Enhancement of the Immune Responses to Ovalbumin in Mice by Oral Administration of the Extract from Radix Cyathulae (RC)

Full Length Research Paper

Enhancement of the immune responses to ovalbumin in mice by oral administration of the extract from Radix cyathulae (RC)

Haibo Feng 1, 2# , Xiaogang Du 1# , Xiaohan Cao 1, Zhenhua Wang 1, 3, Jing Tang 1, Juan Liu 2, Bing Zhao 1, Zhiyu Chen 1 and Xianyin Zeng 1*

1Isotope Research Laboratory, College of Life and Physical Science, Sichuan Agricultural University, Ya’an, Sichuan 625014, PR China. 2Department of Veterinary Medicine, Southwest University, Rongchang, Chongqing, 402460, PR China. 3Department of Animal and Veterinary Science, Chengdu Vocational College of Agricultural Science and Technology, Wen Jiang, Sichuan 611130, P. R. China.

Accepted 12 July, 2012

This study was designed to evaluate the effects of oral administration of the extract from the Radix cyathula (RC) on the immune responses in mice immunized with ovalbumin (OVA). Forty-two Institute of Cancer Research (ICR) mice were randomly divided into six groups with seven animals in each group, and orally administered daily for 4 days at a dose equivalent to 0, 0.05, 0.1, 0.2, 0.4 and 0.8 g of the RC extract, respectively. After that, the mice were subcutaneously immunized twice with OVA at 2 weeks intervals. On day 14 after the second immunization, OVA-specific Immunoglobulin G (IgG) and the IgG subclasses, lymphocyte proliferation response, as well as interleukin-4 (IL-4) and interferon-gamma (IFN-γ) production were measured. Results indicated that the optical density (OD) value of OVA-specific IgG and the IgG subclass were significantly enhanced in mice orally administered RC at the dose of 0.2, 0.4 or 0.8 g, compared to the control group ( p < 0.05). Besides, the splenocytes proliferation response and the production of IL-4 and IFN-γ were also significantly improved ( p < 0.05) after the splenocytes were stimulated by OVA. In addition, the body weight of the mice administered with the RC extract (0, 0.1, 0.2, 0.4 and 0.8 g) was not significantly different compared to the mice administered saline. Collectively, the data suggested that RC extract could improve the immune response of vaccine through up-regulating the humoral immune response and the cellular immune response.

Key words: Radix cyathula , ovalbumin, adjuvant.

INTRODUCTION

Vaccination is the most cost-effective approach for the reversion (Lima et al., 2004). But, the new generation control of infectious diseases. The traditional vaccines, vaccines such as puriﬁed recombinant proteins, synthetic typically including the live attenuated or inactivated whole peptides and plasmid DNA, are unfortunately often much organisms, have been very successful in inducing less reactogenic and immunogenic (Hagan et al., 2001). immune response. However, the safety of the traditional Many strategies are used to improve the immune res- vaccines is questionable due to the risk of the virulence ponse to vaccination including the use of higher vaccine

*Corresponding author. E-mail: [email protected] or [email protected]. Tel: +86 835 2886136. Fax: +86 835 2886136.

#These authors contributed equally to the work. Feng et al. 1273

dose, increasing number of doses, the different route of gradually declined to the room temperature and the light time is up administration (for example, intradermal versus intra- to 12 h/day. All procedures related to the animals and their care muscular administration), accelerating dosing schedule conformed to Guidelines for Keeping Experimental Animals issued by the government of China. and adjuvants such as antigen delivery systems and various immune modulators (Rawer et al., 1987; Donati and Gastaldi, 1988; Quiroga et al ., 1990; Bryan et al., Materials 1992; Jungers et al., 1994; Taglietti, 1995; Disis et al., 1996; Mitwalli et al., 1996; Rey et al., 2000). Among OVA, concanavalin A (ConA), lipopolysaccharide (LPS), RPMI-1640 medium, and goat anti-mouse Immunoglobulin G (IgG) peroxidase them, using the adjuvants maybe is a feasible way to conjugate were purchased from Sigma Chemical Co., Ltd (Saint increase the potency on the appropriate immune Louis, Missouri, USA). The tachypleus amebocyte lysate (TAL) response (Rock et al., 2005; Storni et al., 2005). (sensitivity: λ = 0.125 EU/ml) was bought from Zhanjiang A&C There are many growing evidences that medicinal Biological Ltd. (Zhanjiang, China). The control standard endotoxin herbs and the ingredients can enhance the immune (CSE) and water for the bacterial endotoxin test (BET) were response to vaccines direct against infectious agents (Liu provided by National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Cell counting kit-8 (CCK-8) et al., 1992; Rajput et al., 2007). It has demonstrated that was obtained from Beyotime institute of Biotechonology Haimen the co-administration of vaccines and herbal extracts has (Jiangsu, China). The goat anti-mouse IgG, IgG1, IgG2a and IgG2b increased the antibody response and the T cell conjugated with the peroxidase were gotten from Santa Cruz proliferative response (Hu et al., 2003; Sun et al., 2007). Biotechnology Inc (California, USA). Enzyme-linked immunosorbent Several herbal extracts as adjuvant of vaccines have assay (ELISA) kits of detecting interleukin-4 (IL-4) and interferon- been used in the human and animal clinical trials gamma (IFN-γ) were from Wuhan Boster Biological Technology Co. Ltd (Hubei, China). Fetal calf serum (FCS) was provided by (Scaglione et al., 1990; Scaglione et al., 1994; Scaglione Invitrogen Gibco Biotechnology Inc (California, USA). et al., 1996). Cyathula officinalis (Amaranthaceae family) is a Preparation of RC extract perennial herbaceous plant widely distributed in tropical areas of Asia and Africa, particularly in China, Korea and Dried RC was purchased from Tong Ren Tang Co. Ltd (Ya’an, Vietnam. Its root (“Chuan Niu Xi” in Chinese, C. officinalis China). After cleaning and drying, RC was cut into pieces, and kuan ) is a kind of commonly-used Chinese traditional ground into powder. Then, 100 g of RC powder was saturated in herbal medicine with a wide range of pharmacological 1000 ml of water. After that, the supernatant was collected by activities (National Pharmacopoeia Committee, 2005a). It passing through a filter paper and condensed at a concentration equivalent to 4 g of dried RC per ml by a R502B rotary evaporator is usually used as a tonic, emmenagogue, antiarthritic, (Shenko Tech Co. Ltd., Shanghai, China). diuretic, and antifertility agent to nourish the liver and The diluted preparations were sterilized by pasteurization, and kidneys, strengthen bones and muscles, and invigorate the endotoxin content was determined using the Limulus circulation (National Pharmacopoeia Committee, 2005b). amebocyte lysate test (National Pharmacopoeia Committee, 2005). The modern pharmacological studies demonstrated that When the endotoxin level in RC extract solution was less than 0.5 EU/ml, it was stored at 4°C for us in the future. the root of C. officinalis possessed many functions such as immunostimulant (Ye et al., 2007; Wang et al., 2007), uteri-excitant and antifertility (Li et al., 1990; Li et al., Oral administration of RC extract and immunization 1998), antitumor (Song, 1994; Chen et al., 2003a), Forty-two ICR mice were randomly divided into six groups (n = 7). analgestic, antibacterial, anti-inflammatory (Song and The animals were subcutaneously injected twice with 100 g of Yang, 2001), antisenile (Li et al., 1998). However, to date, OVA with 2 weeks intervals, as described previously (Li et al ., Radix Cyathulae officinalis Kuan (RC) as an adjuvant for 2009). Before each immunization, the mice had been orally vac-cines has not yet been reported. In the present study, administered daily for 4 days with 0.25 ml of 0.9% saline solution we attempted to evaluate the enhancement of the containing RC of 0, 0.05, 0.1, 0.2, 0.4 or 0.8 g (equivalent to 0, immune responses to ovalbumin (OVA) in mice by oral 2.05, 4.1, 8.2, 16.4 or 32.8 g/kg bodyweight). administration of the RC extract. In this study, we demonstrated for the first time that RC can efficiently enhance Enzyme-linked immunosorbent assay (ELISA) the immunogenicity of OVA via improving the humoral and cellular immune responses and the cytokine Blood samples were collected at 2 weeks after the boost, the expression. optical density (OD) value of OVA-specific IgG and isotypes were measured by an indirect ELISA as described by Du (2007). Briefly,

the microtiter plate wells were coated with 100 l of OVA at 50, 25, MATERIALS AND METHODS 50 and 50 g/ml for testing IgG, IgG1, IgG2a and IgG2b antibodies overnight at 4°C. After three washes, the wells were blocked with Animals 5% skimmed milk and incubated for 1h at 37°C. Followed by three times of washing, 100 l of serum (diluted 1:50 in phosphate- Female Institute of Cancer Research (ICR) mice (Grade II, 5 weeks buffered saline (PBS) 5% skimmed milk) was added to each well old) were purchased from Sichuan Laboratory Animal Center and incubated at 37°C for 1 h. The horseradish peroxidase- (SLAC) Co. Ltd. (Sichuan, China), and housed in polypropylene conjugated antibodies against IgG, IgG1, IgG2a or IgG2b were cages with sawdust bedding in hygienically controlled environment. incubated for 1 h at 37°C. After washing, the peroxidase activity Food and water were supplied ad libitum . The temperature was was assayed as following: the substrate solution (10 mg of O- 1274 J. Med. Plants Res.

0.6 a 0.5 b b bc 0.4 c 0.3 c 0.2

OD0.1 value(450 nm) 0 Saline 0.05 0.1 0.2 0.4 0.8 RC (g/dose)

Figure 1. OVA-speciﬁc IgG. Mice were orally administered with RC solution (0, 0.05, 0.1, 0.2, 0.4 or 0.8 g of RC/mouse/day) for 4 days, and then immunized s.c. twice with 100 g of OVA at 2 weeks intervals. Blood samples were collected at the second week after boost for testing the OD value of OVA-speciﬁc IgG by an indirect ELISA. The OD values are presented as mean ± SD (n = 7). Bars with different letters are statistically different ( p < 0.05).

phenylenediamine and 37.5 l of 30% H2O2 in 25 ml of 0.1 M Statistical analysis citrate–phosphate buffer, pH 5.0) was added to each well, and incubated for 10 min at 37°C. The reaction was stopped by 2 M Data analysis was performed with SPSS software (SPSS, Version H2SO 4. The OD was measured in an ELISA reader at 450 nm. Data 11.5, SPSS Inc., Chicago, IL). Analysis of variance (ANOVA) with were expressed as the mean OD value of the samples minus the Bonferroni post-hoc test was used for multiple comparisons mean OD value of the blank control. between groups. Values were expressed as the mean ± standard deviation (SD). p-Values of less than 0.05 were considered statistically signiﬁcant. Splenocyte proliferation assay

Mice were sacrificed on day 14 after the boost, and spleen were RESULTS collected from the mice under aseptic conditions. The spleen was minced and passed through a fine steel mesh to obtain a homogeneous cell suspension. Then, the erythrocytes were lysed Humoral immune response with ammonium chloride solution (0.8%, w/v). After washed three times in PBS, and the splenocytes were suspended incomplete To evaluate the adjuvant effect of RC on the humoral medium (RPMI 1640 supplemented with 0.05 mM 2- immune responses, the OD value of the antigen specific mercaptoethanol, 100 UI/ml penicillin, 100 g/ml streptomycin and IgG and the IgG subclasses were measured by the 10% heat inactivated FCS). The splenocytes were counted with the indirect ELISA. As shown in Figure 1, the OD values of haemocytometer by the trypan blue dye exclusion technique. Splenocytes proliferation was performed as previously described IgG were significantly higher in mice orally administered (Sun et al., 2004). Briefly, splenocytes were seeded into four wells RC at a dose of 0.05, 0.1, 0.2, 0.4 and 0.8 g than that in of a 96-well flat-bottom microtiter plate at 2.5 × 10 6 cell/ml in 100 l mice administered saline solution only ( p < 0.05). OD complete medium, thereafter ConA (5 g/ml), LPS (10 g/ml), OVA value of IgG increased with the dose dependence (5 g/ml), or medium were added giving a final volume of 200 l. pattern. In addition, OD values of IgG1, IgG2a, and The plates were incubated in a humid atmosphere with 5% CO 2 at IgG2b isotypes were higher in mice administered RC 37°C. After 44 h, 20 l of WST-8 solution (5 mg/ml) was added to each well and incubated for another 4 h. The OD at 450 nm was than that in the saline group (Figure 2). But, the determined using a microplate reader. The stimulation index (SI) significantly increased OD values of IgG and isotypes was calculated based on the following formula: SI = the absorbance were observed in mice administered RC at doses of 0.4 value for the stimulation-cultures divided by the absorbance value and 0.8 g ( p < 0.05). for non-stimulated cultures.

Splenocytes proliferation response IFN-γ and IL-4 production by spleoncyte To detect the effects of RC on splenocyte proliferative The single splenocyte suspension was prepared on day 14 after the responses, the proliferation assay was performed by the × 6 boost, and the splenocyte (2.5 10 cells/ml) were incubated with WST method. As shown in Figure 3, SI of the proliferation OVA (ﬁnal concentration 5 g/ml) in 96-well culture plates at 37°C in response to ConA, LPS and OVA was gradually 5% CO 2. After 48 h, the culture supernatant was collected for the detection of IL-4, and IFN-γ levels by the commercial ELISA kits increased in mice administered at 0.2, 0.1 and 0.05 g of according to the manufacturer’s instructions. RC, and signiﬁcantly increased in mice administered at Feng et al. 1275

0.8 saline RC 0.05g a a RC 0.1g 0.7 a a a RC 0.2g RC 0.4g b RC 0.8g 0.6 b bc bc 0.5 bc c c a a 0.4 ab b 0.3 b

OD value (450 nm) (450 value OD c 0.2

0.1

0 IgG1 IgG2a IgG2b IgG isotypes

Figure 2. OVA-speciﬁc IgG isotypes. Mice were orally administered RC solution (0, 0.05, 0.1, 0.2, 0.4 or 0.8 g of RC/mouse/day) for 4 days, and then immunized s.c. twice with 100 g of OVA at 2 weeks intervals. Blood samples were collected at the second week after the boost for testing OD values of OVA-speciﬁc IgG isotypes by an indirect ELISA. The values are presented as mean ± SD (n = 7). Bars with different letters are statistically different ( p < 0.05).

4 a a a Saline RC(0.05g) 3.5 RC(0.1g) RC(0.2g) RC(0.4g) RC(0.8g) 3 b a a a b 2.5 ab ab a 2 ab a b b ab 1.5 b b 1 0.5 Stimulation Indes (SI) Indes Stimulation 0 ConCoAA LPS OVA OVA Mitogens

Figure 3. Splenocyte proliferative responses. Mice were orally administered RC solution (equivalent to 0, 0.05, 0.1, 0.2, 0.4g or 0.8 g of RC/mouse/day) for 4 days, and then immunized s.c. twice with 100 g of OVA at 2 weeks intervals. Splenocytes were prepared at second week after the boost and cultured with ConA (5 g/ml) or LPS (10 g/ml) or OVA (5 g/ml) or RPMI- 1640 for 48 h. Splenocytes proliferation was measured by the WST-8 method as described in in the work and shown as a stimulation index (SI). The values are represented mean ± SD (n = 7). Bars with different letters are statistically different ( p < 0.05).

0.8 and 0.4 g of RC compared to the control group ( p < and IFN -γ production was measured by ELISA. As shown 0.05). in Figure 4A, IL-4 production was signiﬁcantly higher in the mice administered RC (0.8 and 0.4 g) than that in the mice administered saline solution (p < 0.05). Moreover, Effects of RC extract on IL-4 and IFN-γ IFN-γ production was signiﬁcantly higher in the group administered RC (0.8, 0.4, 0.2 and 0.1 g) than that in the To test, RC extract influences the cytokine secretion, IL-4 group administered saline solution (Figure 4B). 1276 J. Med. Plants Res.

400 A a 350 a

300 A 250 bc 200 c c c 150 100 IL-4contens(pg/ml) 50 0 Sline RC0.05g RC0.1g RC0.2g RC0.4g RC0.8g

2000 a B a 1800 b 1600 B 1400 c 1200 1000 d d 800

contents(pg/ml) 600 γ 400

IFN- 200 0 Saline RC0.05g RC0.1g RC0.2g RC0.4g RC0.8g

Figure 4. IL-4 (A) and IFN-γ (B) production. Mice were orally administered RC solution (0, 0.05, 0.1, 0.2, 0.4 or 0.8 g) for 4 days, and then immunized s.c. twice with 100 g of OVA at 2 weeks intervals. Splenocytes were prepared at second weeks after the last immunization and stimulated with OVA (5 g/ml) for 48 h. The supernatants were harvested for determination of IL-4 and IFN-γ production by a capture ELISA. The values are presented as mean ± SD (n = 7). Bars with different letters are statistically different ( p < 0.05).

Effects of RC on the body weight of mice including mineral salts, microorganism-derived adjuvants, cytokines, emulsions and polysaccharides, have been To evaluate the safety of RC extract, the body weight of tested for the research or usage in novel vaccine design mice was measured before and after the first over the last few decades, the vast majority of the immunization and boost. No abnormal behavior and side adjuvants have not been successfully approved for effects were observed in mice throughout the experiment. human use, because of the unacceptable local or syste- Besides, there was no signiﬁcant difference in the body mic toxicity reaction (McCluskie and Weeratna, 2001; weight between the mice administered RC (0, 0.05, 0.1, Spickler et al., 2003; Aguilar and Rodriguez, 2007). 0.2, 0.4 or 0.8 g) and the control group (Table 1) (p > Many natural products have been reported having 0.05). immunomodulatory properties, while their active ingredients are not clear (Banji et al., 2012). It is difficult to purify the herbal extracts, because the co-injection with DISCUSSION the unpurified herbal extracts and antigens can induce the irritation (Qi et al., 2011). The traditional medicinal An efficient adjuvant should have the negligible toxicity herbs are administered by oral route, because the oral and elicit the robust humoral or/and cellular immune res- use of immunomodulators can avoid the side effects ponse against the specific antigen (Marciani et al., 2003). elicited through the parenteral administration (Hu et al., While several hundred different adjuvants, including 2011). For example, oral administration of Rhizoma Feng et al. 1277

Table 1. Effects of oral administration of RC on the mean body weight (mean ± S.D, n = 7).

Gram of RC/mouse Before first immunization Before boost 2 Weeks after boost 0 22.62 ± 0.62 26.08 ± 0.28 26.89 ± 0.57 0.05 23.00 ± 0.96 27.10 ± 0.73 27.62 ± 0.52 0.1 22.37 ± 0.16 26.51 ± 0.42 26.97 ± 0.96 0.2 23.06 ± 0.31 26.66 ± 0.45 27.14 ± 0.70 0.4 23.22 ± 0.14 25.82 ± 0.55 27.40 ± 0.46 0.8 22.92 ± 0.69 26.28 ± 1.05 27.08 ± 0.75

Atractylodis Macrocephalae extract can significantly pathogens such as viruses and the certain bacteria, but increased the immune responses against foot-and-mouth Th2 response mainly targets the extracellular pathogens, disease in mice, no abnormal behavior and side effects such as most bacteria and certain parasites (Chavali et were found in mice throughout the experiment (Li et al., al., 1987; Soren et al., 2000). The higher level of all IgG 2009). Maharaj et al. (1986) and Chavali et al. (1987) subclasses may be explained by increasing both IL-4 and reported that the crude saponin from the bark of the IFN-γ production (Figure 4), suggesting that RC can Quillaja tree by the oral administration had the immuno- enhance both Th1 and Th2 immune responses. potentiating activity with moderated toxicity, however, In addition to the immunomodulatory effects, RC has they were toxic when administered parenterally the anti-inflammatory activity as well. RC together with The cellular immune response plays an important role other herbs has been used to treat the inflammatory in the host response against the intracellular pathogens reaction in the human upper respiratory tract (Ye et al., via limiting their replication and accelerating clearance of 2007). Shi and Zheng, (1988) have found that the OVA- the infected cells (Arama et al ., 2012). Among T- induced the acute inflammatory edema of the foot in rats lymphocytes, the helper T-cells induces B-lymphocytes to was significantly reduced after the oral administration of secrete antibodies, and the cytotoxic T-lymphocytes help the compounds such as polysaccharide extracted from phagocytes to destroy ingested microbes and to clear the the RC. Imbalance of Th1/Th2 lymphocytes may be one intracellular microbes. However, the humoral immunity of the reasons for some inflammatory diseases (Smart et mediated by antibodies, neutralize and eliminate the al., 2002). In this study, RC has been found to stimulate extracellular microbes and microbial toxins (Montomoli et both IFN-γ (Th1-like cytokine) and IL-4 (Th2-like al ., 2011). The capacity to elicit the effective T- and B- cytokine), suggesting that RC can promote the balanced lymphocyte immunity can be shown by the lymphocyte Th1/Th2 response. This may constitutes the anti- proliferation response (Effros, 2007). It is generally known inflammatory mechanism of RC. that ConA stimulates T-cells and LPS stimulates B-cell Adjuvant should be safe enough to induce the minimal proliferation. In this study, RC promoted the ConA-, LPS- adverse effects for use in human and animals. In this and OVA-stimulated splenocyte proliferation in the study, no abnormal behaviors and side effects were immunized mice (Ren et al., 2012). It indicates that RC observed in mice throughout the experiment, and there can significantly improve the humoral immunity and cell- was no significant difference in the body weight between mediated immune response via increasing the activation the mice administered RC and the control mice (Table 1). potential of T- and B-cells. Similar results have been It suggests that oral administration of RC is safe. found in other studies. Jia et al . (2009) reported that oral RC extract contains a lot of bioactive ingredients such administration of the extract from RC for 10 days as saponins and polysaccharide, isoflavones and significantly enhanced lymphocyte transformation in phytoecdysteroids, especially sapoins and polysac- response to ConA stimulation in mice. Xiang et al. (1994) charide, which have been proved to possess the strong have demonstrated that the lymphocyte proliferation in immunomodulatory properties (Hikino et al., 1968; Hikino response to ConA and LPS stimulation and natural killer et al., 1970; Hikino et al ., 1971; Chen et al., 2003b). Jia et (NK) cell activity were enhanced in rats fed by a diet al . (2009) have found that IgM, IgG and IgA in serum supplemented with the RC powder. were significantly increased in chicken orally admini- The cytokines have the pivotal function in regulating the stered with the polysaccharide fractions for 24 days. immune response. Th2 lymphocyte cytokines such as IL- Besides, Li et al . (1998) have reported that the erythro- 4, IL-5 and IL-10 can augment the IgG1 production, while cyte total rosette rate in the mice orally administered of IL-2, TNF-α and IFN-γ produced by Th1 lymphocytes can RC polysaccharide increased significantly compared with improve IgG2a production (Chavali et al., 1987; Soren et the control group. Therefore, the immunomodulatory al., 2000). Oral administration of RC significantly effects found in our study may facilitate to further study increased the level of IgG1, IgG2a and IgG2b (Figure 2). the biofunction of RC extract. In terms of the host response against the infectious In summary, the oral administration of RC significantly diseases, Th1 response primarily targets the intracellular increased the level of IgG and IgG subclass, splenocyte 1278 J. Med. Plants Res.

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