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Evaluation of cytotoxicity and immune modulatory activities of soyasaponin Ab: an in vitro and in vivo study.

ABSTRACT

To improve the immune efficacy of protein subunit vaccines, novel adjuvants are needed to elicit a suitable protective immune response and to promote long term immunologic memory. In this work, soyasaponin Ab, a major constituent among group A soyasaponins in soybeans was purified and prepared from soy hypocotyls. The immunomodulatory effects of soyasaponin Ab both in vitro and in vivo were investigated, and its pro-immunomodulatory molecular mechanism was also studied. For in vitro assays, with mouse macrophage cell line RAW264.7 as the studying model, both cytotoxicity and immune stimulatory activity were investigated to evaluate the potential of soyasaponin Ab as the vaccine adjuvant. The results indicated that soyasaponin Ab could be significantly safer than Quillaja saponins (QS). Soyasaponin Ab showed no toxicities over the tested concentration ranges compared to QS. Soyasaponin Ab was proved able to promote releases of inflammatory cytokines like TNF[alpha] and IL-1[beta] in a dose-dependent manner. Furthermore, NF-[kappa]B signalling was also activated by soyasaponin Ab effectively. In addition, with TLR4 gene expression of RAW264.7 cell inhibited by RNA interference, immune stimulatory effects by soyasaponin Ab dropped down significantly. On the other hand, the in vivo experiment results showed that anti-ovalbumin (OVA) IgG, IgG1, IgG2a, IgG2b were significantly enhanced by the soyasaponin Ab and QS groups (p < 0.05 or p < 0.01). The results suggested that compared to QS, soyasaponin Ab may represent a viable candidate for effective vaccine adjuvant. TLR4 receptor dependent pathway may be involved in immune stimulatory effects of soyasaponin Ab.

Keywords:

Soyasaponin Ab

Adjuvant

Haemolysis

TLR4

OVA

Introduction

Soyasaponins belong to one group of complex and structurally diverse oleanane triterpenoids occurring mainly in soybeans (Glycine max) (Kitagawa et al. 1976, 1985a,b.) (Fig. 2). Soyasaponins can be categorized into two main groups referred to as group A and group B, according to their respective aglycones, soyasapogenol A and soyasapogenol B. Group A soyasaponins have two sugar chains attached to the C-3 and the C-22 of the aglycone. Group B soyasaponins possess a single sugar chain linked to the C-3. Their absolute chemical structures have been elucidated in detail (Kitagawa et al. 1976, 1985a,b). Soyasaponins exert a wide range of pharmacological activities including anticarcinogenic (Xiao et al. 2007; Zhang and Popovich 2008), capable of lowering plasma cholesterol (Lee et al. 2005; Oakenfull and Sidhu 1990), antiviral (Hayashi et al. 1997; Nakashima et al. 1989), hepatoprotective (Kinjo et al. 1998, 2003), antioxidative (Ishii and Tanizawa 2006), and antimutagenic (Berhow et al. 2000; Jun et al. 2002), among them adjuvant activity is gaining more attention (Oda et al. 2000). A correlation between adjuvant activity and amphipathic structure of soyasaponin was completed on an experimental basis using structurally consecutive analogues. The investigation demonstrated that soyasaponins adjuvant activities increased with the hydrophile-lipophile balance (HLB) value, i.e. the length, the number, and the composition of sugar side chains affecting the HLB value would give the overall conformation of each saponin molecule, and the amphipathic structure may define the fundamental adjuvanticity of saponins (Oda et al. 2003).

Soyasaponins can be obtained in large quantities from soybean and many kinds of legume seeds, which could be the great advantage for its practical use as vaccine adjuvants. However, the safety and immune stimulatory activity of soyasaponins have not been evaluated in detail. In the present study, a major constituent among group A soyasaponins in soybeans was isolated, and its in vitro cytotoxicity and immune modulatory effect as well as its in vivo immunoadjuvant activity were investigated.

Materials and methods

Reagents and chemicals

Ovalbumin (OVA, grade V), concanavalin A (conA), lipopolysaccharide (LPS) and saponins from quillaja bark (QS, CFAD-S4521-10G) as partially purified extract with 20-35% of sapogenin content were purchased from Sigma Chemical Co. RPMI-1640 medium was obtained from HyClone, Inc. Foetal calf serum (FCS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. Goat anti-mouse IgG, IgG1, IgG2a or IgG2b horseradish peroxidase (HRP)-conjugates were obtained from Proteintech Group, Inc.

Animals

ICR mice (6-8 weeks old, mean weight 20 g) were purchased from Shanghai Lab. Animal Research Center (Shanghai, China). They were housed in microlon boxes in a controlled environment (temperature 25 [+ or -] 2 [degrees]C) and 12 h dark/light cycle with standard laboratory diet and water ad libitum. The animals were acclimatized to the laboratory conditions for a week prior to the experimentation and randomly divided into six groups of each six animals. All procedures were performed according to the China legislation on the use and care of laboratory animals and according to the guidelines established by the Shanghai Animal Care and Use Committee and university committee for animal experiments.

Plant material and isolation of soyasaponin Ab

Soy hypocotyls (soybean variety Kenfeng15, Heilongjiang Province, China, harvested in 2010 in Heilongjiang Province, China) were provided by Heilongjiang Jiusan Oil & Fat Co., Ltd. (Harbin, China). Prior to pretreatment, the hypocotyls were milled to a powder and sieved (40 mesh). All other chemicals were of analytical reagent grade, and all solutions were used after redistillation.

Soyasaponin Ab was isolated and identified by our previously reported method (Zhao et al. 2012). Briefly, prior to extraction, the ground hypocotyls were defatted by extraction with petroleum ether (30-60[degrees]C) for 4 h under reflux conditions and air-dried after extraction in the fume hood. The extraction was subsequently carried out with 70% (v/v) aqueous ethanol in an ultrasonic bath. Next, the extracts were concentrated under reduced pressure at 40[degrees]C to remove ethanol, and the residual aqueous phase was applied to a Supelpak[TM] XAD-2 (Supelco, Bellefonte, PA, USA) column (i.d. 30 x 450 mm). After washing with Millipore water, the column was eluted with 95% (v/v) aqueous ethanol, and the eluate was evaporated to dryness under vacuum at 40[degrees]C and identified as dried crude extract. The extraction of 100g (dry weight basis) of powdered soy hypocotyls yielded 8.34 g of the crude extract (dry matter) containing 4.88% total soyasaponins after the solid-phase extraction.

Preliminary fractionation of soyasaponin complexes by high-speed counter-current chromatograph (HSCCC) was performed using the same procedure as previously described (Zhao et al. 2012). 100 mg of crude extract was dissolved in 5 ml methanol and loaded on HSCCC. After separation, 22.3 mg soyasaponin complexes with the purity >85% was obtained.

For the final separation of the individual soyasaponins, preparative HPLC (PRE-HPLC) analysis was also carried out as previously described (Zhao et al. 2012). In the step gradient separation, 17 separate fractions were collected, the target compound soyasaponin Ab was obtained in the fraction 6 with the highest yield accounting for 6.32% of the crude extract (18.53% of the loaded soyasaponin complexes).

HPLC, LC-MS and NMR analysis of prepared soyasaponin Ab

The purity of the isolated compound was determined by reversed phase-HPLC using UV, evaporative light scattering detection (ELSD) (Fig. 1) described by Decroos et al. (2005). The result indicated the prepared soyasaponin target component purity was superior to 98%.

For LC-MS analysis of soyasaponins samples, UPLC-MS was performed as described previously (Zhao et al. 2012). The structure was elucidated using LC-MS, [sup.1]H and [sup.13]C NMR spectroscopy (400 MHz [D.sub.2O]), see support information.

LC-MS: m/z 1437.65 for [[M+H].sup.+], the molecular formula of the compound is C67H104033. In this way, by a detailed comparison of the [sup.13]C NMR data with literature (Gurfinkel et al. 2005; Shiraiwa et al. 1991), the structure of the purified compound was identified as the fully acetylated group A soyasaponin Ab (Fig. 2).

Haemolytic activity assay

Citrated BALB/c mouse blood was obtained from the animal facility of the Shanghai Laboratory Animal Centre, CAS (SLACCAS). Red blood cells (RBC) were recovered by centrifugation (2000 rpm for 5 min) and washed three times with sterile phosphate-buffered saline (PBS). A micro haemolytic activity assay was carried out using a 0.5% RBC suspension in PBS. A fixed volume of the suspension (100 [micro]l) was mixed in round bottom microplates with a solution of the product to be tested in saline (100 [micro]l). Soyasaponin Ab was assayed at concentrations ranging from 30 to 1250 [micro]g/ml (30, 40, 50, 62.5,125, 250, 500,1000 and 1250 [micro]g/ml). Haemolytic activity of Quillaja saponin (QS, CFAD-S4521-10G, Sigma) in PBS were also evaluated. Distilled water or PBS buffer was included as positive and negative controls for haemolysis. Microplates were incubated for 30min at 37[degrees]C and centrifuged at 1000rpm for 10min. An aliquot of each supernatant (100 [micro]l) was transferred to a flat-bottom microplates and the optical density (OD) at 405 nm was measured using an ELISA reader (Lab Systems, Finland). Haemolytic activities by saponins were calculated based on the following: percentage of haemolysis = (As - An)/(Ap - An) x 100%. As, An and Ap represented absorbance value of samples, negative control and positive control, respectively. Each sample was tested in triplicate.

Measurement of the cytotoxicity of soyasaponin Ab on RAW264.7

RAW264.7 was purchased from Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cytotoxicity of soyasaponin Ab against RAW264.7 was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) (Mosmann 1983). In short, fresh cells were subcultured in 96 well plate at density of 4 x [10.sup.4] cell/per well for overnight. The Media were absorbed and 200 p.1 of fresh RPMI 1640 media with different concentrations of soyasaponin Ab were added into wells with fresh RPMI 1640 media set up as control. 24 h post treatment, 20 [micro]l MTT solution (5 mg/ml) (Amresco, US) were added into each well and incubated under 37[degrees]C for 4 h. Media in each well were discarded and 150 [micro] of DMSO (Sigma, USA) were added into each well and mixed thoroughly with pipette. After 10 min incubation, optical densities were measured with a microplate reader (Labsystems, Finland) at a wavelength of 570 nm. The percentage of cytotoxicity was calculated by comparing the exposed samples to the controls. To ensure that the control sample could be used as a reference, the viability of control cells was also double-checked by counting the cells in a Burker chamber under a light microscope after staining the cells with trypan blue solution (Sigma Chemical Co., St. Louis, MO).

Immuno-stimulating activity of soyasaponin Ab in RAW264.7

Two different methods were used to detect immunostimulating activity of soyasaponin Ab in mice macrophages. Soyasaponin Ab used for treating RAW264.7 cells in the following experiment were all dissolved and diluted with RPMI 1640 medium (Gibco, USA).

NF-[kappa]B signalling pathway is one of the most important pathways in regulating inflammatory cytokines expression. NF-[kappa]B promoter driven luciferase reporter assay is generally used to detect the activations of the pathway (Battaglia et al. 2008; Bognar et al. 2013). In short, RAW 264.7 cells grown in 24well plates were cotransfected with 0.4[micro]g/well of a NF-[kappa]B promoter driven luciferase reporter plasmid (pGL4.32[luc2P/NF-[kappa]B-RE/Hygro]) (Promega, USA) and 0.08 pg/well of pRL-TK plasmid (Promega, USA) as an internal control for normalization of transfection efficiency. 24 h post transfection, cells were further treated with soyasaponin Ab at concentrations ranging from 20 to 100 [micro]g/ml. RPMI 1640 media were set up as control. 24h later, the cells were lysed and firefly luciferase and Renilla luciferase activities were determined using the Dual-Luciferase reporter assay system (Promega) according to the manufacturer's protocol. The data represent relative firefly luciferase activity normalized to Renilla luciferase activity and are representative of three independently conducted experiments. Data are presented as means [+ or -] standard deviations (SD). Statistical p values < 0.05 were considered significant, and p values of <0.01 were considered highly significant.

Inductions of inflammatory cytokines IL-1[beta], TNF[alpha] were used as the markers to evaluate immuno-stimulatory activities by Ab. RAW 264.7 cells grown in 24-well plates were also treated with different concentrations of Ab ranging from 20 to 100 [micro]g/ml. RPMI 1640 media were also used as control 24 h post treatments, supernatants were collected and levels of cytokines were detected using ELISA kits (Boshide Company, Wuhan, China) according to the manufacturer's instructions.

Expression inhibition of TLR 4 gene with specific siRNA

To evaluate potential role of toll like receptor 4 (TLR4) in activation of inflammatory pathways by soyasaponin Ab. 3 siRNA candidates targeting at different regions of mouse TLR4 gene were chemically synthesized by Shanghai GenePhama Biotech Ltd. based on the reported mouse TLR4 gene coding sequence (Genbank accession number, GI: 118130391) (Table 1). Efficiency of knocking down of mouse TLR4 expression by specific siRNA was identified with western-blotting using anti-TLR4 monoclonal antibody (Santa Cruz, USA). In short, three different siRNAs were transfected respectively into cells using lipofectamine 2000 following the protocol of the manufacturers. 24 h post-transfection, cells were lysed in buffer containing 5% ([beta]-mercaptoethanol, 0.01% NP-40, and 2% sodium dodecyl sulfate (SDS). Scramble siRNA provided by the manufacturer was used as the control. Proteins were separated by 12% SDS polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Thermo Scientific, Rockford, IL, USA) in a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad, Hercules, CA, USA). Rat anti-mouse TLR4 monoclonal antibody was used as the primary antibody at a dilution of 1:200 with horseradish peroxidase-conj ugated goat anti-mouse IgG as the secondary antibody at a dilution of 1:5000 (Santa Cruz, CA, USA). Target bands were observed using West Pico Chemiluminescent Substrate (Thermo Scientific) and exposed to Kodak BioMax MR film (Kodak, Rochester, NY, USA). Detection of mouse GAPDH protein was used normalize sample loadings. Primary monoclonal antibody detecting mouse ([beta] actin (Santa Cruz, USA) was diluted into 1:1000, with horseradish peroxidase-conjugated goat anti-mouse IgG as the secondary antibody at a dilution of 1:5000 (Santa Cruz, CA, USA). Efficiencies of knocking down expression of TLR4 gene by different siRNA were evaluated with software by Tanon, normalized with mouse GAPDH.

Measurement of sera OVA-specific antibody

Groups of six female ICR mice were immunized subcutaneously with either ovalbumin alone in PBS or with OVA mixed with soyasaponin Ab, QS or Alum. Immunisations were performed twice on days 1 and day 15. After two weeks of the second immunization, sera samples were collected to measure the level of anti-OVA IgG, IgG1, IgG2a and IgG2b.

Indirect ELISA was used to detect OVA-specific antibodies in serum as previously described with slight modification (Sun 2006). The absorbance was tested at a wavelength of 450 nm using an ELISA reader. Data were reported as the mean optical density (OD) of the serum samples minus the mean OD of the control. The experiment was performed in triplicate.

Statistical analysis

All results were presented as means [+ or -] standard errors (S.E.) and examined for their statistical significance of difference with Student's t-test and one way ANOVA. p-values of less than 0.05 were considered to be statistically significant.

Results

In contrast with QS saponins, soyasaponin Ab indicated low haemolytic activity and toxicides in vitro

The haemolytic activity of soyasaponin Ab was listed in Table 2. Haemolytic activities of mouse red blood cell treated with soyasaponin Ab were around 0.08%, 0.61%, 3.35% and 3.75% at the concentration of 50 [micro]g/ml, 100 [micro]g/ml, 150 [micro]g/ml and 200 [micro]g/ml respectively, while the haemolytic activities for QS were around 21.1%, 68.8% and 90.2% at the concentration of 10 [micro]g/ml, 20 [micro]g/ml and 30 [micro]g/ml respectively.

Treated with different doses of soyasaponin Ab ranging from 1.0 [micro]g/ml to 20 [micro]g/ml with QS as the control, cytotoxicity of soyasaponin Ab was assessed in RAW264.7 cells based on MTT assays. As it was shown in Fig. 3, soyasaponin Ab did not indicate apparent toxicides against RAW 264.7 cells at all the above doses. Almost 100% of the cells were alive even if the dose was 20 [micro]g/ml. However, when treated doses increased from 1.0 [micro]g/ml to 20 [micro]g/ml, QS indicated significant toxicities against RAW264.7 cells. At dose of 5 [micro]g/ml, around 50% of the cells died. When treated dose was 20 pg/ml, around 95% of the cells died. Based on the data in vitro, soyasaponin Ab was much safer than QS.

Inflammatory cytokines could be stimulated effectively by soyasaponins Ab

Immuno-stimulatory activities by soyasaponin Ab were evaluated with two different assays. One was based on induction of inflammatory cytokines including TNF[alpha] and IL-[beta] as the markers, which could play important roles for host to clear off the invading pathogens. The other method was based on activations of NF-[kappa]B promoter driven luciferase activities. NF-[kappa]B promoter is one of the most important promoter in regulating inflammatory cytokines expressions for immune cells including dendritic cells as well as macrophages.

As it was shown in Fig. 4, in contrast with cells mock treated with RPMI 1640 alone, NF-[kappa]B signalling could be activated and TNF[alpha], IL-1[beta] could be induced by soyasaponin Ab significantly, which also indicated a dose dependent manner. When treated at dose of 20[micro]g/ml, TNF[alpha] and IL-1[beta] could arrive at 250pg/ml or 25 pg/ml respectively. At dose of 100 [micro]g/ml, TNF[alpha] and IL-1[beta] could be 28 pg/ml or 500 pg/ml respectively. In addition, with increasing of treated doses of soyasaponin Ab from 20 to 100 [micro]g/ml, the relative activities of NF-[kappa]B promoter driven luciferase could increase steadily from 25 to 30. Based on the studies, it demonstrated that soyasaponin Ab possessed the capabilities to stimulate inflammatory pathways in antigen presenting cells like macrophages.

TLR4 receptor involved in the immuno-stimulatory activities by soyasaponin Ab

Antigen presenting cells like dendritic cells and macrophages can recognize pathogens PAMPs via PRR on the surface followed by stimulation of the anti-pathogen immune signalling and release of inflammatory cytokines. Due to the amphiphile characteristics of soyasaponin Ab, it was hypothesized that TLRs might be involved in the stimulation of inflammatory pathways via NF-[kappa]B signalling, because TLRs are the most important receptors for antigen presenting cells to recognize conserved structures in microbe including those with amphiphile structures (Kawai and Akira 2010).

Potential involvement of TLR4 was assessed based on inhibition of its expression in RAW264.7 cells. Silencing efficiencies by three different siRNA targeting at different sites of mouse TLR4 mRNA were evaluated. As it was shown in Fig. 5, TLR4 could be knocked down by all of the three siRNA candidates, with siRNA 2 indicated the highest efficiency at 75%. In scramble siRNA transfected cells, TLR4 could be expressed at the same level as normal RAW264.7. Therefore, siRNA 2 was used to detect significance of TLR4 in immunostimulatory activities by soyasaponin Ab.

In cells transfected with scramble siRNA, when treated with soyasaponin Ab at concentration of 100[micro]g/ml, TNF[alpha] and IL-1[beta] level were around 550 pg/ml and 30[micro]g/mL respectively (Fig. 5). However, in siRNA 2 transfected cells, the levels of TNF[alpha] and IL-1[beta] dropped down to 350 or 15 pg/ml, respectively. Consistently, treated with soyasaponin Ab at concentrations of 100 [micro]g/ml, NF-[kappa]B promoter driven luciferase reporter activities decreased from 29 to 17 folds in scramble siRNA group compared with siRNA 2 group. Based on these results, with TLR4 receptor expression knocked down, both the inflammatory cytokine expression as well as NF-[kappa]B signalling could be reduced nearly 50% which suggested that TLR4 might be involved in the immune stimulation by soyasaponin Ab.

Effect of soyasaponin Ab on the OVA-specific serum antibody response

The effect of soyasaponin Ab on the induction of humoral immune responses in OVA-immunized mice was measured 2 weeks after the secondary immunization. The OVA-specific serum IgG, IgG1, IgG2a and IgG2b levels were detected using indirect ELISA and the results are shown in Fig. 6. Significant enhancements to all antibody levels were observed in the group of QS-immunized mice compared with OVA control, OVA/Alum and OVA/soyasaponin Ab groups (p<0.05). OVA immunized mice given different doses of soyasaponin Ab groups showed a various increase in the secretion of IgG, IgG1, IgG 2a and IgG2b. Significant enhancements in total serum IgG and IgG1 levels were observed in OVA/soyasaponin Ab-immunized mice at the doses of 50, 250 and 500 [micro]g compared with OVA alone (p < 0.05). In addition, the serum IgG2a level in OVA/soyasaponin Ab (250, 500 and 1000 [micro]g)-immunized mice was significantly higher than those in the Alum-immunized mice (p < 0.05). Meanwhile, the serum IgG2b level was also significantly enhanced by soyasaponin Ab at doses of 50 and 250 [micro]g as compared with OVA/Alum and OVA control group (p < 0.05 or p < 0.01). Moreover, Alum group could only enhance the serum IgG and IgG1 responses, however, no significant differences (p > 0.05) of titres for IgG2a and IgG2b were observed, which indicated that Alum do not favour its induction of Th1-type immune response. The achieved values are observed to be consistent with previous results and theoretical predictions.

Discussion

Saponins from various plant sources are known to exhibit a variety of biological and pharmacological activities (Osbourn et al. 2011). It was demonstrated that many saponins from traditional Chinese medicinal herbs possess the potential to promote both humoral and cellular immune responses (Sun et al. 2009). Saponins have been found to enhance phagocytosis, promote IL-1[beta] production by peritoneal macrophages, and stimulate secretion of cytokine such as IL-2,1L-4, IL-6, IL-10, IFN-[gamma] and TNF-[alpha] (Song and Hu 2009). Such broad range of cytokines is consistent with the mixed Thl/Th2 responses as observed in an evaluation of ginseng saponins in stimulation of the immune response to PPV vaccines in mice (Rivera et al. 2005). These findings suggest that saponins may exert their adjuvant activities by activating innate immunity.

The critical criteria for ideal adjuvants depend on safety as well as immuostimulatory efficacy. Most studies on the immunostimulatory activities of saponins have been carried out with preparations derived from Q, saponaria (Song and Hu 2009). However, previous studies demonstrated that the QS could lead to toxicides against mammalian cells (Song and Hu 2009). As the traditional food for mankind with definite safety, as expected, saponins prepared from soybeans are much safer than saponins from Q. saponaria. in this study, it indicated that haemolytic activity due to soyasaponin Ab could be less than 4% even treated with dose at concentration of 200 [micro]g/ml (Table 2). In contrast, QS could cause more than 90% of mice red blood cells haemolysis even at dose of 30 [micro]g/ml. In consistent with the result, cytotoxicities caused by soyasaponin Ab in RAW264.7 cells could also be significantly lower than QS did. In addition, it was proved by the study that soyasaponin Ab possessed the capabilities to induce expression of inflammatory cytokines and activate NF-[kappa]B signal pathways in a doses dependent way, which are important indicators of activations of antigen presenting cells.

For many decades, very little progress was made in understanding the mechanism of action of adjuvants, until recently, several significant breakthroughs have occurred in this area. The binding of pathogen-derived molecules to different immune sensors, including Toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors (NLR), and retinoic acid-inducible gene (RIG)-1-like receptors (RLR), activates important innate immune pathways and provides not only an understanding of how current vaccines and adjuvants work, but also potential targets for novel adjuvant development. The adjuvant activities of soyasaponin Ab may be mediated by interaction of their moieties with the cell membrane, possibly via specific receptors. Amphipathic structure of soyasaponin Ab suggested that TLRs especially TLR4 might be involved, because it recognizes lipopolysaccharide, which also with amphipathic structure. With TLR4 gene expression knocked down via specific siRNA interference, production of TNF-[alpha] and Il-1[beta] and activation of NF-[kappa]B signalling due to treatment of soyasaponin Ab could be significantly inhibited. It suggested that soyasaponin Ab binds to TLR4 might invoke a cascade of intra-celiular signalling pathways that induce the production of cytokines in inflammation and immunity.

Lee et al. (2011) has investigated soyasaponin Ab anti-inflammatory effects in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice and LPS-stimulated peritoneal macrophages, and the authors concluded that soyasaponin Ab may ameliorate colitis by inhibiting the binding of LPS to TLR4 on macrophages. Apparently, our data seem to direct in an opposite way, in our study, soyasaponin Ab promoted releases of inflammatory cytokines like TNF[alpha] and IL-1[beta] in a dose-dependent manner by a murine macrophage cell line (RAW264.7). However, in their investigations, the LPS and soyasaponin Ab were co-treated to cell line or animal model. In addition, no direct data has been shown in their study whether soyasaponin Ab possesses immune stimulatory efficacy of its own. There existed the possibility that soyasaponin Ab might directly interact with LPS, thus inhibiting binding of LPS with TLR4. Another possible mechanism could also be that LPS and soyasaponin Ab compete to combine with TLR4 receptor, and soyasaponin Ab exert higher affinity to TLR4 receptor than LPS. Therefore, in the presence of LPS at a constant concentration, with the increase of soyasaponin Ab concentration, the net result was observed in the manner of dose-dependent reduction for the stimulated cytokines. Furthermore, as above mentioned, the in vivo experiment results already indicated that soyasaponin Ab could enhance the immune response as an adjuvant. So, in our study, we re-evaluated the potentials roles of soyasaponin Ab alone in innate immune stimulations as well as the role of TLR4 singling involved. As a result, as mentioned above, our preliminary in vitro data support our hypothesis that the TLR4 receptor dependent pathway may be involved in immune stimulatory effects of soyasaponin Ab.

T lymphocyte-mediated immunity plays an important role in combating intracellular microbe infections. Among the T lymphocytes, helper T cells induce B-lymphocytes to secrete antibodies. Helper T cells can be divided into two subsets of effector cells, namely Th1 and Th2 cells. The Th1 response against malignant cells and intracellular pathogens is superior to the Th2 response (Sun et al. 2008). The capacity to elicit an effective T cell immunity can be shown by the stimulation of lymphocyte proliferation response. Previous studies demonstrated that the most commonly used adjuvants (water/oil emulsions and Alum) elicit only a Th2 immune response (Cox and Coulter 1997). Our previous investigation demonstrated that after stimulated by ConA and LPS, the splenocyte stimulation index (SI) was significantly enhanced in ICR mice by soyasaponin Ab at appropriate doses (0.01-10 [micro]g/ml) compared with the control and Alum groups (Qiao et al. 2014). Our previous results also indicated that soyasaponin Ab and QS could significantly increase the activation potential of T and B cells in ICR mice, while Alum demonstrates poor adjuvant activity at inducing cellular immune responses (Qiao et al. 2014).

In this study, it is consistent with our previous observation that soyasaponin Ab is effective on both Th1 and Th2 cells, as shown with an enhancement of IgG1, IgG2a and IgG2b levels. However, compared with QS, soyasaponin Ab showed weak immunological activity (Fig. 6). QS is a representative adjuvant with stronger adjuvant activity compared to many other plant saponins. It was reported that quillaja saponin extract expressed higher antibody activity at a dose of 20 [micro]g compared to ARS saponins with doses of 100 [micro]g (Sun et al. 2008). Oda et al. (2003) demonstrated a positive correlation of QS between the effect to activate IgG subclass and injection doses at a range of 0-100 [micro]g. Moreover, QS (50 [micro]g) exhibited a much higher activation of the production of IgG and IgG subclass compared to soyasaponin Ab (50 [micro]g) in this study. The high HLB value of QS may account for its strong adjuvanticity on the basis that several saponins reported to be strong adjuvants demonstrated higher HLB values (Bomford et al. 1992; Nagai et al. 2001; Oda et al. 2003). Furthermore, additional consideration of the other chemical structure factors might secondarily explain its adjuvant activity (Oda et al., 2000). On the other hand, as mentioned above, dose-response studies showed that at a suitable dose of soyasaponin Ab could enhance serum antibody production in mice immunized with OVA. Our data suggest that the appropriate soyasaponin Ab dosage appear to be 50-250 [micro]g on the induction of both cell-mediated and humoral immune responses against OVA in mice.

In summary, this study reports the in vitro cytotoxicity and immune stimulating potential of purified soyasaponin Ab, a major constituent among group A soyasaponins in soybeans from native soy hypocotyls which contain the highest content of soyasaponins compared to the other parts of the soybean. In addition, its immune adjuvant activity is also evaluated in vivo in a mouse model of immunization with the prototype antigen, OVA. In general, in our conditions, both the in vivo and in vitro experimental results indicated the high adjuvant activity of soyasaponin Ab with a significant enhancement in the specific antibody and cellular response. Our results indicated that the assayed soyasaponin Ab showed very weak or no haemolytic activity over the tested concentration range compared to QS. The compound is soluble in water and, thus, it yield homogenous preparations when combined with aqueous antigen solutions. Soyasaponin Ab could activate the expression of NF-[kappa]B in RAW264.7, TLR4 may play an important role in the immune effects of soyasaponin Ab. From the results of four different assays applied to study the immunomodulatory activity, it is clear that soyasaponin Ab is potent immunostimulant. Further studies should be carried out to confirm and characterize the activity of soyasaponin Ab together with other soyasaponin components so that these molecules can be utilized as immunomodulators in adjuvant therapy.

ARTICLE INFO

Article history:

Received 25 March 2014

Received in revised form 2 August 2014

Accepted 13 September 2014

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 31071630/C200303). We are grateful to the analysis centre in the Shanghai Zizhu Industrial Park for assisting with our experiments.

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Tao Sun (a,c,) Xinbin Yan (b), Wenxiu Guo (b), Dayun Zhao (b,d,) *

(a) Department of Animal Science, Shanghai Jiao Tong University, Shanghai 200240, China

(b) Department of Food Science and Technology, Shanghai Jiao Tong University, Shanghai 200240, China

(c) Shanghai Municipal Veterinary Key Laboratory, 800 Dongchuan Road, Shanghai 200240, China

(d) Bor S. Luh Food Safety Research Center, Shanghai Jiao Tong University, Shanghai 200240, China

* Corresponding author at: Department of Food Science and Technology, Shanghai Jiao Tong University, P.O. Box 45, Agricultural Building, 800 Dongchuan Road, Minhang District, Shanghai 200240, China. Tel.: +86 2134205711; fax: +86 21 34205711.

E-mail addresses: dyzhao@sjtu.edu.cn, dayunzhao@hotmail.com (D. Zhao).

http://dx.doi.Org/10.1016/j.phymed.2014.09.002

Table 1
Candidate siRNA sequences targeted at mouse
TLR 4 gene expression.

siRNA                      Sequences

siRNAl                     5'-CUAUAGCUUCUCCAAUUUTT-3' (forward)
                           5'-AAAUUGGAGAAGCUAUAGCn'-3' (reverse)
siRNA2                     5'-GGACAGCUUAUAACCUUAATT-3' (forward)
                           5'-UUAAGGUUAUAAGCUGUCCrr-3' (reverse)
siRNA3                     5'-CCUCCAUAGACUUCAAUUATT-3' (forward)
                           5'-UAAUUGAAGUCUAUGGAGGTT-3'(reverse)
Scramble siRNA negative    5'-UUCUCCGAACGUGUCACGUTT-3' (forward)
  control
                           5'-ACGUGACACGUUCGGAGAATT-3' (reverse)

Table 2
Haemolytic activities of soyasaponin Ab.

Group              Concentration    Haemolytic percent (%)
                   ([micro]g/ml)

Distilled water    ND               100.00 [+ or -] 1.56
PBS                ND                0.000 [+ or -] 0.66
Ab QS              200                3.75 [+ or -] 1.02
                   150                3.32 [+ or -] 0.88
                   100                0.61 [+ or -] 0.66
                   50                 0.08 [+ or -] 0.51
                   30                 90.2 [+ or -] 2.5
                   20                 68.8 [+ or -] 3.7
                   10                 21.1 [+ or -] 5.0

ND: not detectable.
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Author:Sun, Tao; Yan, Xinbin; Guo, Wenxiu; Zhao, Dayun
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Nov 15, 2014
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