Dose-dependent Effects of Strontium Ranelate on Ovariectomy Rat Bone Marrow Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells

Abstract

In clinic, strontium ranelate (SrR) is a useful drug to treat osteoporosis by orally taken method, but some side effect appeared in recent years. The aim of this study is to evaluate the effectiveness and safety of SrR on cells by direct application, to study the possibility of local application of this drug. Qualitative ALP staining, quantitative ALP activity assay, alizarin red staining, realtime PCR and westernblot assay were used to evaluate the osteogenesis ability of SrR under normal or osteogenic induction environment of ovariectomy bone marrow mesenchymal stem cells (OVX-BMSCs). The angiogenesis ability of SrR was studied by immunofluorescence staining of CD31 and vWF of OVX-BMSCs under angiogenesis induction environment, transwell, tubeformation and realtime PCR assay of HUVECs. Signaling pathway of PI3K/AKT/mTOR was also studied. The result demonstrated that SrR could enhance proliferation and osteogenic differentiation of OVX-BMSCs. The osteogenesis effect of SrR has been proved by the better performed of ALP activity, alizarin red staining and the remarkable up-regulation of ALP, Col-I, Runx2, OCN, BMP-2, BSP, OPG of the OVX-BMSCs, and reduction of RANKL. In addition, SrR promotes angiogenesis differentiation of both OVX-BMSCs and HUVECs. Higher intensity of immunostaining of CD31 and vWF, better result of transwell and tubeformation assay could be observed in SrR treated group, and increasing mRNA levels of VEGF and Ang-1 in the OVX-BMSCs, VEGF in HUVECs were learnt. Signaling pathway assay showed that PI3K/AKT/mTOR signaling pathway was involved in this SrR triggered angiogenesis procedure. The thrombosis marker ET-1, PAI-1 and t-PA were up-regulated, but no significant differences for low concentration (<0.5mM). The concentration between 0.25-0.5mM may be more appropriate for local application, and locally application of SrR could be considered as a promising way for bone regeneration.

Keywords: Angiogenesis.; HUVECs; OVX-BMSCs; Osteogenesis; Strontium Ranelate.

Figure 1

Immunofluorescence assay of the isolated OVX-BMSCs (×100). The cells were positively stained with CD44, CD90 and CD105, while negative stained with CD34 and CD45. The cell nucleus was stained with DAPI.

Figure 2

MTT assay for OVX-BMSCs treated with different concentrations of SrR. The experiment was repeated for at least 3 times, and the data were expressed as mean ± SD. The OD 570 values were measured at the specific time points to draw curves.

Figure 3

Osteogenesis assay of OVX-BMSCs without osteogenic induction. A, the ALP staining assay of OVX-BMSCs treated with SrR (0.125-2.0mM) for 7 days (×100); B, Quantitative assay of ALP activity at day 4, 7 and 10. C, mRNA levels of osteogenesis- and angiogenesis-related genes in the OVX-BMSCs treated with SrR(0.125-1.0mM) at day 1, 4 and 7. The experiment was repeated for at least 3 times, and the data were expressed as mean ± SD *p<0.05 versus Control.

Figure 4

Osteogenesis assay of OVX-BMSCs under osteogenic induction. A, the ALP staining assay of OVX-BMSCs treated with SrR (0.125-2.0mM) in osteogenic induction cultural medium for 7 days (×100); B, Alizarin Red staining assay of OVX-BMSCs treated with SrR (0.125-2.0mM) in osteogenic induction cultural medium for 21 days (×100); C, Quantitative assay of ALP activity at day 4, 7 and 10. D, Quantitative assay of Alizarin Red Staining. E, mRNA levels of osteogenesis- and angiogenesis-related genes in the OVX-BMSCs treated with SrR(0.125-1.0mM) in osteogenic induction cultural medium at day 4, 7. The experiment was repeated for at least 3 times, and the data are expressed as mean ± SD *p<0.05 versus Control.

Figure 5

Angiogenesis assay of OVX-BMSCs under angiogenesis induction. The left two rows of pictures showed the Dli-acLDL uptake assay (×100), and the right three rows were the immunofluorescence staining of vWF and CD 31, DAPI showed the position of nuclei (×100).

Figure 6

Western blotting assay of HIF-1α, VEGF and SDF-1 proteins in the OVX-BMSCs treated with SrR (0.125-2.0 mM). All experiments were performed in triplicates, and the data were expressed as mean ± SD. *p<0.05 versus control.

Figure 7

HUVECs angiogenesis assay. A, Tube formation assay of the HUVECs treated with SrR (0.125-2.0mM)(×100); B, Transwell assay of the HUVECs treated with SrR (0.125-2.0mM)(×100). All the cells passed through the transwell plates were stained in purple; C, Quantitative assay of tube formation assay, and data were expressed as mean ± SD. ^*p<0.05 versus Control; D, Quantitative assay of transwell. The OD value of stained crystal violet were tested and expressed as mean ± SD. ^*p<0.05 versus Control. E, Real-time PCR analysis of angiogenesis- and thrombosis-related genes expression in the HUVECs treated with SrR (0.125-2.0mM) at day 4, 7 and 10. Data were presented as mean ± SD. ^*p<0.05 versus Control.

Figure 8

Signaling Pathway assay of HUVECs. A, Western Blot assay showed the protein level of AKT, p-AKT, mTOR and p-mTOR after treated with SrR at 0.25mM. A rising level of phosphorylated protein could be observed. B, Western blot assay of protein level of AKT, p-AKT, mTOR and p-mTOR with or without inhibitor LY294002 and SrR. C, Tube formation assay (a)without LY294002 or SrR; (b)without LY294002, with SrR; (c)with LY294002, without SrR; (d)with LY294002 and SrR. Magnification×100. D, The qualitative result of tube formation assay.