried to yield Water-soluble Astragalus Polysaccharides (WAP).
2.3. Physiochemical Analysis of WAP
The concentrations of carbohydrate, uronic acid, and monosaccharide composition were determined as previously reported [12
]. Protein was determined using a Dumas nitrogen analyzer (NDA 701) [15
]. The molecular weight was estimated with high-performance gel permeation chromatography (HPGPC) on a TSK-gel G-3000PWXL column (7.8 mm × 300 mm, TOSOH, Tokyo, Japan) coupled with a Shimadzu HPLC system.
The FT-IR spectrum was recorded with a KBr pellet among wave lengths 500 and 4000 cm−1 on a NEXUS670 FT-IR spectrophotometer.
The 13C NMR spectrum was obtained on a Bruker AVIII spectrometer at 600 MHz. The sample (30 mg) was dissolved in D2O (1 mL, 99.8%), and the spectra were recorded at 25 °C. Acetone was used as an internal standard.
WAP was added to a silicon plate with a thin-layer gold sputter-coated, and then synthesized hydro gel (CMH and NMH3) was freeze-dried and covered with gold before the analysis of scanning electron microscope (SEM). The three-dimensional structure of WAP was characterized on XL 30 ESEM (Philips).
2.4. Animals and Treatment
The present study was reviewed and approved by the Animal Care and Use Committee of the Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences (Ethical approval code: TCS2017021, January 2017). We used a total of 24 male Wistar rats (180 ± 20 g), which were purchased from Changsheng Laboratory Animal Technology Co., Ltd. (Beijing, China). Rats were maintained at a temperature of 22 ± 0.5 °C, a humidity of 50 ± 5%, and light: dark cycles of 12 h:12 h, and had free access to standard laboratory pellets and water. All animals were treated following the Guidelines for the Care and Use of Laboratory Animals recommended by the Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences and the Chinese Legislation on Laboratory Animals. The well-being of all animals was ensured throughout the study, and a minimal number of animals were used.
2.5. Experimental Design
The rats were acclimatized for 7 days and then randomly divided into four groups (n
= 6/group): the control (C) group, antibiotic-associated diarrhea (DM) group, natural recovery (NR) group, and WAP treatment (WAP) group. To establish the ADD model, rats on the DM, NR, and WAP groups received lincomycin hydrochloride (10 mL/kg) twice a day for 4 days by gavage [16
], whereas rats in the C group received an equivalent amount of physiological saline. The rats of the DM group were anesthetized with isoflurane using a small animal anesthesia machine. Blood samples were collected and centrifuged (1500 rpm, 10 min) to obtain the serum. Fecal contents (>0.5 g) were collected under sterile conditions and stored at −80 °C. Colon samples were fixed in 10% neutral formalin. Afterward, the rats were euthanized with CO2
The rats of the WAP group continued to receive WAP (100 mg/kg) once a day for 7 days, whereas the rats of NR and C groups received an equivalent volume of physiological saline. After recovery, blood and colon samples, as well as fecal contents, were collected as described above.
2.6. Histological Analysis
Colon samples were fixed in 10% formalin, dehydrated in ethanol, embedded in paraffin, sectioned (4–5 μm), and stained with hematoxylin and eosin (HE). The sections were observed under an Olympus BH22 Microscope (Tokyo, Japan).
2.7. Microbiota Analysis
DNA extraction, PCR amplification of the V3–V4 regions of 16S rRNA genes, and sequence data analyses were performed as previously reported [17
Operational taxonomic units (OTUs) were compared using the quantitative insights into microbial ecology (QIIME) platform, R software (ver. 3.2.0), and the Greengenes database [18
]. Alpha diversity, including Chao 1 and Shannon indices, were calculated to compare the richness and diversity between groups [19
]. Beta diversity was calculated using nonmetric multidimensional scaling (NMDS) to identify variations in the microbial communities [21
]. Metastats Software was used to compare the abundances of taxa at phylum, class, order, family, and genus levels between samples or groups [22
]. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis was used to identify the microbial communities based on high-quality sequences [23
], in order to obtain the results of functional prediction of gut microbiota. All raw sequences were deposited into the NCBI Sequence Read Archives (SRP238192).
2.8. Measurement of SCFAs
The fecal contents of each rat were collected, and SCFAs were analyzed as previously reported [17
]. The caecal content (100 mg) of each rat was placed into a centrifuge tube and then dissolved with 10 μL of 15% ortho-phosphoric acid, 100 μL of adipic acid (50 μg/mL, internal standard) solution and 400 μL of ether. The mixture was whirled for 1 minute and centrifuged (12,000 rpm/min, 10 min) at 4 °C, and then the supernatant was filtered through a 0.45 μm organic sample compatible membrane filter and used for the assay. Standard solutions of acetate, propionate and butyrate at different concentrations were prepared in ether. All assays were performed using the Agilent 6890N/5975BGC-MS System (Agilent, Santa Clara, CA, USA). The separation of each compound was achieved using an Agilent HP-INNOWAX capillary column (30 m × 0.25 mm, 0.25 μm). The initial oven temperature was 90 °C, which was maintained for 3 min and then increased to 120 °C by 10 °C/min, 150 °C by 5 °C/min, and finally 250 °C by 25 °C/min, then it was maintained for 2 min. The temperature of the ion source and injection port was set at 230 °C and 250 °C, respectively. One microliter solution was injected. The flow rate of helium was 1 mL/min with a 10:1 split ratio. Electron bombardment ionization (EI) source with a full scan and a sim scanning mode were adopted for mass spectroscopy, while the electron energy was 70 ev.
2.9. Statistical Analysis
All data were expressed as means ± standard deviation (S.D.). Statistical analyses were performed using Prism 5 Software. Comparisons between groups were performed using one-way analysis of variance (ANOVA) with Duncan’s range tests. Differences were considered significant when p < 0.05.
For the past 2000 years, plants in China have been used as herbal supplements and medicines to improve human health and treat various diseases [24
]. The multiple uses, components, and targets of these plants have attracted the interest of investigators [25
]. However, the active components of many plants remain unknown. The study of interactions between the active components of plants and gut microbiota has opened many new insights into the exploration and functions of natural resources. Several studies have focused on the effects of chemical components on the gut microbiota of the mammalian host in health and disease models [28
]. Gut microbiota is important for host health since it affects several processes, including the host’s metabolism, shapes systemic immunity, maintains gastrointestinal tract homeostasis, and affects brain function and behavior. Gut microbiota can also metabolize various active components and produce metabolites with variable bioactivity or toxicity that can affect microbial communities in the gut.
Polysaccharides are widely distributed in plants and have been reported to modulate the gut microbiota under various conditions [31
]. Polygonatum kingianum
polysaccharides can improve the gut microbial environment in diabetic rats [22
]; Ganoderma lucidum
polysaccharides can affect the gut microbial environment in mice with chronic pancreatitis [32
]; Panax ginseng
] polysaccharides can modulate the gut microbial environment in mice with antibiotic-associated diarrhea; and Dictyophora indusiata
] can promote recovery from antibiotic-driven intestinal dysbiosis and improve gut epithelial barrier function in mice.
is used as a traditional medicinal plant in China. In this study, polysaccharides from Astragalus membranaceus
were obtained, and one AAD rat model was established to investigate the effects of Astragalus
polysaccharides on AAD. At the genus level, there was a significant decrease in the richness of Adlercreuzia
, and Treponema
, but an increase in the richness of Clostridium
, and Pseudomonas
in the DM group compared with the C group. WAP treatment could significantly improve the richness and diversity of the gut microbiota in AAD rats. Compared with the DM group, WAP increased the richness of Bacteroidetes and Proteobacteria but decreased the richness of Firmicutes, which suggested that WAP could restore microbial species at the phylum level. Compared to physiological saline treatment, the relative abundance of Pseudomonas
was significantly increased, whereas that of Allobaculum
was decreased in the WAP group. Both physiological saline and WAP could reduce the abundance of Pseudomonas
compared with AAD rats. However, WAP could reduce the richness of Allobaculum
to a level lower than in NR rats, and closer to that in C rats, which could maintain the healthy status of gut microbiota. It can be concluded that WAP can improve AAD by modulating the composition and diversity of the gut microbiota. Compared with the polysaccharides we have extracted from Schisandra chinensis
], which could increase the relative abundance of Blautia
, and Lachnospiraceae
, but decrease the relative abundance of Ruminococcus-1
, and Erysipelatoclostridium
at the genus level, suggesting that different polysaccharides can alter the gut microbiota differently, which is possibly due to the variations of monosaccharide composition, fine structure, and space conformation. Although the mechanisms responsible for balancing the gut microbiota by WAP, WSP, or other polysaccharides from plant resources need more research, our results might offer some data and references on similar research.
The changes in the colon structure and SCFA concentrations were also associated with antibiotic treatment, and they reflected the status of the gut environment. In this study, rats of the DM group showed an abnormal colon structure and decreased concentrations of acetate, propionate, butyrate, and total SCFAs, suggesting that the AAD rat model was successfully established. Compared to the NR group, WAP could restore the colon structure and increase the concentrations of propionate, butyrate, and total SCFAs. These results indicated that WAP promoted a healthy gut structure and normal SCFA concentrations. Many studies have investigated the concentrations of SCFAs, which are derived from polysaccharides [31
] and mainly composed of acetate, propionate, and butyrate. SCFAs have been reported to participate in the host metabolism as important signaling molecules [35
]. They are also important to the gut microbial environment and closely involved in immune, anti-tumor, and anti-inflammatory activities [36
]. Generally, SCFAs have beneficial effects on hosts. We found that WAP significantly improved the concentrations of propionate, butyrate, and total SCFAs in the caecum of treated rats compared to NR rats. These findings were different from those on Schisandra chinensis
polysaccharides, which increased the concentrations of acetate, propionate, and total SCFAs. Therefore, variable polysaccharide structures can affect the fermentative activity of different bacteria in the intestinal tract to produce different SCFAs. Our results also indicate that WAP could improve the gut environment by producing SCFAs.
Gut dysbiosis is associated with many diseases and metabolic syndromes [38
], such as diabetes, obesity, inflammatory bowel disease, fatty liver disease, and liver cirrhosis. Therefore, it is crucial to maintain a healthy gut microbial environment. Polysaccharides can alter the composition of the intestinal microbial community, and this phenomenon may offer new insights into the mechanisms involved and how natural active components can treat disease. Therefore, the purification of WAP was carried on in our research group in order to obtain a homogeneous polysaccharide fraction for further study of the underlying molecular mechanisms and structure-activity relationship.
In conclusion, WAP was mainly composed of glucose, galactose, arabinose, and glacturonic acid. There were glucan, arabinogalactan and RG-I regions in WAP, and it showed a loosely irregular sheet conformation. WAP exerts beneficial effects on rats with AAD by restoring the gut structure, improving the diversity, composition, and metabolic function of the gut microbiota, and increasing the production of SCFAs. Compared to rats of the NR group, WAP adjusted the relative abundance of Pseudomonas, Allobaculum, and Coprococcus at the genus level; significantly increased the concentrations of propionate, butyrate and total SCFAs; and recovered the metabolic processes of gut microbiota. These results indicate that WAP could serve as a potential natural product to ameliorated AAD, and the effects might be associated with WAP’s modulating effects on gut microbiota.