Ovalbumin (OVA), concanavalin A (ConA) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (Merck KGaA). Goat anti-mouse IgG (cat. no. 1036-05), IgG1 (cat. no. 1070-05) and IgG2a (cat. no. 1080-05) horseradish peroxidase conjugates were purchased from SouthernBiotech. The RPMI-1640 medium was purchased from Hyclone (GE Healthcare Life Sciences). Fetal bovine serum (FBS) was supplied by Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. Aluminum hydroxide gel (alum) was supplied by Zhejiang Wanma Pharmaceutical Co., Ltd. Cell Counting kit-8 (CCK-8) was purchased from Vazyme. All other chemicals were of analytical reagent grade. The ELISA kits (IFN-γ, cat. no. F10660; TNF-α cat. no. F11630; IL-2 cat. no. F10780; IL-4 cat. no. F10810; and IL-5 cat. no. F10820) were from Shanghai Westang Biotechnology Co., Ltd. FITC/phycoerythrin (PE) rat anti-mouse CD4/CD8 antibodies (cat. no. FMD001-050) were purchased from Beijing 4A Biotech Co., Ltd. RNAiso plus^® (cat. no. 9108), First Strand cDNA Synthesis kit (cat. no. RR036A) and SYBR^® Premix Ex Taq™ were purchased from Takara Biotechnology Co., Ltd. Pathogenic porcine Escherichia coli (E. coli) (19) was kindly provided by Dr Jingxuan Ni (Longyan University). RCIE (cat. no. NAT-177) was purchased from Naturalin Bio-Resources Co., Ltd and standardized to obtain a concentration of 10% isoflavone (consisting of 10.2% formononetin, 9.6% biochanin A, 0.32% genistein and 0.08% daidzein). The solubility of RCIE was improved by treating with NaOH at pH 12 and 60°C for 6 h as previously reported (20,21).

A total of 100 female ICR (CD-1) mice (age, 6 weeks, grade II; weight range, 18–22 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (certificate no. SCXK2016-0003). All mice were maintained under the conventional housing conditions for one week prior to experiments. They were housed under controlled conditions, specifically at 24±2°C, with 50±10%, humidity with a 12 h light/dark cycle, and were provided free access to food and drinking water.

The experimental protocol for testing the immune adjuvant response induced by RCIE combined with OVA was as described in previous studies (3,22). Briefly, the mice were divided into 6 groups (n=10 mice/group): Saline group was defined as blank control; other groups of mice were subcutaneously immunized with 100 µg OVA alone or 100 µg OVA + adjuvant (200 µg alum or 50, 100 or 200 µg RCIE) on day 1. Mice received a booster injection after 2 weeks. Splenocytes, serum and peripheral blood were collected 2 weeks following the booster injection. After immunization, the mental state (23), diet and activity of the mice was monitored every day.

A challenge test using RCIE combined with E. coli vaccine was conducted in mice as previously described (19,21). Pathogenic E. coli was inactivated using 0.2% formaldehyde at 65°C for 8 h. Following a sterility test, the live E. coli vaccine was prepared to a final concentration of 1×10⁸ CFU/ml.

A total of 40 mice were randomly divided into the following four groups (n=10/group): i) Vehicle group, which was subcutaneously injected with 0.2 ml saline; ii) saline + E coli group, which was subcutaneously injected with 0.1 ml E. coli vaccine (containing 1×10⁷ CFU) and 0.1 ml saline; iii) alum + E. coli group, which was subcutaneously injected with 0.1 ml E. coli vaccine (containing 1×10⁷ CFU) and 0.1 ml alum (200 µg/0.1 ml); and iv) RCIE + E. coli group, which was subcutaneously injected with 0.1 ml E. coli vaccine (containing 1×10⁷ CFU) and 0.1 ml RCIE (100 µg/0.1 ml). Each mouse was subsequently injected with 0.2 ml (2×10⁷ CFU) pathogenic E. coli 3 days following immunization. Mouse mortality was recorded 2 h following bacterial challenge, and every 6 h thereafter. The survival rate (%) was calculated as follows: (The number of surviving mice/total number of mice) ×100.

For the present study, humane endpoints were established and applied at the earliest experimental timepoint without adversely affecting scientific objectives. The endpoints for the animal experiments were 2 weeks following the booster injection and 48 h after challenge with pathogenic E. coli bacteria, following which all animals were humanely euthanized. Firstly, each mouse was anesthetized with 2–5% isoflurane by inhalation and anesthesia was subsequently confirmed by blink reflex examination. Blood samples were then extracted by orbital sinus puncture following anesthesia, after which the mice were sacrificed by cervical dislocation. To minimize animal suffering, the experimental design was optimized such that alternatives were considered, pain and the number of animals used were kept to a minimum, and only qualified personnel were permitted to perform the experiments. All experiments were performed in accordance with the guidelines of the Animal Ethics Committee of Fujian province (Fujian, China) and were approved by the Institutional Animal Care and Use Committee of Longyan University (Longyan, China).

Splenic tissues were collected from the mice under sterile conditions. The spleens were transferred to a 200-mesh cell strainer and ground to obtain cell suspensions in PBS. Following erythrocyte lysis, the splenocytes were cultured in RPMI-1640 medium supplemented with 10% FBS in an incubator with 37°C and 5% CO₂. Next, the splenocytes (100 µl/well) were seeded into 96-well plates at a density of 1×10⁷ cell/ml before ConA (5 µg/ml), LPS (5 µg/ml), OVA (10 µg/ml) or media were added to the wells to a final volume of 200 µl. After 72 h incubation, cell viability was measured using the CCK-8 assay, according to the manufacturer's protocols. The stimulation index (SI) was calculated using the following formula: SI = absorbance value at OD 450 nm for mitogen-activated cultures/absorbance value for non-stimulated cultures.

The levels of OVA-specific IgG, IgG1 and IgG2a antibodies in mouse serum were measured using an indirect ELISA method as previously described (5). Briefly, microtiter plate wells were first coated with 100 µl OVA solution (50 µg/ml dissolved in 50 mM carbonate-bicarbonate buffer, pH 9.6) for 24 h at 4°C. The wells were then washed three times with PBS containing 0.05% Tween-20 and then blocked with 5% bovine serum albumin (cat. no. ST023; Beyotime Institute of Biotechnology) at 37°C for 2 h. The serum was diluted 1:10 with 0.5% BSA/PBS. Following a further three washing steps, 100 µl diluted serum sample or 0.5% BSA/PBS (control) was added (in triplicate) to the wells and incubated for 2 h at 37°C. Next, after three washing steps, 100 µl horseradish peroxidase (HRP)-conjugated antibody (IgG, 1:10,000; IgG1, 1:4,000; IgG2a, 1:4,000) or 0.5% BSA/PBS (control) was added to each well. The plates were further incubated for 1 h at 37°C. After washing, the wells were incubated with TMB chromogenic substrate in 37°C for 30 min. The substrate reaction was stopped by the addition of 50 µl of 2 M sulfuric acid. The absorbance value at 490 nm was measured for each well using a microplate reader.

Serum levels of IL-2, IFN-γ, TNF-α, IL-4 and IL-5 were quantified using commercial ELISA kits according to the manufacturers' protocols.

Blood was collected from the mice by retroorbital exsanguination into tubes containing the anticoagulant heparin. The number of peripheral blood cells in serum isolated from the mice immunized with OVA was detected using flow cytometry. A total of 10 µl FITC-conjugated CD4 (0.25 µg) and 5 µl PE-conjugated CD8 (0.25 µg) monoclonal antibodies were added to 50 µl anticoagulant blood. This mixture was mixed and incubated gently at room temperature for 30 min in the dark. 1.8 ml hemolysin solution was added to each tube, and incubated at 37°C for 10 min. Following centrifugation at 1,500 × g, the pelleted cells were rinsed using fluorescence-activated cell sorting (FACS) buffer (2% FBS in PBS) before resuspension in 0.5 ml FACS buffer. FACS data acquisition was performed on the BD FACS Calibur™ system (BD Biosciences), and the data was analyzed on Novo express 1.3 software (ACEA Biosciences, Inc.).

Splenocytes were seeded (5×10⁶ cells/ml, 1 µl/well) into 24-well plates, and ConA (5 µg/ml) was then immediately added. After 12 h treatment, cells were harvested and total RNA was extracted using TRIzol^® according to the manufacturer's protocol. The RNA was reverse-transcribed to cDNA using a First Strand cDNA Synthesis kit according to the manufacturer's protocol. The cDNA samples were stored at −20°C until further use. qPCR was subsequently performed in a 20 µl total reaction volume consisting of 2X SYBR^® Premix Ex Taq™ (10 µl), 0.5 µl each primer (10 µM), 2 µl cDNA and 7 µl ddH₂O with a concomitant reaction condition (10 min at 95°C, then followed by 35 cycles of 30 sec at 94°C, 30 sec at 55°C and 60 sec at 72°C). The sequences of the primers used are shown in Table I. Relative expression levels of mRNAs using β-actin as an internal control were analyzed using the 2^−ΔΔCq method (24).

Values are expressed as the mean ± SEM. Differences between the experimental and control groups were analyzed using SPSS 20.0 software (IBM Corp.). One-way ANOVA followed by Student-Newman-Keuls multiple comparison test was used for multigroup analyses. Survival rates were analyzed using the log-rank test in GraphPad Prism software (version 6; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

No notable stress responses, including visible changes in daily appetite, mental state or motor ability were observed in the mice following the first immunization and booster immunization 2 weeks later. In addition, no differences in body weight were observed after immunization between the adjuvant groups (OVA + RCIE, OVA + alum) and control groups (saline and OVA alone) (data not shown).

The effects of RCIE-OVA immunization on splenocyte proliferation in mice are shown in Fig. 1. Splenocyte viability in the OVA + RCIE (50 or 100 µg) and OVA + alum groups was significantly higher compared with that of the OVA control group following OVA stimulation (P<0.05 or P<0.01). Splenocytes isolated from mice immunized with OVA + RCIE (100 and 200 µg) exhibited significantly higher cell viability following treatment with LPS compared with those from mice immunized with OVA alone (P<0.05). Splenocytes isolated from OVA + RCIE (100 µg)- and OVA + alum-immunized mice stimulated with ConA displayed significantly increased cell viability compared with those from the OVA control group (P<0.05). However, no significant differences were observed between the OVA and OVA + RCIE (50 or 200 µg) groups following ConA stimulation. The results demonstrated the effect of RCIE on improving the cellular immune response.

The levels of OVA-specific IgG, IgG1 and IgG2a antibodies are shown in Fig. 2. Serum IgG levels were significantly higher in the OVA + alum and OVA + RCIE (50, 100 and 200 µg) groups compared with the OVA group (P<0.05 or P<0.01). In addition, serum IgG1 levels were significantly higher in all RCIE-immunized groups of mice compared with the OVA group. No significant differences were observed in the total serum IgG1 levels between the OVA + alum group and the mouse groups immunized with OVA + RCIE. RCIE (50 µg) and RCIE (100 and 200 µg) also significantly increased total serum IgG2a levels in OVA-immunized mice (P<0.05 or P<0.01, respectively), but no significant differences in serum IgG2a levels were identified between the OVA + alum and OVA alone groups. Therefore, these observations suggest that alum only significantly enhanced the levels of Th2-type antibodies, whereas the levels of Th1-type antibodies were significantly increased by RCIE compared with alum at appropriate levels.

The levels of the cytokines IFN-γ, TNF-α, IL-2, IL-4 and IL-5 were next measured using ELISA kits and the results are shown in Fig. 3. The serum levels of IFN-γ, IL-2 and IL-4 in mice immunized with OVA + RCIE (100 µg) were significantly higher compared with those in the OVA group (P<0.05). Although increases were observed in the levels of TNF-α and IL-5 in the OVA + RCIE group compared with the OVA group, no statistically significant differences were detected. This suggests that RCIE can significantly enhance the levels of Th1 and Th2 cytokines in OVA-immunized mice.

The effects of RCIE on the levels of CD4⁺ and CD8⁺ T cells in the blood from OVA-immunized mice are summarized in Fig. 4. Compared with the OVA group, the proportion of CD4⁺ T cells in mice immunized with OVA + RCIE (100 µg) was significantly increased (P<0.05). No significant differences were detected in the proportion of CD4⁺ T cells between the OVA + RCIE (50 µg) and OVA + alum groups. Additionally, the proportion of CD8⁺ T cells in the OVA-immunized mice was not significantly affected by RCIE.

The levels of IL-2, IFN-γ, T-bet, IL-10, GATA-3 and IL-4 mRNA expression in ConA-stimulated mouse splenocytes are shown in Table II. Following ConA treatment, the mRNA expression levels of the Th2-associated cytokines IL-4, IL-10 and transcription factor GATA-3, as well as those of Th1-associated cytokines IL-2, IFN-γ and transcription factor T-bet were significantly increased in splenocytes from the OVA + RCIE groups at certain concentrations compared with those in the OVA group (P<0.05 or P<0.01). However, only IL-4, IL-10 and GATA-3 mRNA expression levels were observed to be significantly higher in the OVA + alum group compared with the OVA group (P<0.05 or P<0.01). These results suggest that the inclusion of RCIE during immunization induced the gene expression of Th1/Th2 cytokines and transcription factors in splenocytes following stimulation with ConA.

Three days following inoculation with E. coli vaccine, the mice were challenged with pathogenic porcine E. coli. In the vehicle group, the mice began to exhibit symptoms associated with infection 4 h following bacterial challenge including curling, diminished movement, increased depression, ruffled fur, shortness of breath and death within 6 h, with no mice surviving beyond 24 h. In the RCIE + E. coli group, deaths were observed commencing at 18 h following bacterial challenge with no additional deaths observed 24 h after challenge. Since statistical significance could already be observed in the mortality rates between the four experimental groups at 48 h after bacterial challenge, all experiments were terminated at 48 h post-challenge. The survival rates of immunized mice were found to be 80% (8/10) for the RCIE group, 60% (6/10) for the alum + E. coli group and 30% (3/10) for the saline + E. coli group (Fig. 5). Compared with the vehicle and saline + E. coli groups, the survival rate in the RCIE + E. coli group was significantly higher (P<0.05). These results suggest that RCIE can significantly enhance the efficacy of E. coli vaccine.