Ginsenoside Rg1 interferes with the progression of diabetic osteoporosis by promoting type H angiogenesis modulating vasculogenic and osteogenic coupling

Figures

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  FIGURE 1. Identification of osteoprogenitors by flow cytometry.

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  FIGURE 2. Ginsenoside Rg1 can promote osteogenic differentiation of osteoprogenitors. (A) ALP staining and quantitative analysis in CON group, Inducer group and Inducer + Rg1 group. (B) Alizarin red staining and quantitative analysis in CON group, Inducer group and Inducer + Rg1 group. (C) Immunofluorescence staining of Osterix in the CON group, Inducer group and Inducer + Rg1 group. (D) Quantification of the mean relative levels of Osterix in osteoprogenitors in each group. Scale bar: 20 µm. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS: not significant. The above experiments were independently performed three times.

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  FIGURE 3. Ginsenoside Rg1 can promote (under high glucose exposure) osteoprogenitor proliferation and inhibit apoptosis. (A) MTT assays were used to assess the proliferation of osteoprogenitors by OD values in each group at 24 h, 48 h, and 72 h. (B) Flow cytometry was used to detect the apoptosis of osteoprogenitors in each group at 48 h. (C) The apoptosis rate of the osteoprogenitors in each group at 48 h. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS: not significant. The above experiments were independently performed three times.

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  FIGURE 4. Ginsenoside Rg1 promotes VEGF secretion from osteoprogenitors. (A) qRT‒PCR was used to detect the expression level of VEGF mRNA in osteoprogenitors in each group. (B) Western blotting was used to detect the expression level of VEGF protein in osteoprogenitors in each group. The housekeeping gene GAPDH served as an internal control. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS: not significant. The above experiments were independently performed three times.

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  FIGURE 5. Ginsenoside Rg1 promotes the proliferation of HUVECs and their secretion of Nocth1 and Noggin in high glucose culture. (A) MTT assays were used to detect the proliferation of HUVECs by the OD values in each group at 24 h, 48 h, and 72 h. (B) Flow cytometry was used to detect the apoptosis of HUVECs in each group at 72 h. (C) The apoptosis rate of HUVECs in each group at 72 h (D) qRT‒PCR was used to detect the levels of the Nocth1 and NOG mRNA expressed by HUVECs in each group. (E) Western blotting was used to detect the levels of the Notch1 and Noggin protein secreted by the HUVECs in each group. The housekeeping gene GAPDH served as an internal control. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS: not significant. The above experiments were independently performed three times.

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  FIGURE 6. Ginsenoside Rg1 could ameliorate bone mass reduction and microstructural damage in GK rats. (A) Micro-CT images of the tibiae of Wistar and GK rats before treatment. (B) BMD of the tibia of Wistar and GK rats before treatment. (C) Two-dimensional micro-CT images of the tibia of rats in each group after 12 weeks of treatment. (D) Three-dimensional micro-CT images of the trabecular bone of rats in each group after 12 weeks of treatment. (E–J) Quantitative analysis of BMD (E), BV/TV (F), Tb. N (G), SMI (H), Tb. Sp (I), and Tb. Th (J) in the proximal tibia of rats in each group after 12 weeks of treatment. (K) H&E staining of the tibial metaphysis in each group. Scale bar: 50 µm. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS: not significant. n = 3. H&E staining experiments were performed independently three times.

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  FIGURE 7. Ginsenoside Rg1 promotes type H angiogenesis and osteogenesis related factor secretion in GK rats. (A) Immunofluorescence staining images of CD31 and Emcn in bone tissue of GK rats in each group. (B) Osterix immunofluorescence staining images in bone tissue of GK rats in each group. (C–E) Quantification of the mean relative levels of CD31 (C), Emcn (D), and Osterix (E) in bone tissue of GK rats in each group. (F) qRT‒PCR was used to detect the expression levels of the NOG, Notch1, and VEGF mRNA in the bone tissue of GK rats in each group. (G) Western blotting was used to detect the protein expression levels of Noggin, Notch1, and VEGF in the bone tissue of GK rats in each group. The housekeeping gene GAPDH served as an internal control. Data are expressed as the mean ± SD. Scale bar: 20 μm *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS: not significant. n = 3. The above experiments were independently performed three times.