Pharmacology and metabolomic reveal Polysaccharides from polygonatum sibiricum ameliorate the diabetic osteoporosis in zebrafish

Fig. 1. Experimental design

Fig. 2. Effects of metformin (Met) and Polygonatum sibiricum polysaccharide (PSP) on mortality of zebrafish. (A-B) Healthy adult zebrafish were transferred to liquid medium containing 0, 50, 100, 200, 300, 400, and 500 µg/mL Met, or 0, 75, 150, 300, 400, 500, and 600 µg/mL PSP for 9 days. Death rates of zebrafish were recorded. Data were means ± SE of three independent experiments with at least three biological replicates for each individual experiment

Fig. 3. Effects of metformin (Met) and Polygonatum sibiricum polysaccharide (PSP) on the fasting blood glucose of zebrafish under model treatment. (A) Healthy adult zebrafish were injected with streptozocin (STZ) by microinjection needle. Control zebrafish were injected with the citric acid buffer. After the injection, zebrafish were put back into liquid medium for 14 days, and the fasting blood was detected. (B) Healthy adult zebrafish were injected with STZ for 3 days, and then transferred to liquid medium containing 300 µg/mL Met, or 75, 150, or 300 µg/mL PSP for another 3 days. Afterwards, the fasting blood were detected. Data were means ± SE of three independent experiments with at least three biological replicates for each individual experiment. Within each set of experiments, columns with different letters denoted significant differences at P < 0.05 according to Duncan’s multiple range test

Fig. 4. Effects of metformin (Met) and Polygonatum sibiricum polysaccharide (PSP) on the caudal fin regeneration of zebrafish under model treatment. Healthy adult zebrafish were injected with streptozocin (STZ) by microinjection needle for 3 days. Then, the tail fin was cut off according to uniform standards, and transferred to liquid medium containing 300 µg/mL Met, or 75, 150, or 300 µg/mL PSP for another 3 days. (A) The caudal fin pictures of zebrafish were respectively taken by EVOS cell imaging system. (B) The caudal fin regeneration length was measured by Image Pro Plus 6.0. Scale bars = 400 μm. Control zebrafish were injected with the citric acid buffer. Within each set of experiments, columns with different letters denoted significant differences at P < 0.05 according to Duncan’s multiple range test

Fig. 5. Classification, principal component analysis (PCA), and hierarchical cluster analysis (HCA) of the metabolites in the four groups of zebrafish. (A) Classification; (B) PCA score plot; (C) HCA heatmap of all metabolic profiles. The upregulated and downregulated metabolites were indicated by different shades of red and blue, respectively

Fig. 6. Venn diagram analysis and number of metabolites of five comparisons. (A) Venn diagram illustrating the overlapping and specific differential metabolites for the five comparisons. (B) Number of specific differential metabolites of five comparisons (Model/Control, Met/Control, PSP/Control, Met/Model, PSP/Model)

Fig. 7. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations and enrichment results of the differentially accumulated in the comparisons Model/Control (A), Met/Model (B), PSP/Model (C). The p value calculated based on the hypergeometric distribution, which the number of differential substances KEGGID in the enrichment table of this pathway and the number of metabolites of the pathway itself

Fig. 8. Changes in the expression levels of TNF-α and IL-1β genes related serum inflammatory cytokines. Healthy adult zebrafish were injected with STZ for 3 days, and then transferred to liquid medium containing 300 µg/mL Met, or 75, 150, or 300 µg/mL PSP for another 3 days. Afterwards, the expression levels of TNF-α and IL-1β genes were analyzed using RT-qPCR. Relative TNF-α and IL-1β expression in the control samples was set as 1. The expression levels of corresponding genes were normalized against β-actin, an internal control gene. Data were means ± SE of three independent experiments with at least three biological replicates for each individual experiment. Within each set of experiments, columns with different letters denoted significant differences at P < 0.05 according to Duncan’s multiple range test