Changes in BMC, BMD, and serum bone metabolism markers after IR supplementation
To examine changes in rat bone health, we measured the BMC and BMD of the whole body, femur, and tibia by dual-energy X-ray absorptiometry (DXA; Fig. 2a). As shown in Fig. 2a, the whole-body BMC (n = 6, p = 0.000895) and tibia BMC (n = 6, p = 0.00149) increased significantly in normal rats after supplementation with IR light. Although the femoral BMC failed to show a significant difference, it showed an increasing trend (n = 6, p = 0.0575). For ovariectomized rats, the BMC of the whole body (n = 6, p = 2.55e–05) increased significantly after IR supplemental light, with the tibia BMC (n = 6, p = 0.0522) also showing an increasing trend, while the femur BMC did not change significantly. This indicates that long-term, low-energy IR supplementary light has a positive effect on whole body bone and tibial bone mass in both normal and ovariectomized rats.
The rat BMD measurements are provided in Fig. 2b. Inconsistent with the BMC results, the changes in femoral BMD were the most apparent; all significantly increased after IR supplementation (n = 6, Normal: p = 0.00229, Ovx: p = 0.00149). In contrast, the tibial BMD exhibited an increasing trend after IR supplementation, but was significant only in the normal rat group (n = 6, p = 0.000276). No significant changes in whole-body BMD were observed. These data indicate that long-term, low-energy IR supplementation has a positive effect on the local BMD, in both normal and ovariectomized rats, and can promote bone formation. Through micro-CT scanning to obtain the cross-sectional image of the distal femur, we can visualize the increase in trabecular bone density after infrared supplementation (Fig. 2c).
We also examined the changes in serum bone metabolism markers in the rats. The ovariectomized rats exhibited significant increases in 1,25(OH)2D3 (n = 6, p = 0.0324) and BALP (n = 6, p = 2.26e–06) concentrations after exposure to the IR supplementary light (Fig. 2d). There was also an increasing trend in the normal rats, indicating that IR supplementation can increase the concentration of 1,25(OH)2D3 and BALP in rats and has a positive effect on vitamin D synthesis and bone metabolism. Meanwhile, we observed increases in TRACP concentrations in both the normal + IR and ovariectomized +IR groups, suggesting that IR supplementation can increase the bone resorption rate in rats, regardless of the health of the bone.
Effects of IR supplementation on alpha diversity, beta diversity, and the microbial network
To evaluate the effects of IR irradiation on the richness and diversity of the gut microbiota, we compared the richness index (Chao1) and diversity index (Shannon_e) in each treatment group. The species abundance of the gut microbiota in both normal and ovariectomized rats significantly decreased after irradiation with the IR light (Fig. 3a). The Shannon_e index estimation indicates that IR irradiation and bone loss did not affect the gut microbiota diversity of the rats (Fig. 3b).
Based on the unweighted UniFrac distances of the OTU levels, we used Principal Coordinate Analysis (PCoA) to determine the effect of IR supplementation on the distribution of gut microbiota. As shown in Fig. 3c, we observed a separation of the microbiota between the white light-irradiated rats and IR-supplemented rats, whereas there was no apparent separation between the ovariectomized rats and normal rats. Similarity analysis between the different groups showed that IR supplementation had a highly significant effect on the composition of gut bacterial communities (ANOSIM, R = 0.8433, p = 0.001), and the effect is far greater than that of ovariectomy (ANOSIM, R = –0.011, p = 0.44).
We selected OTUs with relative abundance > 100 and used Spearman correlation coefficients to construct microbial interaction networks. Species analysis indicates that the four groups of networks are composed of bacteria from 4 phyla: Bacteroidetes, Firmicutes, Saccharibacteria, and Proteobacteria (Fig. 4). Table 1 presents the structural characteristics of the microbial networks in the different treatment groups. The microbial networks in both the normal and ovariectomized rats, after supplementation of IR, were significantly increased in degree and closeness centrality (Additional file 1: Figure S1). Meanwhile, supplementation with IR resulted in a larger average aggregation coefficient and a smaller average path length in both the normal and ovariectomized rats.
Taxon-based analysis of bacterial communities
To understand the types of bacteria in the rat intestine that could be affected by IR exposure, we analyzed the composition of the gut microbiota at the phylum and family levels (Fig. 3d, e). As shown in Fig. 3d, nine phyla of bacteria were detected in all four groups of rats, regardless of bone metabolism and IR exposure. Specifically, bacteria such as Firmicutes and Bacteroides were found as the dominant flora in the rat intestine. Saccharibacteria were found to be dramatically reduced in both normal and ovariectomized rats (Ovx: p = 0.005, Normal: p = 0.002) exposed to the IR light. The ratio of Firmicutes to Bacteriodetes showed a tendency to decrease (Additional file 1: Figure S2a). A total of 44 families have been annotated, of which 13 significantly changed. Fig. 3e shows the proportions of gut microbiota in each group at the family level. Interestingly, IR supplementation had a substantial impact on the gut microbiota, while ovariectomy did not (Additional file 1: Figure S3). We found that the relative abundance of Clostridiaceae_1, Erysipelotrichaceae, Peptostreptococcaceae, Porphyromonadaceae, and Alcaligenaceae all increased significantly after exposure to IR, while the relative abundance of Desulforibrionaceae decreased significantly (Additional file 1: Figure S2b). Meanwhile, there was a greater abundance of Prevotellaceae and Lactobacillaceae after exposure to IR, although these differences did not reach statistical significance.
We also assessed significant difference in genus after exposure to infrared. The IR exposure in both the normal and ovariectomized groups led to a decrease in Candidatus_Saccharimonas and an increase in Clostridium_sensu_stricto_1, Turicibacter and ParabacteroidesFig. 3f and g).
Functional prediction of metabolic pathway
The metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human disease pathways associated with the rat gut microbiota compositions were examined using the Tax4Fun software (Fig. 5, Additional file 2: Supplemental Table S1). The pathways annotated to metabolism accounted for the vast majority, of which Energy metabolism, Lipid metabolism, and metabolism of terpenoids and polyketides changed significantly in both ovariectomized and normal rats (p < 0.05). Next, we selected the top 60 pathways of relative abundance in metabolism, according to the KEGG pathway database (Additional file 1: Figure S4). IR supplementation significantly enriched the Butanoate metabolism, Propanoate metabolism, and Fatty acid biosynthesis pathways.