Prenylated Isoflavonoids-Rich Extract of Erythrinae Cortex Exerted Bone Protective Effects by Modulating Gut Microbial Compositions and Metabolites in Ovariectomized Rats

Abstract

Flavonoids, found in a wide variety of foods and plants, are considered to play an important role in the prevention and treatment of osteoporosis. Our previous studies demonstrated that Erythrina cortex extract (EC) rich in prenylated isoflavonoids exerted bone protective effects in ovariectomized (OVX) rats. The present study aimed to investigate the interactions of gut microbiota with the EC extract to explore the underlying mechanisms involved in its beneficial effects on bone. Sprague-Dawley female rats of 3-months-old were ovariectomized and treated with EC extract for 12 weeks. EC extract reversed ovariectomy-induced deterioration of bone mineral density and bone microarchitecture as well as downregulated cathepsin K (Ctsk) and upregulated runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) in the tibia of OVX rats. Its protective effects on bone were correlated with changes in microbial richness and the restorations of several genera. EC increased the serum circulating levels of acetate and propionate in OVX rats. We conclude that the bone protective effects of EC extract were associated with the changes in microbial compositions and serum short chain fatty acids (SCFAs) in OVX rats.

Keywords: Erythrinae cortex; gut microbiota; ovariectomy rats; prenylated flavonoids; short chain fatty acids.

Figure 1

Effects of Erythrinae Cortex (EC) extract on bone specific mRNA expression in rat tibia. Three-month old SD rats were subjected to the following treatment for 12 weeks after ovariectomy: Sham, Sham-operated, vehicle-treated; OVX, ovariectomized, vehicle-treated; PR, ovariectomized, Premarin-treated (0.13 mg/kg); EC, ovariectomized, EC extract-treated (600 mg/kg). (A) OCN, osteocalcin; (B) Runx2, runt-related transcription factor 2; (C) OPG/Rankl, OPG, osteoprotegerin; Rankl, receptor activator of nuclear factor κB ligand; (D) ALP, alkaline phosphatase; (E) Ctsk, cathepsin K; (F) Trap, tartrate-resistant acid phosphatase 5; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Results were expressed as mean ± SEM (n = 6). # p < 0.05, ## p < 0.01 vs. Sham; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. OVX.

Figure 2

Changes in fecal microbial diversities upon different treatments measured by α-diversity on observed operational taxonomic unit (OTU). (A) Observed species; (B) Chao index; (C) Ace index; (D) Shannon index; (E) Simpson index. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test (n = 6), and ### p < 0.001 vs. Sham; *** p < 0.001 vs. OVX.

Figure 3

PCA and PCoA plots based on operational taxonomic unit (OTU) abundance. (A) Principal component analysis (PCA) plot. (B) Principal coordinate analysis (PCoA) plot of the unweighted UniFrac distance matrix. Number in brackets represents contributions of principal components (A) or coordinate (B) to differences among samples. A dot represents each sample, and different colors represent different groups.

Figure 4

Community pie-plot analysis and microbiota alternation at Phylum level in Sham, OVX, EC and PR-treated OVX rats. n = 6 in each group. The dashes in the scalar diagram indicate means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Extended error bar plot identifying the significant differences of phylotypes between mean proportions of top 20 bacterial taxa at genus level. (A) Sham vs. OVX; (B) OVX vs. EC; (C) Sham vs. OVX. Data were showed as relative abundance (%). n = 6 in each group. Statistical analysis was performed by the Wilcoxon rank-sum test, and * p < 0.05, ** p < 0.01.

Figure 6

The correlation between relative abundance (%) of top 50 bacterial taxa and bone related indicators at genus level. (A) correlation heatmap; (B) the changes of 5 genera that closely correlated with bone indicators in Sham, OVX and EC-treated OVX rat microbiotas. The correlation coefficient was determined by the Spearman index. Red, positive correlation; Green, negative correlation. * p < 0.05, ** p < 0.01 *** p < 0.001 for (A). # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Sham; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. OVX for (B).

Figure 7

The effects of Erythrinae Cortex (EC) extract treatment on the serum levels of short chain fatty acids (SCFAs). (A) the levels of acetate; (B) the levels of propionate. Values represent the mean SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test (n = 10–12). # p < 0.05 vs. Sham and * p < 0.05 vs. OVX.