The improvement effects of edible bird’s nest on proliferation and activation of B lymphocyte and its antagonistic effects on immunosuppression induced by cyclophosphamide

Figures & data

Table 1 Amino acid distribution (mg/g) of EBNE

Name	Total amino acid (T)	Free amino acid (F)	(F/T) ×100
Aspartic acid	41.8	0	0
Threonine	33.1	0.12	0.36
Serine	35.4	0.19	0.54
Glutamic acid	34.6	0.40	1.16
Proline	40.3	0	0
Glycine	17.6	0.16	0.91
Alanine	20.2	0.20	0.99
Valine	31.5	0.14	0.44
Methionine	2.7	0.13	4.81
Isoleucine	14.2	0.14	0.99
Leucine	32.3	0.17	0.53
Tyrosine	33.2	0.18	0.54
Phenylalanine	28.9	0.15	0.52
Histidine	14.7	0	0
Lysine	15.1	0	0

Notes: EBN was dried at 50°C for 24 hours and then ground. The ground EBN was kept in PBS at 30°C for 24 hours. This suspension was then fully ground using a tissue grinder, and after centrifugation for 10 minutes at 3,000× g, the supernatant was collected and subjected to 0.2 μm filtration. The amino acid distribution (mg/g) of the EBNE was determined using an L-8900 automatic amino acid analyzer.

Abbreviations: EBNE, EBN extract; EBN, Edible bird’s nest; PBS, phosphate buffered saline.

Figure 1 SDS–PAGE analysis of EBNE protein.

Notes: The EBN was dried for 24 hours at 50°C and then ground. The ground EBN was kept in PBS at 30°C for 24 hours. The suspension was fully ground by Tissue Grinders, and then, the supernatant was obtained after centrifugation for 10 minutes at 3,000× g and subjected to 0.2 μm filtration. Protein concentration of EBN extract determined using the BCA protein assay kit was 0.75 mg/mL, amino acid distribution (mg/g) of EBNE was determined using L-8900 automatic amino acid analyzer. Add 200 μL SDT (4% SDS, 100 mM DTT, 150 mM Tris–HCl pH 8.0) into EBN protein extracts, then emerge into a boiling bath for 5 minutes. After centrifugation for 5 minutes, 10 μL of supersonic lysate was analyzed by SDS–PAGE.
Abbreviations: SDS–PAGE, SDS–polyacrylamide gel electrophoresis; EBNE, EBN extract; BSA, bovine serum albumin; EBN, Edible bird’s nest; PBS, phosphate buffered saline.

Figure 2 Effects of EBNE on the proliferation of lymphocytes.

Notes: Mouse lymphocytes were incubated at 10⁵ cells/well for 48 hours with 2.5 μg/mL ConA and EBN extract at varying concentrations. Proliferation was determined by CCK-8 kit. Data are mean ± SD (n=3/treatment). **Significant difference from control group (P<0.01). ^##Significant difference from ConA group (P<0.01).
Abbreviations: EBNE, EBN extract; EBN, Edible bird’s nest; ConA, concanavalin A; SD, standard deviation; EBNL, EBN low dose; EBNM, EBN medium dose; EBNH, EBN high dose.

Figure 3 Effects of EBNE on the proliferation of B-cell.

Notes: CFSE-labeled splenocytes were incubated with 5 μg/mL LPS or EBNE at varying concentrations for 72 hours at 37°C with 5% CO₂, then stained with CD19 for 30 minutes at 37°C in darkness. The cells were washed twice with PBS, and cell proliferation was measured by flow cytometry and analyzed with FlowJo Version 7.6.5 analysis software.
Abbreviations: EBNE, EBN extract; EBN, Edible bird’s nest; CFSE, carboxyfluorescein diacetate succinimidyl ester; CTRL, control; LPS, lipopolysaccharide; EBNL, EBN low dose; EBNM, EBN medium dose; EBNH, EBN high dose; CFDA-SE(PE), carboxyfluorescein diacetate-succinimidyl ester (phycoerythrin); PBS, phosphate buffered saline.

Figure 4 Effects of EBNE on the proliferation of T-cell.

Notes: CFSE-labeled splenocytes were incubated with 5 μg/mL ConA or EBNE at varying concentrations for 72 hours at 37°C with 5% CO₂, then stained with CD3 for 30 minutes at 37°C in darkness. The cells were washed twice with PBS, and the cell proliferation was measured by flow cytometry and analyzed with FlowJo Version 7.6.5 analysis software.
Abbreviations: EBNE, EBN extract; EBN, Edible bird’s nest; CFSE, carboxyfluorescein diacetate succinimidyl ester; ConA, concanavalin A; CTRL, control; EBNL, EBN low dose; EBNM, EBN medium dose; EBNH, EBN high dose; CFDA-SE(PE), carboxyfluorescein diacetate-succinimidyl ester (phycoerythrin); PBS, phosphate buffered saline.

Table 2 Effects of EBN extracts on the activation of B-cells

Group	CD19^+CD69^+/CD19^+ (%)	CD19^+CD25^+/CD19^+ (%)	CD19^+CD71^+/CD19^+ (%)
CTRL	21.6±0.95	13.37±2.65	34.9±3.00
LPS	83.9±2.84**	79.30±1.48**	89.7±0.68**
EBNL	41.3±1.93**,##	27.63±2.54**,##	48.6±1.04**,##
EBNM	51.1±2.43**,##	34.60±2.95**,##	59.0±3.33**,##
EBNH	64.4±1.42**,##	46.87±0.93**,##	72.0±1.92**,##

Notes: Cultures were incubated with 20 μL LPS (5 μg/mL) or different concentrations of EBNE (0.19 mg/mL, 0.38 mg/mL, or 0.75 mg/mL) and were harvested for 6 hours, 24 hours, or 48 hours. The cells were then double stained with antibodies against CD19 and the CD69, CD25 or CD71 antigen for 30 minutes at 37°C in darkness. The cells were washed and phenotyped by flow cytometry. The data are the mean ± SD (n=3/treatment).

** Significant difference from control group (P<0.01).

## Significant difference from LPS group (P<0.01).

Abbreviations: EBN, Edible bird’s nest; CTRL, control; LPS, lipopolysaccharide; EBNL, EBN low dose; EBNM, EBN medium dose; EBNH, EBN high dose; EBNE, EBN extract; SD, standard deviation.

Table 3 Determination of B-lymphocyte proliferation by cytometry-based cell counting

Group	PI (without polymyxin B)	PI (with polymyxin B)
Control	1.08±0.005	1.08±0.005
LPS	1.59±0.026&&	1.19±0.02**,$$
EBNL	1.12±0.011&&	1.16±0.012**,$$
EBNM	1.15±0.01&&	1.19±0.012**,$$
EBNH	1.14±0.005&&	1.24±0.02**,$$

Notes: A total of 10 μg polymyxin B/mL and 10 μg LPS/mL or EBNE (0.19 mg/mL, 0.38 mg/mL, or 0.75 mg/mL) were dissolved in media and incubated with cells for 72 hours at 37°C with 5% CO₂. Following incubation, the B lymphocytes were stained with anti-CD19 for 30 minutes at 37°C in darkness. The samples were analyzed by flow cytometry. The data are the mean ± SD (n=3/treatment).

&& or

** Significant difference from control group (P<0.01).

$$ The PI (with polymyxin B) significant difference from the PI (without polymyxin B) (P<0.01).

Abbreviations: LPS, lipopolysaccharide; EBNL, EBN low dose; EBNM, EBN medium dose; EBNH, EBN high dose; EBNE, EBN extract; PI, proliferation index; SD, standard deviation; EBN, Edible bird’s nest.

Table 4 Effect of EBN on the production of IgE, IgA, and IgM

Group	IgE	IgA	IgM
Control	4.08±0.13	8.74±0.71	31.35±4.83
LPS	5.43±0.1*	20.33±1.0**	84.37±16.39*
EBN	5.28±1.1*	17.28±1.0**	79.24±16.03*

Notes: Cells were plated at 3×10⁶ cells/well. A total of 20 μg LPS (5 μg/mL) or EBNE (0.38 mg/mL) was dissolved in media and incubated with the cells for 72 hours at 37°C with 5% CO₂. Following incubation, the concentrations of IgE, IgA, IgM in the supernatant were measured using a CBA Mouse Immunoglobulin Isotyping Kit. The data are the mean ± SD (n=3/treatment).

* Significant difference from control group (P<0.05).

** Significant difference from control group (P<0.01).

Abbreviations: LPS, lipopolysaccharide; EBN, Edible bird’s nest; EBNE, EBN extract; SD, standard deviation; CBA, cytometric bead array.

Table 5 Effect of EBN on the production of IgG3, IgG1, IgG2a, and IgG2b

Group	IgG3	IgG1	IgG2a	IgG2b
Control	4.47±0.14	5.37±0.71	4.83±0.025	4.63±0.56
LPS	6.36±0.72**	8.35±1.21*	9.04±2.73*	10.63±4.91*
EBN	5.85±0.63*	7.79±1.63	6.87±1.14	6.51±1.32

Notes: Cells were plated at 3×10⁶ cells/well. A total of 20 μg LPS (5 μg/mL) or EBNE (0.38 mg/mL) was dissolved in media and incubated with the cells for 72 hours at 37°C with 5% CO₂. Following incubation, the concentrations of IgG3, IgG1, IgG2a, and IgG2b in the supernatant were measured using a CBA Mouse Immunoglobulin Isotyping Kit. The data are the mean ± SD (n=3/treatment).

* Significant difference from control group (P<0.05).

** Significant difference from control group (P<0.01).

Abbreviations: LPS, lipopolysaccharide; EBN, Edible bird’s nest; SD, standard deviation; CBA, cytometric bead array.

Table 6 Effect of orally administered EBN on the secretion of intestinal sIgA

Group	Intestinal sIgA (µg/mL)
Control	13.83±0.99
Model	8.26±1.09**
EBNL + model	10.50±1.80**,##
EBNM + model	12.87±1.38##
EBNH + model	11.04±1.82**,##

Notes: The lumen of the proximal 10 cm of the small intestine was washed with 3 mL PBS. After centrifugation (4°C, 3,000× g for 10 minutes), the supernatant was frozen at −80°C until use. Measurement of total IgA was performed using a sandwich-type ELISA. The data are the mean ± SD (n=6/treatment).

** Significant difference from control group (P<0.01).

## Significant difference from CY group (P<0.01).

Abbreviations: EBN, Edible bird’s nest; EBNL, EBN low dose; EBNM, EBN medium dose; EBNH, EBN high dose; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; PBS, phosphate buffered saline; CY, cyclophosphamide.

Table 7 Effects of EBNE on CD3⁺/CD19⁺ lymphocytes among PP cells

Group	CD3^+ (%)	CD19^+ (%)
Control	29.03±1.23	53.90±3.63
Model	56.06±5.60**	28.81±4.08**
EBNL + model	44.15±5.56**,##	42.80±5.87**,##
EBNM + model	35.73±4.47**,##	47.41±6.39**,##
EBNH + model	39.59±4.96**,##	41.31±5.66**,##

Notes: After cell counting, 10⁶ cells were placed in flow cytometry tubes for staining of cells. Single-cell suspensions prepared as described earlier were incubated with 1 μg of either anti-CD3 or anti-CD19 antibody for 30 minutes at 37°C in darkness. The cells were then pelleted, and the supernatant was decanted, after which the cells were washed in PBS twice and resuspended in 1.2 mL plastic tubes containing 500 μL PBS. Data were acquired on a BD FACSCalibur flow cytometer and analyzed with FlowJo Version 7.6.5 analysis software. The data are the mean ± SD (n=6/treatment).

** Significant difference compared with the control group (P<0.01).

## Significant difference compared with the CY group (P<0.01).

Abbreviations: EBNL, EBN low dose; EBNM, EBN medium dose; EBNH, EBN high dose; PP, Peyer’s patch; PBS, phosphate buffered saline; EBN, Edible bird’s nest; EBNE, EBN extract; CY, cyclophosphamide.