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

Abstract

Edible bird's nest (EBN) is regarded as an immune-enhancing food in the People's Republic of China. The aim of this study is to demonstrate the efficiency of EBN in improving the immunity of mouse both in vivo and in vitro. We observed the effects of EBN on spleen lymphocytes proliferation and activation, as well as immunoglobulin isotypes as indicators. In addition, we evaluated the content of total sIgA in the intestinal juice to assess mucosal immunity. The results showed that EBN could promote the proliferation and activation of B-cells and increase IgE, IgA, IgM, and IgG3 levels. We also found that EBN extract can promote the secretion of sIgA in the small intestine. Using cyclophosphamide (CY), we established an immunosuppressed mouse model in which we identified a reversal influence on the ratio of CD3(+)/CD19(+) cells, which indicates that EBN also protects B-cells from the damage induced by CY. We also applied polymyxin B to exclude the interference of lipopolysaccharide throughout the experiment. In conclusion, we found that EBN can reduce the intestinal immune injury induced by CY by accelerating the proliferation and activation of B-cells and enhancing antibody secretion of B-cells.

Keywords: EBN; chemotherapy; immunological enhancement; intestinal mucosal immune.

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.