The herb residue of Jianweixiaoshi tablets was obtained from Jiangsu Yangtze River Pharmaceutical Group Company, Ltd. (Taizhou, China) and homogenized for 2 h using a pulper. Bacillus subtilis (isolated from Douchi and stored in Professor Chen's laboratory at Nanchang University), Aspergillus oryzae (isolated from Douchi and stored in Professor Chen's laboratory as above) and Lactobacillus plantarum M3 (isolated from sourdough and stored in in Professor Chen's laboratory as above) bacteria (all 10⁸ colony forming U/ml) were used as the inoculum for the preparation of the herb residue fermentation supernatant. B. subtilis and A. oryzae were added to the herb residue for 24 h, and L. plantarum M3 was added for 24–36 h at 37°C. Fermented products were subsequently centrifuged for 30 min at 1,000 × g at 4°C in a refrigerated centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd., Changsha, China) to obtain the fermentation supernatant.

The present study was approved by the Ethical Committee of the Second Affiliated Hospital of Nanchang University (Nanchang, China) and all methods were performed in accordance with the approved guidelines.

A total of 30 specific pathogen free 6–8-week-old male C57BL/6 mice weighting 20–30 g were housed and fed a commercial diet with water ad libitum. All mice were purchased from the Hebei Center for Disease Control and Prevention (Hebei, China), housed five per cage, on a 12-h light/dark cycle at 23±2°C and at 50±10% relative humidity. To establish the spleen-deficient mice model, 20 ml/kg/day 100% rhubarb decoction (supplied by Jiangxi Provincial Hospital of Traditional Chinese Medicine, Jiangxi, China) was administered intragastrically to mice for 7 days. All control animals were treated with the equivalent volume of PBS. Subsequently, model mice were divided into 3 groups: i) The modeling group (n=10), in which mice were only administered PBS; ii) the probiotics + drug residues group (n=10), in which mice were administered herb residue fermentation supernatant (0.1 ml/20 g); and iii) the Jianweixiaoshi tablets group (n=10), in which mice were administered Jianweixiaoshi tablets (0.1 ml/20 g). Mouse feces were collected at four time-points: i) During the control stage, on day 0, prior to treatment; ii) during the modeling stage, on day 7, following treatment with rhubarb decoction; iii) during the treatment stage, on day 14, following drug treatment; and iv) during the recovery stage, on day 21. In each group, feces of three mice from the modeling, probiotics + drug residue and Jianweixiaoshi tablets groups were randomly selected and used for denaturing gradient gel electrophoresis (DGGE) analysis.

A total of 24 h following the final drug administration, five animals in each group were sacrificed by decapitation. Tail vein method was used to obtain the whole blood, and serum was obtained by the centrifugation of clotted blood at 2,500 × g for 15 min at 4°C, and interleukin (IL)-2, IL-4 and interferon-γ (IFN-γ) were determined using ELISA kits for IL-2 (88-7024-88; Mouse IL-2 ELISA Ready-SET-Go!), IL-4 (88-7044-22; Mouse IL-4 ELISA Ready-SET-Go!) and IFN-γ (BMS606; Mouse IFN-γ Platinum ELISA; all from eBioscience; Thermo Fisher Scientific, Inc., Waltham, MA, USA) (15). Spleen and thymus were harvested from mice and weighed immediately. Thymus and spleen indices were calculated according to the following formula: Thymus or spleen index = [(weight of thymus or spleen)/body weight] × 100.

Fresh fecal samples were subjected to treatment within 2 h following collection. All samples were serially diluted 10-fold with saline solution and 300-µl solutions resulting from each dilution were separately plated on a brain-heart infusion agar supplemented with 10% sterile skimmed milk (Beijing Land Bridge Technology Co., Ltd., Beijing, China) for total anaerobic bacteria, and incubated anaerobically at 37°C for 36 h. De Man, Rogosa and Sharpe agar (Oxoid; Thermo Fisher Scientific, Inc.) was used for anaerobic culture of Lactobacilli at 37°C for 24 h. Enterococci were aerobically cultured at 37°C for 24 h on Slanetz-Bartley medium agar and MacConkey agar (both from Oxoid; Thermo Fisher Scientific, Inc.) was used for aerobic culture of Enterobacteria at 37°C for 24 h (16–18).

DNA was isolated using bead-beating method (19). Following phenol-chloroform extraction, DNA was precipitated with 75% ethanol and resuspended in 50 µl TE buffer (10 mM Tris-Cl, 1 mM EDTA; pH 7.6). Primers, including 357 forward (5′-TACGGGAGGCAGCAG-3′) and 519 reverse (5′-ATTACCGCGGCTGCTGG-3′), were used to amplify total bacterial DNA, and Lac1 (5′-AGCAGTAGGGAATCTTCCA-3′) and Lac2 (5′-ATTYCACCGCTACACATG-3′) were used to amplify DNA from Lactobacillus species. GC clam-in primers were selected to generate GC-enriched polymerase chain reaction (PCR) products suitable for separation by DGGE. Subsequently, a PCR was performed using the Taq DNA Polymerase kit (Takara Biotechnology Co., Ltd., Dalian, China) in a Biosci PCR system, with 30 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 60 sec. Amplicons of 16S ribosomal RNA were used for sequence-separation by DGGE, as previously described (20,21). DGGE was performed on 8% polyacrylamide gels containing acrylamide, bisacrylamide, formamide and a gradient of 35–65% urea. Tris-HCl (40 mM; pH 8.0) was used as the electrophoresis buffer in a Bio-Rad DGGE system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Electrophoresis was initiated by a pre-run for 5 min at 220 V followed by run at a fixed voltage of 85 V for 16 h at 60°C. Gels were stained with AgNO₃ and developed following electrophoresis. Subsequently, gels were covered with cellophane membranes and dried overnight at 4°C. DGGE patterns were subsequently normalized and analyzed using Bionumeric software (version 2.0; Applied Maths, Sint-Martens-Latem, Belgium). During the processing, lanes were defined and the background was subtracted. In the process of normalization, differences in the intensity of the lanes were compensated and the association matrix was calculated. Clustering was performed using the Pearson correlation and unweighted pair group method with arithmetic mean (UPGMA) methods.

Bands of interest were excised from the gel using a sterile blade and incubated overnight at 4°C in Tris-EDTA buffer (pH 8.0) to allow for DNA diffusion out of the polyacrylamide matrix. The solution was used directly for further amplifications. For sequencing, eluted DNA was amplified using the same primer pairs and conditions as described above, without the GC clamp. PCR products for sequencing were purified using a QIAquick PCR purification kit (Qiagen GmbH, Hilden, Germany). PCR products were subcloned using the pMD18-T vector system I (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. E. coli DH5α cells (Beijing Tiangen Biochemical Technology Co., Ltd., Beijing, China) were subjected to electrotransformation with recombinant plasmids. Selection of transformants was performed on lysogeny broth agar (Oxoid; Thermo Fisher Scientific, Inc.) containing 100 mg/ml ampicillin. Transformants were randomly selected and sequenced by Invitrogen (Thermo Fisher Scientific, Inc.) (22–24).

Data are presented as the mean ± standard deviation. Data were analyzed using SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA) using one-way analysis of variance with the least significant difference post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Compared with the control group, treatment with rhubarb reduced body weight, spleen index and thymus index (Fig. 1) in the modeling group, and presented depressive behavior, constipation, unkempt fur and poor appetite. Treatment with herb residue fermentation supernatant and Jianweixioashi tablets resulted in an increase in body weight, spleen index and thymus index, although these differences were not statistically significant.

Treatment with rhubarb decreased the levels of IL-2, IL-4 and IFN-γ in the modeling group, compared with the control group. Administration of probiotics with herb residues significantly increased levels of IL-2, −4 and IFN-γ, compared with the modeling group (all P<0.05; Fig. 2). Jianweixioashi tablets significantly increased levels of IL-4, compared with the modeling group (P<0.05).

The effects of the fermentation supernatant on the viable number of Enterococci, Enterobacteria and Lactobacilli were determined in the present study. Viable cell counts indicated that, during the treatment stage, fermentation supernatant and Jianweixioashi tablets increased the number of Lactobacilli, and reduced the number of Enterococci and Enterobacteria, compared with the modeling group (all P<0.05; Fig. 3). During the recovery stage, fermentation supernatant significantly inhibited the growth of Enterococci and Enterobacteria, and promoted the growth of Lactobacilli, compared with the modeling group (P<0.05).

The results of the DGGE analysis indicated that an uncultured bacterium, L. murinus, and Enterococcus sp. were the dominant bacteria in all groups throughout the experiments. In addition, the DGGE profile indicated that treatment with rhubarb markedly reduced the band number, while the administration of fermentation supernatant and Jianweixiaoshi tablets enhanced the bacterial diversity in the treatment and recovery stages (Fig. 4; Table IA). However, the results of the UPGMA analysis demonstrated low levels of similarity between bacteria in the control, treatment and recovery stages in the probiotics + drug residues and Jianweixiaoshi tablets treatment groups.

As demonstrated by Bacillus DGGE profiles, treatment with rhubarb induced an effect on L. murinus, and uncultured bacteria d and f (Fig. 5). L. murinus was the dominant bacterium in all groups and during all stages. In addition, treatment with rhubarb eliminated uncultured bacterium d during the modeling stage in the probiotics + drug residue treatment group, while treatment with fermentation supernatant recovered the growth of uncultured bacterium d during the treatment and recovery stages (Fig. 5; Table IB).