Protective effect of polysaccharides fractions from Sijunzi decoction in reserpine-induced spleen deficiency rats

Abstract

This work focuses on exploring the active polysaccharide fraction from Sijunzi decoction (SJZD), which is a classical herbal prescription in traditional Chinese medicine (TCM). Three polysaccharide fractions (S-1, S-2, S-3) were obtained from SJZD, the effects of which on the immune system (organic, cellular and molecular levels) and intestinal microbiota, as well as their metabolites (short chain fatty acids, SCFA) were investigated in reserpine-induced Wistar rat models. The results revealed that the spleen index, IL-2 and IFN-γ levels of S-3 group were significantly decreased compared to the model group, and the CD4+/CD8+ ratio displayed a significant increase in the S-3 group, indicating the immune enhancing effect of polysaccharide fraction S-3. Moreover, the disturbance of gut microbiota induced by reserpine was restored after administration of polysaccharide fraction S-3, indicated as the Shannon's diversity index and similarity coefficient index both increased significantly in S-3 group. In SCFA analysis, the content of acetic acid, propionic acid and butyric acid in faeces of S-3 group rats was also significantly increased after treated with S-3. All the results consistently indicated that polysaccharide fraction S-3 from SJZD could mitigate effect in the reserpine-induced rats and potentially treat spleen deficiency. The comprehensive screening strategy established in this study may provide a new idea to achieve further understanding of TCM.

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1. Introduction

Polysaccharide is a biological macromolecule which consists of many monosaccharides that are assembled by glycosidic bond polymerization. It is well known that polysaccharides have a variety of biological functions such as anti-tumor,¹ anti-inflammatory² and gastrointestinal diseases alleviating effects.³ The biological mechanism of these effects were found to be associated with the immune system. Recently more and more studies focus on the immune regulation effect of polysaccharide, particularly in the intestinal tract.^4,5 Extensive evidences have demonstrated that polysaccharide from herbs plays a role on gut mucosal immune regulation, which will affect the percentage of lymphocytes and the expression of intestinal cytokines.⁶

In addition, polysaccharide from diet enforces the microbial diversity in human intestine gut, which is important for maintaining the normal physiological state of the human body.⁷ It has been reported that Atractylodes macrocephala Koidz polysaccharide is primarily metabolized in the intestinal tract.⁸ The degradation of polysaccharide in the intestinal requires several functional microbe groups that interact with each other's nutrition chain to transform polysaccharide molecules to short chain fatty acids (SCFA) and gas.⁹ SCFA can enhance intestinal immunity by activating both intestinal immune switch and immune T cells;^10,11 however, due to poor bioavailability of polysaccharide, the mechanism of polysaccharide-mediated immune responses has not been fully understood and remains unknown.

Sijunzi decoction (SJZD), a traditional Chinese herbal prescription, is a classical prescription for curing spleen deficiency in traditional Chinese medicine (TCM). It is well known for treating disorders of endocrine immune system manifested in poor appetite, indigestion and watery stools or diarrhea.¹² It is also used as a tonic supplement for health maintenance.¹³ The ingredients of SJZD include four kinds of Chinese herbs, Ginseng Radix, Atractylodes Macrocephalae Rhizoma, Poria and Glycyrrhizae Radix. Polysaccharide is considered as the most abundant as well as the major effective component in SJZD.¹⁴ Polysaccharides from SJZD can affect the reaction of wounded intestinal epithelial cells,^12,15 improve the intestinal flora in spleen-deficient rats, and induce the local immune response in the intestinal mucosa. Ginseng fruits polysaccharide has a backbone mainly consisting of (1 → 6)-linked-Galp, (1 → 3, 6)-linked-Galp and (1 → 3, 6)-linked-Glcp residues, shows immune modulating activities in rats with Lewis lung carcinoma.¹⁶ Polysaccharide from Atractylodes Macrocephalae Rhizoma is known to regulate the gut microbes and is widely used in the treatment of chronic intestinal disease.⁸ The main component of Poria is polysaccharide β-(1 → 3)-D-glucan, shows better antioxidant activity.¹⁷ Polysaccharides from the roots of Glycyrrhizae Radix are strong antiadhesive systems, which may be used as potent tools for a further development of cytoprotective preparations with anti-infectious potential.¹⁸ However, the detailed spectrum of active polysaccharides from SJZD remains unclear.

Reserpine, one of the earliest drugs to be effective as central tranquilizing agent, was employed mainly for its action in the treatment for hypertension.¹⁹ Reserpine inhibits the vesicular monoamine transporter (VMAT2), and depletes the brain monoamines such as dopamine by interfering with storage capacity, and produces depression-like syndrome, low body temperature, bad appetite, inactiveness and diarrhea, gastric mucosal injury in animals. Because of its numerous adverse-effects, nowadays reserpine is rarely used in human treatments.²⁰ However, it has been proven to be a useful tool to produce the spleen deficiency animal models in TCM studies.²¹ In the theory of TCM, spleen deficiency is a common clinical syndrome and described as symptoms such as epigastralgia, flatulence after meal, lack of appetite, wilted complexion, loose stool, lassitude, fatigue, etc. It is a comprehensive manifestation of decreases in multiple systems, including digestion, absorption, energy conversion, and the immune system.²² Here, the spleen is a functional unit and a comprehensive conception of structure and function that includes not only the spleen in modern anatomy but also the pancreas and lymphatic system.

As mentioned above, the interaction between polysaccharides, intestinal microbiota and host immune system is important to the pharmacological efficacy of SZJD. In this study, we introduced a comprehensive method, which covers the assessment of immune system regulation, intestinal microbiota and SCFA, to screen for the active polysaccharide fraction from the SJZD by reserpine-induced rats. This comprehensive screening strategy would be useful for further understanding the physiological effects and therapeutic benefits of polysaccharide.

2. Materials and methods

2.1 Preparation and separation of total polysaccharide from SJZD

Ginseng Radix, Atractylodes Macrocephalae Rhizoma, Poria and Glycyrrhizae Radix were mixed in a ratio of 3 : 3 : 3 : 2 by weight. The botanical origins of the above Chinese herbs were identified by the corresponding author. Voucher specimens were deposited at the School of Pharmacy, Shanghai Jiao Tong University. SJZD was prepared as previously described²³ with following modifications. Dried herbs were boiled twice in distilled water and concentrated to 1 g mL⁻¹. The supernatant was centrifuged, and ethanol was then added to precipitate the crude polysaccharide. The precipitate was collected by centrifugation and deproteinated by the Sevage method.²⁴ The crude polysaccharide precipitate was shaken vigorously with chloroform and n-butanol (4 : 1) for five times and washed with ethanol, acetone, and ether sequentially. Finally, the total polysaccharides (SJZPS) were dissolved in distilled water and lyophilized.

2.2 Purification of polysaccharides in cellulose chromatography column (DEAE-52)

The crude the polysaccharides were dissolved and filtered through 0.45 μm filters, put into a DEAE-52 cellulose chromatography column and eluted with deionized water, 0.1 M NaCl, 0.2 M NaCl and 0.3 M NaCl at the flow rate of 1 mL min⁻¹. The proportion of eluents was determined using the phenol-sulfuric acid method²⁵ with glucose as the standard. The eluents were dialyzed in 3000 Da MWCO tubing in deionized water and concentrated to dryness.

2.3 Determination analysis of S-1, S-2 and S-3

Total carbohydrate content of fractions S-1, S-2 and S-3 was determined by phenol–sulfuric acid colorimetric method using glucose as the standard.²⁵ The protein content was measured by the Lowry method²⁶ with BSA as the standard. Uronic acid content was determined by m-hydroxybiphenyl colorimetric procedure with D-glucuronic acid as the standard,²⁷ and monosaccharide composition of S-1, S-2 and S-3 was determined by GC-MS.²⁸

2.4 Induction of spleen deficiency in rats

56 male Wistar rats (200 ± 20 g) were purchased from the SLAC Laboratory Animal Co., Ltd (Shanghai, China). Rats were kept in SPF-grade Experimental Animal Houses (Shanghai Jiao Tong University, China) with free access to food and water under standard temperature conditions (22 °C) and a 12 h light/dark cycle. All protocols involving animals were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University.

The rats were randomly divided into 7 groups (n = 8). Rats in the groups of model, atropine, S-1, S-2, S-3 and SJZTP were injected intraperitoneally with reserpine (purchased from Sigma-Aldrich St. Louis, MO) at 0.5 mg kg⁻¹ d⁻¹ for 10 days to induce spleen deficiency.²¹ Rats in the control group were injected with the same volume of saline. After induction, rats in S-1, S-2, S-3 and SJZTP groups were intragastrically given S-1, S-2, S-3 and SJZTP (200 mg kg⁻¹) for 7 days, respectively. Rats in atropine treated group were injected intraperitoneally atropine at 0.5 mg kg⁻¹ d⁻¹. Rats in control and model groups received distilled water.

About 1.0 g of fecal pellets per rat was directly collected from the anus into sterile plastic tubes on the 1^st, 10^th, and 17^th days of the experiment and stored at −20 °C. Rats were weighed and sacrificed by cervical dislocation on day 17, and spleens and thymuses were immediately removed and weighed. The thymus index (expressed as the thymus weight relative to the body weight), and spleen index (expressed as the spleen weight relative to the body weight) were measured.²⁹ Intraepithelial lymphocytes (IEL) were isolated from the proximal 2/3 of the small intestine by a modified version of the method described by Kearsey and Montufar-Solis.^30,31

2.5 IEL T cell subgroup and cytokines measurement

For IEL T cell subgroup measurement, IEL were incubated for 30 min at 4 °C with anti-CD4 or anti-CD8 antibodies (Abcam, Cambridge, UK). Subsequently, the cells were washed twice with PBS and resuspended in PBS. CD4+ and CD8+ T cell counts were determined by flow cytometry (FACSCalibur, Becton Dickinson, USA).

Additionally, IEL (1 × 10⁶ mL⁻¹) isolated from rats were seeded onto a 96-well plate in the presence of ConA (10 μg mL⁻¹) and cultured at 37 °C in 5% CO₂. After incubation for 4 h, cellular supernatants were collected and analyzed for levels of IL-2 and INF-γ using enzyme-linked immunosorbent assay (ELISA) kit (ShangHai Joyee Biotechnics Co. Ltd, China) according to the manufacturer's instructions.

2.6 16S rDNA PCR-DGGE

Total DNA of intestinal bacteria from faeces was extracted as previously described.³² The V3 region of the 16S rDNA gene for all bacteria was amplified with primers P3-GC-f (5′-CGCCCGCCG CGCGCGGC GGGCGGG GGGGG CACG GGGGG CCTACGGG AG GCAGCAG-3′) and P2-r (5′-ATTAC CGCGG CTGCTGG-3′). The PCR solution included 2 μL of purified DNA, 4 μL of each primer, 0.8 μL of Taq DNA polymerase, 4 μL MgCl₂, 4 μL of dNTP's (the biological reagents were obtained from Sangon Biotech Co., Ltd. Shanghai, China) and sterile water in a final volume of 50 mL. The PCR procedure was comprised of an initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s and extension at 72 °C for 30 s. Final extension was done at 72 °C for 10 min. Equivalent amounts of PCR products were run in 8% acrylamide gels in the DCode Universal Mutation Detection System (Bio-Rad, Hercules, CA). A denaturing gradient of 35–60% was prepared with the gradient delivery system (Model 475, Bio-Rad). Gels were run at 60 °C for 8 h at 120 volts and then stained with rapid silver staining method.

PCR-DGGE fingerprint of rats were analyzed by Tanon GIS2010 gel image processing system. Assessment of fingerprints was carried out by using the Shannon's diversity index and similarity coefficients.³³ Further, principal component analysis³⁴ was performed by the multivariate statistical analysis software package (multi-variate statistical package, MVSP) 3.13 package.

2.7 Gas chromatography for SCFA analysis

Standard solutions of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid and caproic acid were prepared at 1, 0.4, 0.2, 0.1, 0.05, 0.01, and 0.005 mg mL⁻¹ with ether (including internal standard 0.1 mg mL⁻¹), respectively. Faeces (0.4 g) was blended in water by vortex stirring grinder and centrifugalized at 5000 rpm for 20 min. Centrifugalized supernatant fluid (0.8 mL) was extracted from each sample. 0.2 mL 50% H₂SO₄ and 1 mL internal standard solution were added after vortex blending for 1 min, and the mixture was centrifugalized at 12 000 rpm for 10 min and placed in 4 °C for 30 min. The upper ether solution was taken for GC analysis.

Analysis was performed on a Shimadzu gas chromatograph (GC-2010, Shimadzu, Japan) equipped with a flame ionization detector and an Agilent DB-FFAP column (30 m × 0.25 mm × 0.25 μm). The instrument was monitored, and the chromatograms were acquired using GC solution software (Shimadzu). Based on the acquired GC data including standard curves, the SCFA in wet fecal matter is expressed as “micromoles of SCFA per gram of wet fecal matter”.

2.8 Statistical analysis

All data were expressed as mean ± standard deviation. Statistical analysis was performed with one-way analysis of variance (ANOVA) or paired t-test. Values of p < 0.05 were considered to be statistically significant.

3. Results

3.1 Physico-chemical characterization and monosaccharide component of major fractions from SJZD

As the complex structure of polysaccharide, the parameters such as total sugar content, percentage of uronic acid, total protein and monosaccharide composition are commonly used as quality index to control the quality of polysaccharide fractions. As shown in Table 1, the yield rate of major fractions S-1, S-2 and S-3 was 36.6%, 11.8% and 20.4% respectively, based on the dried SJZPS used. The total sugar content determined by phenol-sulfuric acid colorimetric was 99%, 87.5% and 67.5%, respectively; the percentage of uronic acid determined by m-hydroxybiphenyl colorimetric was 0.5%, 1.7% and 10%, respectively; and the total protein determined by the Lowry method was 0.2%, 3.4% and 11.2% for S-1, S-2 and S-3, respectively. Monosaccharide compositions determined by GC analysis showed that S-1 contained four types of monosaccharides (Ara, Man, Glc and Gal with the molar ratio of 0.8 : 4.4 : 93.1 : 1.6) and the other two acidic polysaccharides were composed of more than four kinds of monosaccharides. S-2 were composed of Rha, Ara, Man, Glc and Gal (1.4 : 5.3 : 1.6 : 83.5 : 8.1), and the proportions in S-3 were Rha, Ara, Xyl, Man, Glc and Gal with the molar ratio of 7.3 : 10.1 : 2.6 : 5.2 : 55.6 : 19.1. In addition, infrared absorption spectra of S-1, S-2 and S-3 were also measured. Please see ESI files (Fig. S1†).

Table 1 Physico-chemical characterization and monosaccharide component of major fractions from SJZD

Fraction	Yield (%)	Total sugar (%)	Uronic acid (%)	Total protein (%)	Rha	Ara	Xyl	Man	Glc	Gal
S-1	36.6	99.0	0.5	0.2	—	0.8	—	4.4	93.1	1.6
S-2	11.8	87.5	1.7	3.4	1.4	5.3	—	1.6	83.5	8.1
S-3	20.4	67.5	10	11.2	7.3	10.1	2.6	5.2	55.6	19.1

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3.2 Effects of polysaccharides fractions from SJZD on reserpine-induced rats

The animal model of spleen deficiency was established 10 days after the injection of reserpine in rats. The characteristic symptom of watery stools was clearly observed in the model rats, while other symptoms that were similar to the symptoms of spleen deficiency in human were also observed, such as humped back, narrow eyes, listlessness, inappetence and weight loss. After treated with polysaccharides (SJZTP, S-1, S-2, S-3), the rats showed relieved symptoms, suggesting that the positive influence of polysaccharides on reserpine-induced spleen deficiency in rats.

Moreover, Table 2 indicated the changes of rat weight at different periods during the experiment. Weight of rats in reserpine treated groups showed a significant decrease compared to that in control group from day 5 (p < 0.05). Administration of polysaccharides could impair this tendency of body weight decrease. At day 17, weight of rats in polysaccharide groups (SJZTP, S-1, S-2, S-3) increased significantly compared to that before polysaccharide administration (p < 0.05), while the weight loss in model group remained unchanged. These results revealed the effect of polysaccharides from SZJD on rats suffering from spleen deficiency.

Table 2 Weight of rats at the different periods of experiment (n = 8)

Group	1^st day (g)	5^th day (g)	10^th day (g)	17^th day (g)
a p < 0.05.b p < 0.01 compared with control group.c p < 0.01, compared with 10^th day in the same group.
Control group	194.12 ± 6.26	227.37 ± 8.31	255.50 ± 13.56	270.75 ± 13.85
Model group	195.68 ± 4.13	215.75 ± 9.48^a	217.18 ± 20.36^b	220.87 ± 21.95
Atropine group	192.62 ± 10.83	202.31 ± 15.02^b	193.93 ± 23.45^b	220.18 ± 16.22^c
Total (SJZTP treatment group)	197.06 ± 4.32	210.25 ± 19.01^b	206.56 ± 28.15^b	230.12 ± 26.11^c
S-1 treatment group	193.93 ± 4.59	213.25 ± 8.44^b	212.43 ± 16.01^b	234.31 ± 10.16^c
S-2 treatment group	193.56 ± 9.92	206.62 ± 11.85^b	196.62 ± 13.26^b	224.56 ± 9.87^c
S-3 treatment group	198.00 ± 10.30	212.50 ± 12.27^a	207.87 ± 14.97^b	235.56 ± 17.09^c

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3.3 Effects on immune system regulation in reserpine-induced rats of polysaccharides fractions from SJZD

The changes of immune organ index were listed in Fig. 1A. The spleen index of S-3 group was significantly decreased compared to the model group (p < 0.05). Although the index of SJZTP, S-1 and S-2 groups was lower than the model group, the differences were not statistically significant. There was no significant difference of thymus index among the groups, though there was a tendency of increase in the polysaccharides and the atropine administration groups compared to the model group.

Fig. 1 (A) Thymus index and spleen index in reserpine-induced rats. (B) CD4+ and CD4+/CD8+ ratio, (C) IL-2 and (D) INF-γ of small intestine endothelial lymphocyte in reserpine-induced rats. *p < 0.05, **p < 0.01 compared with model group. Control, control group; model, model group; atropine, atropine group; total, SJZTP treatment group; S-1, S-1 treatment group; S-2, S-2 treatment group; S-3, S-3 treatment group.

As shown in Fig. 1B, the percentage of CD4+ T-lymphocytes and the ratio of CD4+/CD8+ in model rats were both significantly lower compared with the control group (p < 0.05), indicating the disruption of immune response by reserpine injection. The percentage of the CD4+ T-lymphocyte in rats from S-1 and S-3 groups showed a significant increase than that in rats from the model group (p < 0.05). In addition, the CD4+/CD8+ ratio in S-3 group was significantly upregulated. These results indicated that the effect of polysaccharide fraction S-3 on immune cells was stronger than that of S-2 and atropine because there was only a minor tendency but not statistical significance of the restoration in S-2 and atropine groups.

The secretion of IL-2 and INF-γ from IEL in different group of rats was shown in Fig. 1C and D. The production of IL-2 and INF-γ from IEL in reserpine-induced rats was significantly increased (p < 0.05) compared with those in the control group. After treatment, the concentration of INF-γ in atropine and S-3 groups was significantly lower than that in model group (p < 0.05). Similarly, the concentration of IL-2 in S-2, S-3 and atropine groups was significantly lower than that in model group (p < 0.05), which indicated the regulating effect of S-3 on the secretion of IL-2 and INF-γ.

3.4 Effects of polysaccharides fractions from SJZD on intestinal microbiota in reserpine-induced rats

A remarkable difference in 16S rDNA PCR-DGGE fingerprint was found between normal rats and rats with spleen deficiency induced by reserpine. The number of bands was decreased after reserpine-induction, and the Shannon's index of the disordered model (2.25 ± 0.22, n = 8) was significantly lower than that of normal ones (2.76 ± 0.19, p < 0.05, n = 8). The altered profile and less diverse fingerprints indicated the status of spleen deficiency. The similarity (Cs) of 16S rDNA PCR-DGGE fingerprints of the same rat before and after reserpine induction decreased to approximately 45% in reserpine-treated groups, whereas it was 67% in control group that received distilled water (p < 0.05). The difference suggested that the constitution of intestinal bacterial community in reserpine-treated rats was significantly changed from that in normal rats. After polysaccharide treatment, Shannon's index of Total, S-1, S-2 and S-3 groups significantly increased compared with that before treatment; meanwhile, both polysaccharide treatment groups and S-3 groups showed significantly increased similarity coefficient than other groups (Fig. 2).

Fig. 2 (A) Shannon's diversity index and (B) similarity coefficient of 16S rDNA PCR-DGGE fingerprinting of different group rats.

*p < 0.05, **p < 0.01, compared with before reserpine intraperitoneal. ^#p < 0.05, ^##p < 0.01, compared with after reserpine intraperitoneal. ^☆p < 0.05, ^☆☆p < 0.01 compared with control group, ^★p < 0.05, ^★★p < 0.01 compared with model group. Control, control group; model, model group; atropine, atropine group; total, SJZTP treatment group; S-1, S-1 treatment group; S-2, S-2 treatment group; S-3, S-3 treatment group.

Moreover, multivariate statistical analysis was adopted to 16S rDNA PCR-DGGE fingerprint with principal component analysis (PCA). PCA results of 56 rat fecal samples collected after polysaccharide treatment (Fig. 3) showed that the samples from control group gathered in the third quadrant, the samples from model group concentrated in the fourth quadrant, and samples from other treatment groups dispersed in the control and model quadrant. According to the analysis, S-3 group was the most similar to the control group, indicating that the disturbance of gut microbiota induced by reserpine was restored after administration of polysaccharides fraction S-3.

Fig. 3 Principal component analysis (PCA) of fecal bacterial 16S rDNA PCR-DGGE fingerprint from 56 treatment rats.

Control, control group; model, model group; atropine, atropine group; total, SJZTP treatment group; S-1, S-1 treatment group; S-2, S-2 treatment group; S-3, S-3 treatment group.

3.5 Effect of polysaccharides fractions from SJZD on SCFA in feces of reserpine-induced rat

After reserpine injection (Table 3), the SCFA content (mg mL⁻¹) was found to decrease in rat feces; the level of acetic, propionic acid, butyric acid was significantly lower than that before injection. After polysaccharide treatment (17^th day), the SCFA content in feces tended to increase compared with that collected at 10^th day before treatment. Only acetic acid and butyric acid contents increased after S-2 administration, but the level of acetic acid, butyric acid and propionic acid all went up significantly after S-3 treatment, indicating a stronger effect of S-3 on SCFA regulation in gut.

Table 3 SCFA content of rat feces (mg mL⁻¹)^a

Group	Acetic acid			Propionic acid			Butyric acid
	1^st day	10^th day	17^th day	1^st day	10^th day	17^th day	1^st day	10^th day	17^th day
a Statistical significance of differences was calculated by paired t-test, the data are expressed as mean ± SD.b p < 0.05 compared with 1^st day.c p < 0.05 compared with 10^th day.
Control	0.24 ± 0.02	0.24 ± 0.01	0.24 ± 0.02	0.15 ± 0.04	0.18 ± 0.02	0.17 ± 0.04	0.24 ± 0.05	0.25 ± 0.01	0.20 ± 0.02
Model	0.25 ± 0.05	0.10 ± 0.06^b	0.14 ± 0.02	0.16 ± 0.02	0.07 ± 0.04^b	0.08 ± 0.02	0.24 ± 0.02	0.11 ± 0.03^b	0.14 ± 0.04
Atropine	0.23 ± 0.02	0.13 ± 0.02^b	0.12 ± 0.03	0.16 ± 0.02	0.08 ± 0.02^b	0.11 ± 0.03	0.22 ± 0.01	0.10 ± 0.01^b	0.18 ± 0.02
Total	0.25 ± 0.04	0.14 ± 0.03^b	0.13 ± 0.02	0.15 ± 0.02	0.09 ± 0.01^b	0.10 ± 0.02	0.25 ± 0.02	0.11 ± 0.01^b	0.21 ± 0.03
S-1	0.25 ± 0.03	0.14 ± 0.03^b	0.15 ± 0.04	0.15 ± 0.02	0.09 ± 0.02^b	0.11 ± 0.03	0.25 ± 0.03	0.11 ± 0.01^b	0.23 ± 0.02^c
S-2	0.21 ± 0.01	0.14 ± 0.01^b	0.18 ± 0.01^c	0.13 ± 0.01	0.09 ± 0.01^b	0.10 ± 0.02	0.25 ± 0.02	0.10 ± 0.03^b	0.23 ± 0.01^c
S-3	0.22 ± 0.02	0.13 ± 0.01^b	0.19 ± 0.01^c	0.13 ± 0.01	0.08 ± 0.02^b	0.15 ± 0.02^c	0.26 ± 0.04	0.12 ± 0.02^b	0.24 ± 0.01^c

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4. Discussion

Reserpine administration can exhaust catecholamines in vivo, which suppresses the activity of adrenergic nerve and activate the parasympathetic nerve. As a result, reserpine administration in rats can induce depressive behavior, remarkable hypothermia and impaired parasympathetic function.^21,35 These changes are often accompanied by intestinal imbalance, metabolic disorders and immune function decline, which are similar to spleen deficient patterns. Therefore, reserpine was often used to induce the spleen deficient model in TCM. It is reported that Sijunzi formula reversed the increase of TNF-α and decrease of TGF-β in jejunum of spleen deficiency rats induced by reserpine to normal level significantly.⁶ In our study, we utilized the rats with reserpine induced spleen deficiency to investigate the active polysaccharide fraction from the SJZD, using a comprehensive screening strategy covering the assessment of immune system regulation, intestinal microbiota and SCFA.

Spleen and thymus are important immune organs in mammals. They are the sites for the growth and proliferation of immunological cells. The development status of immune organs directly affects the immune function and the ability of the body to resist disease. Therefore, changes of the spleen and thymus index can reflect the development of immune organs and the health of immune function.³⁶ The results of our study showed that the spleen index in S-3 group rats was significantly lower than that in the model group, which indicated the regulating effects of polysaccharide fraction S-3 on the development of immune organs. Moreover, S-3 was demonstrated to affect CD4+ and CD4+/CD8+ IEL of reserpine treated rats and reduce the levels of INF-γ and IL-2 in IEL. It is reported that high level of CD4+ and CD4+/CD8+ IEL are correlated with the enhanced immune system.³⁷ IEL seem to play an important role in the immune surveillance of the intestine, although their function is not fully understood yet.^38,39 Our results suggested that polysaccharide fraction S-3 could enhance the function of immune system, through either immune organ or molecular level and cellar level of intestinal immune function.

More than a thousand kinds of microorganisms are present in human digestive tract, and they play an important role in promoting the growth and development of the host, supporting vitamin synthesis and metabolism, balancing the immunity and inhibiting pathogenic bacteria colonize.⁴⁰ The injection of reserpine disturbed the balance of intestinal flora after ten days, resulting in reduction of Shannon's index and similarity coefficient of rat fecal microbiota. After the treatment of polysaccharide fraction S-3, Shannon's index and similarity coefficient of rat fecal microbiota both increased, suggesting that the disturbance of intestinal flora caused by reserpine was restored. In addition, SCFA analysis in our study demonstrated that polysaccharide fraction S-3 could significantly upregulate the level of acetic acid, propionic acid and butyric acid content in faeces samples from reserpine treated rat, which are produced by intestinal probiotics and good for health. SCFA are organic acids with 1–6 carbon atoms, which is mainly composed of anaerobic bacteria glycolysis indigestible carbohydrates in lumen, with acetic acid, propionic acid, butyric acid as main ingredients. Clinical studies have shown that SCFA is essential to maintain the intestinal morphology and function, and SCFA is used to ease or treat various intestinal diseases.⁴¹ The intestinal imbalances can result in the reduced level of butyric acid, and subsequently lead to the reduced expression of epithelial tight junction protein and increased epithelial permeability. Dysfunction of epithelial barrier will lead to increased bacteria migration through lamina and decreased concentration of IgA and other defensing elements.⁴² Yukihiro Furusawa found that bacteria can be induced to produce butyrates, which are considered as the epigenetic switch to activate immune T cells and enhance immunity function, by carbohydrates in dietary intestinal.¹⁰ According to our results and the literature, we could hypothesize that polysaccharide fraction S-3 from SJZD increases the amount of intestinal probiotics, and then upregulates SCFA including acetic acid and butyric acid produced by intestinal probiotics. The SCFA, acting as the intestinal immune molecular switch, will initiate the intestinal immune response and enhance the body's immune function.

5. Conclusion

In conclusion, an active polysaccharide fraction (S-3) from SJZD, which can improve the immune function of intestinal endothelial lymphocyte, up-regulate the level of SCFA in faeces and mitigate the spleen deficiency symptoms in reserpine-induced rats, was identified using the novel comprehensive screening strategy. Therefore, this fraction could potentially treat spleen deficiency syndrome.

6. Declaration of interest

The authors report no declarations of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 30973962 and 81473318).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra06361f

‡ Contributed to this paper equally.

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