Asperosaponin VI promotes bone marrow stromal cell osteogenic differentiation through the PI3K/AKT signaling pathway in an osteoporosis model

Abstract

Asperosaponin VI (ASA VI), a natural compound isolated from the well-known traditional Chinese herb Radix Dipsaci, has an important role in promoting osteoblast formation. However, its effects on osteoblasts in the context of osteoporosis is unknown. This study aimed to investigate the effects and mechanism of ASA VI action on the proliferation and osteogenic differentiation of bone marrow stromal cells isolated from the ovariectomized rats (OVX rBMSCs). The toxicity of ASA VI and its effects on the proliferation of OVX rBMSCs were measured using a CCK-8 assay. Various osteogenic differentiation markers were also analyzed, such as ALP activity, calcified nodule formation, and the expression of osteogenic genes, i.e., ALP, OCN, COL 1 and RUNX2. The results indicated that ASA VI promoted the proliferation of OVX rBMSCs and enhanced ALP activity and calcified nodule formation. In addition, while ASA VI enhanced the expression of ALP, OCN, Col 1 and RUNX2, treatment with LY294002 reduced all of these osteogenic effects and reduced the p-AKT levels induced by ASA VI. These results suggest that ASA VI promotes the osteogenic differentiation of OVX rBMSCs by acting on the phosphatidylinositol-3 kinase/AKT signaling pathway.

Figure 1. 3D reconstructions of femoral metaphyseal trabecular structure were observed by micro-CT.

(A) BMD (bone mineral density) of SHAM group and OVX group was determined by micro-CT. (B) BV/TV (Percent bone volume) of SHAM group and OVX group was determined by micro-CT. (C)Trabecular Sepa ration/Spacing of SHAM group and OVX group was determined by micro-CT. (D) The ALP expression of the two groups of BMSCs were observed on 7d and 14d. The OVX group was significantly lower than that of the SHAM group. (E) The calcium deposit formation of the two groups were determined by Alizarin red staining. The OVX group was significantly lower than that of the SHAM group. (F) The data are represented as the mean ± SD. *P < 0.5 vs the SHAM group.

Figure 2. The effect of ASA VI on OVX rBMSC proliferation was analyzed at 1, 4, and 7 days, respectively.

Cell proliferation was evaluated by a CCK-8 assay. The data are represented as the mean ± SD. **P < 0.01 vs the controls. ***P < 0.001 vs the controls.

Figure 3. ALP activity was assessed using a commercial ALP kit after cells were cultured with various concentrations of ASA VI for 5 or 10 days.

The data are represented as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs the controls; ^#P < 0.05 and ^##P < 0.01 vs ASA VI 10⁻⁵M.

Figure 4. The mineralization of OVX rBMSCs.

(A) OVX rBMSCs were seeded in 24-well plates at a cell density of 5 × 10³ and were treated with ASA VI (10⁻⁵ M) and/or LY294002 (50 μM); after 21 days, the formation of mineralized nodules was measured by alizarin red S staining. (A) After imaging the nodules, 10% cetylpyridinium chloride was used to dissolve the nodules, and mineralization was quantified using a Bio-Rad microplate reader. (B) The data are represented as the mean ± SD. **P < 0.01 vs the controls; ^#P < 0.05 and ^##P < 0.01 vs ASA VI 10⁻⁵M.

Figure 5. Cultured cells were treated with ASA VI 10⁻⁵M for 48 hours with or without LY294002 (50 μM).

Total RNA was then extracted for quantitative PCR to assess the mRNA levels of osteogenesis-related genes, including (A) ALP, (B) OCN, (C) COL 1, and (D) RUNX2. The data are represented as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs the controls; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.01 vs ASA VI 10⁻⁵M.

Figure 6. The effects of ASA VI on p-AKT expression in OVX rBMSCs.

(A) Western blotting was used to analyze p-AKT expression after a 48-hour ASA VI 10⁻⁵M treatment, with or without LY294002. (B) The Western blotting results were analyzed using Quantity One Bio-Rad software. The data are represented as the mean ± SD. **P < 0.01 vs the controls; ^###P < 0.001 vs ASA VI 10⁻⁵M.