Nodakenin Ameliorates Ovariectomy-Induced Bone Loss by Regulating Gut Microbiota

Abstract

Disordered gut microbiota (GM) structure and function may contribute to osteoporosis (OP). Nodakenin has been shown to ameliorate osteoporosis; however, its anti-osteoporotic mechanism is unknown. This study aimed to further reveal the mechanism of the anti-osteoporotic action of nodakenin from the perspective of the microbiome and metabolome. An osteoporosis model was induced in mice through ovariectomy (OVX), with bone mass and microstructure assessed using μCT. Subsequently, ELISA and histologic examination were used to detect biochemical indicators of bone conversion and intestinal morphology. Using metabolomics and 16S rRNA sequencing, it was possible to determine the composition and abundance of the gut microbiota in feces. The results revealed that nodakenin treatment improved the bone microstructure and serum levels of bone turnover markers, and increased the intestinal mucosal integrity. 16S rRNA sequencing analysis revealed that nodakenin treatment decreased the relative abundance of Firmicutes and Patescibacteria, as well as the F/B ratio, and elevated the relative abundance of Bacteroidetes in OVX mice. In addition, nodakenin enhanced the relative abundance of Muribaculaceae and Allobaculum, among others, at the genus level. Moreover, metabolomics analysis revealed that nodakenin treatment significantly altered the changes in 113 metabolites, including calcitriol. A correlation analysis revealed substantial associations between various gut microbiota taxa and both the osteoporosis phenotype and metabolites. In summary, nodakenin treatment alleviated OVX-induced osteoporosis by modulating the gut microbiota and intestinal barrier.

Keywords: gut microbiota; intestinal barrier; metabolomics; nodakenin; osteoporosis.

Figure 1

Effect of nodakenin on femoral bone microarchitecture in OVX mice. (A) Schematic diagram of in vivo experiments. (B) Micro-CT scanning representative image. (C) Statistics of bone morphometric parameters, bone mineral density (BMD), bone volume fraction (BV/TV), number of bone trabeculae (Tb.N), and structural modeling index (SMI). Mean ± SEM (n = 6). * p < 0.05; ** p < 0.01.

Figure 2

Nodakenin improves bone turnover marker expression in OVX mice. (A–D) Observation of the changes in B-ALP, BGP, TRAP, and RANKL in the serum of each group. (E) Representative pictures of TRAP staining. Scale bars are 100 μm. (F) ImageJ statistics of osteoclast number (Oc.N)/bone surface area (BS). * p < 0.05; ** p < 0.01.

Figure 3

Clustering and diversity analysis of gut microbiota in different groups of mice. Changes in fecal α-diversity of gut microorganisms: Chao 1 estimator (A) and Shannon index (B). (C) Plot-based UniFrac PCoA. (D) Cluster analysis of gut microbiota among samples. Mean ± SEM (n = 6). * p < 0.05, ns means no significant difference.

Figure 4

Relative abundance analysis of the gut microbiota at the multispecies level for different groups. (A) Composition of gut microbiota at the phylum level. (B) Gut microbiota are significantly different at the phylum level. (C) Abundance ratio of F/B. (D) Composition of gut microbiota at the phylum level. (E) Gut microbiota significantly differ at the genus level. (F) Histogram of the distribution of LDA values. (G) Evolutionary branching diagram of LEfSe analysis. Mean ± SEM (n = 6). * p < 0.05; ** p < 0.01.

Figure 5

Analysis of correlations between gut microbiota and bone turnover indicators. (A) Heat map of Spearman’s r correlation between gut microbiota and bone biological parameters at the phylum level. (B) Heat map of Spearman’s r correlation between gut microbiota and bone biological parameters at the genus level.* p < 0.05; ** p < 0.01.

Figure 6

Composition and differences in gut metabolites and analysis of metabolic pathways. (A) OPLS-DA analysis of the OVX group versus the nodakenin-treated group. (B) Volcano plot of differential metabolites. (C) VIP value plot of differential metabolites (top 20 VIP values). (D) KEGG differential metabolic pathway plot (top 20 metabolic pathway hits).

Figure 7

Nodakenin prevents OP by accumulating calcitriol. (A) ELISA detection of calcitriol in mice serum. (B–E) qRT-PCR was performed to determine the levels of VDR, ALP, Col-1 and OCN mRNA expression. mean ± sem (n = 6). * p < 0.05, ** p < 0.01.

Figure 8

Heatmap of Spearman-analyzed bone-related metrics, gut microbiota, and top 20 metabolites. * p < 0.05, ** p < 0.01, *** p < 0.001 and *** p < 0.0001.

Figure 9

Nodakenin ameliorates intestinal barrier impairment in OVX mice. (A) Representative images of hematoxylin and eosin staining. Scale bars are 100 μm. (B–D) Image J measured the height of small intestinal villi and depth of crypts and analyzed the V/C ratio. (E–H) qRT-PCR was performed to determine the levels of Occludin, ZO-1, IL-1β and TNFα mRNA expression. mean ± sem (n = 6). * p < 0.05, ** p < 0.01.