https://orcid.org/0000-0003-4175-7870
Figures & data
Table 1. The primer sequences used in quantitative qRT-PCR.
Table 2. Antibodies for western blot, immunofluorescence staining and immunohistochemistry staining.
Figure 1. HPLC chromatogram of LWDH. A. Paeonol; B. Alisol B 23-acetate; C. Moroniside and Loganin. D. Paeoniflorin.
Figure 2. LWDH attenuates the bone loss caused by OVX. (A) 3D reconstruction of representative images of the coronal and cross-sectional surfaces of the distal femur at 8 weeks after surgery in each group. The highlighted area of the Coronal row is region of interest (ROI). (B-G) Micro-CT analyses of bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th), trabecular separation (Tb. Sp) and midshaft cortical thickness (Ct. Th). (n = 6, *p < 0.05 vs. Sham; #p < 0.05 vs. OVX by one-way ANOVA test.)
Figure 3. LWDH enhances osteogenic capacity and inhibits lipogenic capacity in OVX rats. LWDH enhances osteogenic capacity and inhibits lipogenic capacity in OVX rats. (A) HE staining images of the sections from the distal femur. (B) Immunohistochemical staining images of the osteogenic factor Runx2. (C) Histological evaluation of the Goldner trichome staining in each group, green-stained mineralized bone (MB) and red-stained unmineralized bone (UB). (D) TRAP staining of femur sections. (E) Representative HE staining of femur sections exhibiting the marrow adipose tissue. (F) The quantification of Runx2 in rats from different groups. (G) Quantification of osteoblasts in different groups of rats. (H) Number of fat cells in the femur (n = 6, *p < 0.05 vs. Sham; #p < 0.05 vs. OVX by one-way ANOVA test).
Figure 4. LWDH delays the senescence of BMSCs in OVX rats. (A, B) Representative images of aging-related β-galactosidase staining in different groups of rat BMSCs and quantitative analysis of positive cells (n = 6). (C) CCK8 assay was used to evaluate cell viability (n = 6). (D) Cell cycle distribution (G0/G1, S, G2/M) was detected by flow cytometry (n = 3). (E, F) BMSCs of each group were subjected to qRT-PCR to detect the expression of p53 and p21 mRNA (n = 6). (G-I) BMSCs were lysed and prepared to measure the expression levels of p53 and p21 by WB (n = 3). *p < 0.05 vs. Sham; #p < 0.05 vs. OVX by one-way ANOVA test.
Figure 5. LWDH enhances BMSCs osteogenic capacity and inhibits lipogenic capacity. (A) Osteogenic differentiation was assessed by ALP staining. (B) Representative images of Alizarin Red staining. (C) Representative images of oil red O staining. (D) ALP activity assays. (E) Calcium deposition was determined by measuring optical density. (F) quantitative analysis of oil red O staining. (G-I) qRT-PCR analyses of the expression of Runx2, Osterix and Ocn under osteogenic condition. (J-L) qRT-PCR analyses of the expression of Adipoq, PPARγ and Cebpα under adipogenic condition (n = 6). *p < 0.05 vs. Sham; #p < 0.05 vs. OVX by one-way ANOVA test.
Figure 6. LWDH can maintain BMSCs autophagy levels in OVX rats. (A-C) BMSCs of each group were subjected to qRT-PCR to detect the expression of LC3II, p62 and Beclin1 mRNA (n = 6). (D-F, H) The expression and quantitative analysis of LC3II, p62 and Beclin1 at the protein level in each group were detected by WB (n = 3). (G, I) The expression and quantitative analysis of YAP at the protein level in each group were detected by WB (n = 3). (J) BMSCs of each group were subjected to qRT-PCR to detect the expression of YAP mRNA (n = 6). (K, L) Quantitative and statistical analysis of immunohistochemical (IHC) staining of YAP-positive cells in distal femoral tissues (n = 6). *p < 0.05 vs. Sham; #p < 0.05 vs. OVX by one-way ANOVA test.
Data availability statement
The data that support the findings of this study are available from the corresponding author Lan Yang upon reasonable request.