Modulatory effects of feeding pregnant and lactating mice Rhodiola kirilowii extracts on the immune system of offspring

Abstract

Plants of Rhodiola genus are medicinal herbs that have a number of therapeutic properties, including anti-inflammatory and immunomodulatory activity. The present study aimed to determine whether the use Rhodiola kirilowii as an immunostimulant during pregnancy has an adverse effect on the development of the offspring immune system. Following mating, pregnant mice were placed in three groups that were fed during pregnancy and lactation with R. kirilowii aqueous extract (RKW; 20 mg/kg), R. kirilowii 50% hydro-alcoholic extract (RKW-A; 20 mg/kg) or water (control group), receiving water. Following birth, offspring were given six weeks to develop prior to evaluation of their immune system. Morphometric and morphological examination of the spleen did not reveal any abnormalities or differences between the experimental and control groups. However, both RKW and RKW-A splenic lymphocytes presented a diminished proliferative response to concanavalin A. RKW spleen lymphocytes demonstrated increased metabolic activity following phytohaemagglutinin (PHA) stimulation, which was associated with a higher percentage of cluster of differentiation 4 positive spleen cells and lower interleukin-17a (IL-17a) serum concentration. The RKW-A group exhibited a diminished proliferative response of spleen lymphocytes to PHA and lipopolysaccharide (LPS), and increased serum concentrations of IL-10 and tumor necrosis factor-α (TNF-α). The progeny of mice fed with RKW-A extract demonstrated a significantly lower level of anti-SRBC antibody following immunization compared with progeny of the control (P=0.0305) and RKW (P=0.0331) groups. In conclusion, caution is recommended in the use of RKW and RKW-A extracts as immunostimulants in pregnancy.

Keywords: Rhodiola kirilowii; immune response; mice; pregnancy; progeny.

Figure 1.

Cellularity of progeny spleens. Results presented are the mean of cells calculated per gram of spleen ± standard error of the mean. Number of spleens tested, 87 (control, 41; RKW, 24; RKW-A, 22). RKW, Rhodiola kirilowii water extract; RKW-A, Rhodiola kirilowii hydro-alcoholic extract.

Figure 2.

Phenotypes of spleen lymphocytes. Phenotypic analysis was performed by flow cytometry [number of spleens tested, 46 (control, 15; RKW, 13; RKW-A, 18)]. Results are presented as the mean percentage of spleen cells ± SEM. Statistical analysis performed: Unpaired t-test, Shapiro-Wilk normality test, two-way analysis of the variance and Bonferroni correction. SEM, standard error of the mean; CD, cluster of differentiation; RKW, Rhodiola kirilowii water extract; RKW-A, Rhodiola kirilowii hydro-alcoholic extract. *P=0.0004.

Figure 3.

Proliferation index of splenocytes following phytohemagglutinin (PHA) stimulation. Results are presented as the mean RFU or cpm ± SEM, measured by the (A) alamarBlue assay [number of spleens tested, 59 (control, 31; RKW, 16; RKW-A, 12)] or (B) [³H] thymidine incorporation assay following PHA (2 µg/ml) stimulation [number of spleens tested, 27 (control, 9; RKW, 9; RKW-A, 9)]. Statistical analysis performed: Unpaired t-test, Shapiro-Wilk normality test, one-way analysis of the variance and the Tukey comparison test. RFU, relative fluorescence units; SEM, standard error of the mean; cpm, counts per minute; RKW, Rhodiola kirilowii water extract; RKW-A, Rhodiola kirilowii hydro-alcoholic extract. *P=0.0496.

Figure 4.

Proliferation indexes of splenocytes following Concanavalin A (ConA) stimulation. Results are presented as the mean RFU or cpm ± SEM, measured by the (A) alamarBlue assay [number of spleens tested, 59 (control, 31; RKW, 16; RKW-A, 12)] or (B) [³H] thymidine incorporation assay following ConA (5 µg/ml) stimulation [number of spleens tested, 27 (control, 9; RKW, 9; RKW-A, 9)]. Statistical analysis performed: Unpaired t-test, Shapiro-Wilk normality test, one-way analysis of the variance and the Tukey comparison test. RFU, relative fluorescence units; SEM, standard error of the mean; cpm, counts per minute; RKW, Rhodiola kirilowii water extract; RKW-A, Rhodiola kirilowii hydro-alcoholic extract. *P=0.0306 vs. control; **P=0.0356 vs. control.

Figure 5.

Proliferation index of splenocytes following lipopolysaccharide (LPS) stimulation. Results are presented as the mean RFU or cpm ± SEM, measured by the (A) alamarBlue assay [number of spleens tested, 59 (control, 31; RKW, 16; RKW-A, 12)] or (B) [³H] thymidine incorporation assay following LPS (20 µg/ml) stimulation [number of spleens, 27 (control, 9; RKW, 9; RKW-A, 9)]. Statistical analysis performed: Unpaired t-test, Shapiro-Wilk normality test, one-way analysis of the variance and the Tukey comparison test. RFU, relative fluorescence units; SEM, standard error of the mean; cpm, counts per minute; RKW, Rhodiola kirilowii water extract; RKW-A, Rhodiola kirilowii hydro-alcoholic extract.*P=0.0175.

Figure 6.

Concentration of selected cytokines (A) IL-2, (B) IL-4, (C) IL-6, (D) IFN-γ and (E) TNF-α in the sera. n=54 mice (control, 20; RKW, 18; RKW-A, 16). Results presented are the median ± the range of the cytokine in pg/ml. Statistical analysis performed: Unpaired t-test, Shapiro-Wilk normality test, Kruskal-Wallis test and Dunn's test. RKW, Rhodiola kirilowii water extract; RKW-A, Rhodiola kirilowii hydro-alcoholic extract. *P=0.0123 from Mann Whitney test.

Figure 7.

Concentration of (A) IL-17a and (B) IL-10 cytokines in the sera. n=54 mice (control, 20; RKW, 18; RKW-A, 16). Results present the median ± the range of the cytokine in pg/ml. Statistical analysis performed: Unpaired t-test, Shapiro-Wilk normality test and the Kruskal-Wallis test. RKW, Rhodiola kirilowii water extract; RKW-A, Rhodiola kirilowii hydro-alcoholic extract. *P=0.0347.

Figure 8.

Levels of anti-sheep red blood cell (SRBC) antibody production. Results are presented as the mean log titer ± SEM of anti-SRBC antibody production in progeny whose mothers were fed RKW and RKW-A extracts during pregnancy and lactation. n=29 mice (control, 9; RKW, 7; RKW-A, 13). Statistical analysis performed: Unpaired t-test, Shapiro-Wilk normality test, one-way analysis of the variance and the Tukey comparison test. SEM, standard error of the mean; RKW, Rhodiola kirilowii water extract; RKW-A, Rhodiola kirilowii hydro-alcoholic extract. *P=0.0331, **P=0.0305.