Protective effects of Brazilian propolis supplementation on capillary regression in the soleus muscle of hindlimb-unloaded rats

Abstract

The protective effects of Brazilian propolis on capillary regression induced by chronically neuromuscular inactivity were investigated in rat soleus muscle. Four groups of male Wistar rat were used in this study; control (CON), control plus Brazilian propolis supplementation (CON + PP), 2-week hindlimb unloading (HU), and 2-week hindlimb unloading plus Brazilian propolis supplementation (HU + PP). The rats in the CON + PP and HU + PP groups received two oral doses of 500 mg/kg Brazilian propolis daily (total daily dose 1000 mg/kg) for 2 weeks. Unloading resulted in a decrease in capillary number, luminal diameter, and capillary volume, and an increase in the expression of anti-angiogenic factors, such as p53 and TSP-1, within the soleus muscle. Brazilian propolis supplementation, however, prevented these changes in capillary structure due to unloading through the stimulation of pro-angiogenic factors and suppression of anti-angiogenic factors. These results suggest that Brazilian propolis is a potential non-drug therapeutic agent against capillary regression induced by chronic unloading.

Keywords: Anti-angiogenic factors; Brazilian propolis; Capillary regression; Hindlimb unloading; Muscle atrophy; Oxidative stress.

Fig. 1

Transverse sections from each group (a CON, b CON + PP, c HU, d HU + PP) stained for alkaline phosphatase (AP). e The capillary-to muscle fiber (C/F) ratio of the soleus muscle. CON Control group, CON+PP control plus propolis supplementation group, HU hindlimb unloading group, HU+PP hindlimb unloading plus propolis supplementation group. Scale bar: 100 μm. Values are shown as the mean (bars) ± standard deviation of the mean (SEM; error bars). The asterisk (*), dagger (†), and double dagger (‡) indicate a significant difference from the CON, CON + PP, and HU groups, respectively, at P < 0.05

Fig. 2

Representative confocal laser scanning microscopic images of the three-dimensional (3D) capillary architecture of the soleus muscle of a rat in the CON (a), CON + PP (b), HU (c), and HU + PP (d) groups. Scale bar: 100 μm. e, f Mean capillary luminal diameter (e) and capillary volume (f) in the soleus muscle of each group. Values are shown as the mean (bars) ± SEM (error bars). The asterisk (*), dagger (†), and double dagger (‡) indicate a significant difference from the CON, CON + PP, and HU groups, respectively, at P < 0.05

Fig. 3

Frequency distribution of capillary luminal diameter in the CON (a), CON + PP (b), HU (c), and HU + PP (d) groups. Arrows indicate the median of capillary diameter

Fig. 4

Transverse sections from each group (a CON, b CON + PP, c HU, d HU + PP) stained with the fluorescent probe dihydroethidium (DHE) that detects oxidative stress. e, f Mean levels of DHE fluorescence (e) and superoxide dismutase 1 (SOD-1) protein expression (f) in the soleus muscle of each group. Scale bar: 50 μm. Values are shown as the mean (bars) ± SEM (error bars). The asterisk (*), dagger (†), and double dagger (‡) indicate a significant difference from the CON, CON + PP, and HU groups, respectively, at P < 0.05. AU Arbitrary units

Fig. 5

Mean levels of p53 protein expression (a) and mean levels of thrombospondin-1 (TSP-1) mRNA (b) in the soleus muscles of each group. Values are shown as the mean (bars) ± SEM (error bars). The asterisk (*), dagger (†), and double dagger (‡) indicate a significant difference from the CON, CON + PP, and HU groups, respectively, at P < 0.05

Fig. 6

Mean levels of vascular endothelial growth factor (VEGF) protein expression in the soleus muscles of each group. Values are shown as the mean (bars) ± SEM (error bars). The asterisk (*), dagger (†), and double dagger (‡) indicate a significant difference from the CON, CON + PP, and HU groups, respectively, at P < 0.05

Fig. 7

Microscopic images of the soleus muscle stained with cluster of differentiation 31 (CD31; a) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL; b); c merged images of a and b. Scale bar: 50 μm. Arrowheads indicate TUNEL-positive endothelial cells. d Mean number of the TUNEL-positive endothelial cells of the soleus muscle in each group. Values are shown as the mean (bars) ± SEM (error bars). The asterisk (*), dagger (†), and double dagger (‡) indicate a significant difference from the CON, CON + PP, and HU groups, respectively, at P < 0.05