Oral supplementation of diabetic mice with propolis restores the proliferation capacity and chemotaxis of B and T lymphocytes towards CCL21 and CXCL12 by modulating the lipid profile, the pro-inflammatory cytokine levels and oxidative stress

Abstract

Background: Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease caused by the selective destruction of pancreatic β cells, followed by hyperglycemia, oxidative stress and the subsequent extensive impairment of immune cell functions, a phenomenon responsible for the development of chronic diabetic complications. Propolis, a natural bee product that is extensively used in foods and beverages, significantly benefits human health. Specifically, propolis exerts antioxidant, anti-inflammatory and analgesic effects that may improve diabetic complications. To further elucidate the potential benefits of propolis, the present study investigated the effect of dietary supplementation with propolis on the plasma cytokine profiles, free radical levels, lipid profile and lymphocyte proliferation and chemotaxis in a streptozotocin (STZ)-induced type I diabetic mouse model.

Methods: Thirty male mice were equally distributed into 3 experimental groups: group 1, non-diabetic control mice; group 2, diabetic mice; and group 3, diabetic mice supplemented daily with an ethanol-soluble derivative of propolis (100 mg/kg body weight) for 1 month.

Results: First, the induction of diabetes in mice was associated with hyperglycemia and significant decreases in the insulin level and the lymphocyte count. In this context, diabetic mice exhibited severe diabetic complications, as demonstrated by a significant decrease in the levels of IL-2, IL-4 and IL-7, prolonged elevation of the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and reactive oxygen species (ROS) and altered lipid profiles compared with control non-diabetic mice. Moreover, antigen stimulation of B and T lymphocytes markedly reduced the proliferative capacity and chemotaxis of these cells towards CCL21 and CXCL12 in diabetic mice compared with control mice. Interestingly, compared with diabetes induction alone, treatment of diabetic mice with propolis significantly restored the plasma cytokine and ROS levels and the lipid profile to nearly normal levels. Most importantly, compared with untreated diabetic mice, diabetic mice treated with propolis exhibited significantly enhanced lymphocyte proliferation and chemotaxis towards CCL21 and CXCL12.

Conclusion: Our findings reveal the potential immuno-modulatory effects of propolis, which acts as a natural antioxidant to enhance the function of immune cells during diabetes.

Fig. 1

Significant alterations in the levels of ROS in different organs of diabetic mice after supplementation with propolis. The levels of ROS were determined in control mice (open bars), diabetic mice (black bars) and propolis-treated diabetic mice (hatched bars) as described in the Materials and Methods section. The pooled data for 10 mice from each group are expressed as the mean ROS level ± SEM. ^* P < 0.05 for diabetic versus control; ^# P < 0.05 for diabetic + propolis versus diabetic; ⁺ P < 0.05 for diabetic + propolis versus control (ANOVA followed by Tukey’s post-test)

Fig. 2

Propolis restores the lipid profile in diabetic mice. The levels of HDL-C, LDL-C, total cholesterol and MDA were determined in control mice (open bars), diabetic mice (black bars) and diabetic mice supplemented with propolis (hatched bars) as described in the Materials and Methods section. The pooled data for 10 mice from each group are expressed as the mean level of each lipid ± SEM. ^* P < 0.05 for diabetic versus control; ^# P < 0.05 for diabetic + propolis versus diabetic; ⁺ P < 0.05 for diabetic + propolis versus control (ANOVA followed by Tukey’s post-test)

Fig. 3

Altered proliferative capacity of B and T lymphocytes in diabetic mice supplemented with propolis. The proliferative capacity of B lymphocytes in response to stimulation with IL-4 and CD40L and of T lymphocytes in response to stimulation with SEB was evaluated using CFSE assays and flow cytometry. a Representative dot plots showing the gating strategy to obtain viable lymphocytes based on forward and side scatter and to discriminate between the CD45R/B220⁺ cell population (B-lymphocytes) and the CD45R/B220^neg cell population (T-lymphocytes) using an CD45R/B220-PE mAb and flow cytometric analysis. b One representative experiment showing the analysis of CFSE-stained B cells (after gating for viable cells); the percentage of proliferating cells (CFSE-lo) in control, diabetic and propolis-treated diabetic mice is indicated in each panel. c & d The data from the different experiments (n = 10) are expressed as the mean percentage of proliferating cells ± SEM in response to stimulation either with IL-4 and CD40L or with SEB in control mice (open bars), diabetic mice (black bars), or propolis-treated diabetic mice (hatched bars). ^* P < 0.05 for diabetic versus control; ^# P < 0.05 for diabetic + propolis versus diabetic; ⁺ P < 0.05 for diabetic + propolis versus control (ANOVA followed by Tukey’s post-test)

Fig. 4

Modulation of chemokine-mediated chemotaxis by B and T lymphocytes from propolis-treated diabetic mice. PBMCs were subjected to migration assays in response to CCL21 and CXCL12. a Representative dot plots showing the gating strategy to obtain viable lymphocytes based on forward and side scatter and to discriminate between the CD45R/B220⁺ cell population (B-lymphocytes) and the CD45R/B220^neg cell population (T-lymphocytes) using a PE-CD45R/B220 mAb and flow cytometric analysis. The input cells and the migrated cells were stained with the PE-CD45R/B220 mAb. The cells were then counted for 60 seconds via flow cytometry to calculate the percentage of cells that migrated nonspecifically (based on the number of cells that migrated to medium alone) or specifically (based on the number of cells that migrated to medium containing a chemokine). To calculate the percentage of specific migration induced by chemokines, the percentage of cells migrating to medium alone was subtracted from the percentage of cells migrating to the medium containing a chemokine. b & c The data from the different experiments (n = 10) are expressed as the mean percentage of chemokine-mediated specific migration of B and T lymphocytes ± SEM in control mice (open bars), diabetic mice (black bars) or propolis-treated diabetic mice (hatched bars). ^* P < 0.05 for diabetic versus control; ^# P < 0.05 for diabetic + propolis versus diabetic; ⁺ P < 0.05 for diabetic + propolis versus control (ANOVA followed by Tukey’s post-test)