Modified Si-jun-zi granule
Modified Si-jun-zi granule (M-SJZG) is constituted of eight herbs, including Codonopsis pilosula (Franch.) Nannf, Atractylodes Lancea (Thunb.) DC, Wolfiporia cocos, Glycyrrhiza uralensis Fisch. ex DC (Preparata), Astragalus mongholicus Bunge, Bupleurum chinense DC, Citrus × aurantium L. and Crataegus pinnatifida Bunge (fruit) with a dosage ratio of 9:9:9:6:10:6:6:6, all of these were purchased from Sichuan c&y Traditional Chinese Medicine CO., LTD. and accorded with Chinese veterinary drug code(the details are summarized in Supplementary Table 1). Herbs were mixed and wetted with distilled water (1:8, w/v) for 2 h. Then, the mixture was boiled for 3 h and filtered through multi-layer gauze. The filtrate was gathered and residues were decocted twice with water (1:6, w/v) for 3 h. At last, the combined filtrates was concentrated to 1.0 g/mL which measured by raw medicine. The decoction was mixed with starch (1:1, v/w). M-SJZG were prepared by wet granulation with proper concentration of alcohol as adhesive for convenience of daily administration.
The quality control of M-SJZG was analyzed using high-performance liquid chromatography (HPLC) with Corona Ultra detection(CAD) in Agilent HPLC system. M-SJZG (5 g) wasdissolved in10mL Methanol solution (70%) and diluted 10 times. The solution centrifuged (1500 rpm, 15 min) and filtered (0.22 µm), and then, 20µL was injected into an HPLC system for assay. The separation was performed using a reverse-phase column(Intersil ODS-3 5 µm, 4.6 mm × 250 mm I.D.) connected with a guard column (C18, 5 µm, 4.6 mm × 10 mm I.D.). The elution flow rate was 1.0 mL/min with a mobile phase gradient of A-B (A: H2O/H3PO4 = 1000 mL/2 mL;B: CH3CN), which was varied as follows:0 min, 90% A, 10% B; 9 ~ 29 min, 90 ~ 85% A, 10 ~ 15% B; 29 ~ 37 min, 85 ~ 82% A, 15 ~ 18% B; 37 ~ 46 min, 82% A, 18% B; 46 ~ 51 min, 82 ~ 80%A, 18 ~ 20% B; 51 ~ 72 min, 80 ~ 72% A, 20 ~ 28% B; 72 ~ 82 min, 72% A, 28% B; 82 ~ 87 min, 72 ~ 70% A, 28 ~ 30% B; 87 ~ 92 min, 70 ~ 67% A, 30 ~ 33% B; 92 ~ 99 min, 67 ~ 50% A, 33 ~ 50% B; 99 ~ 110 min, 50% A, 50% B; 110 ~ 115 min, 50 ~ 47% A, 50 ~ 53% B; 115 ~ 118 min, 47 ~ 30% A, 53 ~ 70% B; 118 ~ 130 min, 30% A, 70% B; 0 min, 90% A, 10% B; 130 ~ 135 min,30 ~ 90% A, 70 ~ 10% B; 135 ~ 140 min༌90% A, 10% B. The injection volume was 20 µL, and the UV detection wave length was set at 285 nm for saikoside A, liquiritin and hesperidin; 250 nm for glycyrrhizic acid. The fingerprint chromatogram of M-SJZG is shown in Supplementary Fig. 1.
Chemicals and reagents
Glutamine (Gln) was purchased from Huana Chemical Co., Ltd (china), HE stain (Hematoxylin and Eosin) were purchased from Thermo Fisher Co., Ltd (American), all Elisa kits was purchased from Nanjing Jiancheng Bioengineering Institute, FastKing RT kit was purchased from Tiangen Biotech (Beijing) CO., LTD, the others reagents was purchased from ChenDu Chron Chemicals Co., Ltd (china).
Two hundred and eighty Rex rabbits, approximately 45 days of age and 750-950g in weight, were obtained from the Rex rabbit institute, Sichuan Academy of Grassland Sciences. The animals were provided with basal diet and tap water at liberty and maintained in cages under controlled conditions (23±2℃, 12 h light/dark cycle). All experiments and procedures were carried out according to the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of China. The composition and nutrient levels of the basal diet were listed in Table 1.
Growth performance, diarrhea frequency and mortality
One hundred and twenty Rex rabbits were randomly divided into four groups, including control group and M-SJZG (0.5%, 1%, 2%) groups. Except the control group, M-SJZG were administrated as dietary supplement for thirty consecutive days, to evaluate the protective effect by growth performance, diarrhea frequency (Pdiarrhea was calculated as equation 1) and mortality.
Where A represents the the total times of diarrhea, and B represents the sum of survival days of each rabbit in the group.
Tissue collection and measurement
To revealed the protective mechanism of M-SJZG from intestinal development and immunity, the others one hundred and sixty rabbits were randomly divided into five groups, including control group, M-SJZG (0.5%, 1%, 2%) groups and Glutamine (Gln) (0.8%) group. M-SJZG (0.5%, 1%, 2%) or Gln (0.8%) were administrated as a dietary supplement for thirty consecutive days. In each group, the six rabbits were randomly selected, weighted and sacrificed on the 15th and 30th day after treatment, respectively. The intestinal length and relative weight were measured after washing by physiological saline. Then the duodenum, jejunum and ileum were collected and fixed with 4% paraformaldehyde, respectively.
The histopathological changes of various intestinal segments were observed under a high-resolution microscope with photographic facility after being embedded in paraffin and made slides with HE staining. Image pro Plus was used to measure the length of villi and the depth of crypt, then the ratio was calculated as follows (Equation 2).
Where Lvilli and Dcrypts stand for the length of villi and the depth of crypt, respectively.
ELISA analysis of serum biochemical indices and SIgA
The serum and intestinal mucus was collected after experimental period. Then the abundance of serum biochemical indices and SIgA on the intestinal mucus were assayed by ELISA kits according to the manufacturer's instructions.
Immunohistochemical analyses of SIgA
The abundance of SIgA was assessed for paraffin embedded slides after the sections were dewaxed. Endogen biotin and non-specific signals were blocked with appropriated reagents. Antigen retrieval was carried out in a microwave oven (two cycles for 5 min each at 780 W, in citrate buffer, pH 6.0, twice washed in PBS for 5 min each). Then, the treated slides were overnight incubated with primary antibodies at -4℃ in a humidified chamber, washed in PBS, and visualized by biotinylated secondary antibodies followed by stained by DAB kit for 2 min, washed in distilled water. Finally, counterstained with Hematoxylin, dehydrated, transparentized and sealed. At least 10 fields of view from each sample were analyzed with the image analysis software for each protein of interest.
Zo-1, Claudin-1, Occludin, SGLT1 and GLUT2 level assay
Total RNA was isolated from samples of jejunum, ileum and colon with TRIZOL regent (Tiangen Biotech, Beijing) and then treated with DNase I (Tiangen Biotech, Beijing) according to the manufacturer’s instructions. Primers used in this study are presented in Table 2. GAPDH was used as an internal control to normalize expression of target gene transcripts. Real-time PCR was performed as previous studies described(Yin et al., 2015).
The one-way analysis of variance (ANOVA) was used to analyze experimental data by SPSS 25.0 software. All values were presented as the mean ± SD, and P < 0.05 was considered statistically significant.