Xianling Gubao capsule alleviates osteoporosis: Modulation of gut-bone axis and Wnt signaling

Figure 1 The effect of Xianling Gubao capsule on body weight, uterine index, and serum estrogen level in ovariectomized rats. A: Weight changes in each group of rats; B: Uterine index of each group of rats; C: Serum estradiol concentration. Data are presented as the mean ± SD (n = 10 per group). ^aP < 0.05 vs ovariectomy group; ^cP < 0.001 vs ovariectomy group; ^dP < 0.05 vs sham-operated group; ^eP < 0.01 vs sham-operated group. Sham: Sham-operated; OVX: Ovariectomy; E2: Estradiol; XLGB-L: Xianling Gubao capsule-low; XLGB-M: Xianling Gubao capsule-middle; XLGB-H: Xianling Gubao capsule-high.

Figure 2 Xianling Gubao capsule improves the bone microstructure in ovariectomy rats. A: Representative micro-computed tomography images of the distal femur (coronal view); B: Quantitative analysis of bone microstructural parameters, including bone mineral density, bone volume/tissue volume, trabecular separation, trabecular number, and trabecular thickness. Data are presented as the mean ± SD (n = 10). ^aP < 0.05 vs ovariectomy group; ^bP < 0.01 vs ovariectomy group; ^cP < 0.001 vs ovariectomy group; ^eP < 0.01 vs sham-operated group. Sham: Sham-operated; OVX: Ovariectomy; E2: Estradiol; XLGB-L: Xianling Gubao capsule-low; XLGB-M: Xianling Gubao capsule-middle; XLGB-H: Xianling Gubao capsule-high; BMD: Bone mineral density; BV: Bone volume; TV: Tissue volume; Tb.Sp: Trabecular separation; Tb.N: Trabecular number; Tb.Th: Trabecular thickness.

Figure 3 Xianling Gubao capsule regulates serum levels of bone metabolism markers and minerals in ovariectomy rats. A: Serum level of procollagen I N-terminal propeptide; B: Serum level of collagen type I alpha 1; C: Serum level of osteocalcin; D: Serum level of runt-related transcription factor 2; E: Serum calcium concentration; F: Serum phosphorus concentration; G: Serum alkaline phosphatase activity. Data are presented as the mean ± SD (n = 10). ^bP < 0.01 vs ovariectomy group; ^cP < 0.001 vs ovariectomy group; ^dP < 0.05 vs sham-operated group; ^eP < 0.01 vs sham-operated group. Sham: Sham-operated; OVX: Ovariectomy; E2: Estradiol; XLGB-L: Xianling Gubao capsule-low; XLGB-M: Xianling Gubao capsule-middle; XLGB-H: Xianling Gubao capsule-high; PINP: Procollagen I N-terminal propeptide; COL1A1: Collagen type I alpha 1; OCN: Osteocalcin; RUNX2: Runt-related transcription factor 2; Ca: Calcium; P: Phosphorus; ALP: Alkaline phosphatase.

Figure 4 Xianling Gubao capsule ameliorates bone histopathological damage in ovariectomy rats (hematoxylin and eosin staining). Scale bars: 200 μm (5 ×) and 500 μm (2 ×). Sham: Sham-operated; OVX: Ovariectomy; E2: Estradiol; XLGB-L: Xianling Gubao capsule-low; XLGB-M: Xianling Gubao capsule-middle; XLGB-H: Xianling Gubao capsule-high.

Figure 5 Effect of Xianling Gubao capsule on the mRNA expression of key bone metabolism factors in bone tissue of ovariectomized rats. A: Expression level of osteoprotegerin; B: Expression level of receptor activator of nuclear factor-kappaB; C: Expression level of receptor activator of nuclear factor-kappaB ligand. Data are presented as the mean ± SD (n = 3). ^aP < 0.05 vs ovariectomy group; ^bP < 0.01 vs ovariectomy group; ^cP < 0.001 vs ovariectomy group; ^eP < 0.01 vs sham-operated group. Sham: Sham-operated; OVX: Ovariectomy; E2: Estradiol; XLGB-L: Xianling Gubao capsule-low; XLGB-M: Xianling Gubao capsule-middle; XLGB-H: Xianling Gubao capsule-high; OPG: Osteoprotegerin; RANK: Receptor activator of nuclear factor-kappaB; RANKL: Receptor activator of nuclear factor-kappaB ligand.

Figure 6 Effects of Xianling Gubao capsule-middle on the diversity and composition of gut microbiota in ovariectomy rats. A: Abundance-based coverage estimator index for community richness (P = 0.069); B: Chao1 index for community richness (P = 0.075); C: Shannon index for community diversity (P = 0.065); D: Simpson index for community diversity (P = 0.21). E: Principal coordinate analysis plot based on Bray-Curtis distances; F: Non-metric multidimensional scaling plot; G: Linear discriminant analysis effect size identifying differentially abundant taxa across groups. Data are presented as the mean ± SD (n = 5). Sham: Sham-operated; OVX: Ovariectomy; XLGB-M: Xianling Gubao capsule-middle; ACE: Abundance-based coverage estimator.

Figure 7 Effects of Xianling Gubao capsule on intestinal microbiota composition and metabolic pathways in ovariectomized rats. A: Phylum-level composition abundance; B: Genus-level species composition abundance; C: Spearman correlation between the top 10 most abundant phyla and bone formation indicators; D: Spearman correlation between the top 15 most abundant genera and bone formation indicators. Data are presented as the mean ± SD (n = 5). ^aP < 0.05 vs ovariectomy group; ^bP < 0.01 vs ovariectomy group; ^cP < 0.001 vs ovariectomy group. Sham: Sham-operated; OVX: Ovariectomy; XLGB-M: Xianling Gubao capsule-middle; OCN: Osteocalcin; PINP: Procollagen I N-terminal propeptide; BMD: Bone mineral density; BV: Bone volume; TV: Tissue volume; E2: Estradiol; Ca: Calcium; ALP: Alkaline phosphatase; Tb.Th: Trabecular thickness; Tb.Sp: Trabecular separation; Tb.N: Trabecular number.

Figure 8 Multivariate statistical analysis and model validation of fecal metabolomics in ovariectomy rats treated with Xianling Gubao capsule. A: Orthogonal projections to latent structures discriminant analysis (OPLS-DA) score plot in negative ion mode; B: OPLS-DA score plot in positive ion mode; C: Permutation test validating the stability and predictive ability of the OPLS-DA model in positive ion mode; D: Permutation test validating the stability and predictive ability of the OPLS-DA model in negative ion mode. The data are expressed as means ± SD (n = 5). XLGB: Xianling Gubao capsule; OVX: Ovariectomy; Sham: Sham-operated.

Figure 9 Volcano plots of differential metabolites in fecal samples from rats in each group. A: Differential metabolites between the Sham group and the ovariectomy (OVX) group in negative ion mode; B: Differential metabolites between the sham-operated group and the OVX group in positive ion mode; C: Differential metabolites between the Xianling Gubao capsule-middle group and the OVX group in negative ion mode; D: Differential metabolites between the Xianling Gubao capsule-middle group and the OVX group in positive ion mode. The data are expressed as means ± SD (n = 5). OVX: Ovariectomy; Sham: Sham-operated; XLGB-M: Xianling Gubao capsule-middle.

Figure 10  Cluster heatmap analysis of potential fecal biomarkers across experimental groups. A: Differential metabolite profile between the sham-operated group and ovariectomy group; B: Differential metabolite profile between the Xianling Gubao capsule-middle and ovariectomy groups. The data are expressed as means ± SD (n = 5). Sham: Sham-operated; OVX: Ovariectomy; XLGB: Xianling Gubao capsule.

Figure 11  Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differential metabolites based on the MetaboAnalyst platform. KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 12  Xianling Gubao capsule activates the Wnt/β-catenin signaling pathway and suppresses peroxisome proliferator-activated receptor gamma expression in bone tissue of ovariectomy rats. A: MRNA expression level of wingless-type MMTV integration site family, member 3A in bone tissue; B: MRNA expression level of glycogen synthase kinase-3 beta in bone tissue; C: MRNA expression level of β-catenin in bone tissue; D: MRNA expression level of runt-related transcription factor 2 (RUNX2) in bone tissue; E: Representative western blot bands (left) and quantitative analysis (right) of peroxisome proliferator-activated receptor gamma protein expression in femoral tissue; F: Representative immunohistochemical staining images (20 × magnification) showing the protein expression and localization of RUNX2 and β-catenin in rat femoral sections; G: Quantitative analysis of RUNX2 and β-catenin protein expression based on immunohistochemical staining. The data are expressed as means ± SD (n = 3). ^bP < 0.01 vs ovariectomy group; ^cP < 0.001 vs ovariectomy group. Wnt3a: Wingless-type MMTV integration site family, member 3A; Sham: Sham-operated; OVX: Ovariectomy; XLGB-M: Xianling Gubao capsule-middle; RUNX2: Runt-related transcription factor 2; GSK3β: Glycogen synthase kinase-3 beta.