The role of the ERK1/2 pathway in simvastatin-loaded nanomicelles and simvastatin in regulating the osteogenic effect in MG63 cells

Abstract

Objectives: The present study aimed to clarify the role of the ERK1/2 pathway in simvastatin (SV)-loaded nanomicelles (SVNs)- and SV-mediated promotion of cell osteogenic differentiation and explore the molecular mechanisms by which SVNs exhibited a greater efficacy in promoting osteogenic differentiation than SV.

Materials and methods: SVNs were synthesized using a dialysis method. MG63 cells were treated with 2.5, 0.25, and 0.025 μmol/L of the drug. The optimal drug dosage was determined by examining the proliferative activity and ALP activity of the MG63 cells. Subsequently, Western blot analysis was performed to analyze the levels of the phosphorylated ERK1/2 proteins in each experimental group at various time points. Finally, the inhibitor PD98059 was used to effectively inhibit the ERK1/2 pathway. The resulting changes in the proliferative activity of MG63 cells and the osteogenesis-related markers were analyzed.

Results: The SVNs synthesized in the present study had a mean diameter of 27 nm. The encapsulation and drug-loading efficiencies were 52.03% ± 4.05% and 9.42% ± 0.66%, respectively. SVNs and SV exhibited optimum osteogenesis-promoting effects when the drugs were administered at a concentration of 0.25 μmol/L. The drug-induced activation of the ERK1/2 pathway reached a peak at 15 minutes after administration and then declined rapidly. From 24 hours to 7 days, SVNs and SV exerted an inhibitory effect on the ERK1/2 pathway rather than an activating effect. Throughout the whole experimental process, the regulatory effect of SVNs on the ERK1/2 pathway was significantly greater than that of SV. Inhibition of the ERK1/2 pathway by PD98059 markedly reduced the proliferative activity of the cells in all experimental groups. In addition, the ALP activity and the expression levels of the osterix (OSX) and osteocalcin (OC) proteins were drastically increased.

Conclusion: SVNs significantly increased the effect of SV-induced osteogenic differentiation by strongly inhibiting the ERK1/2 pathway.

Keywords: ERK1/2 pathway; dental implant restoration; osteogenic effect; simvastatin-loaded nanomicelles.

Figure 1

The characteristics of simvastatin-loaded nanomicelles. Notes: (A) The particle size of simvastatin-loaded nanomicelles was measured using a Zetasizer instrument. (B) Simvastatin-loaded nanomicelle morphology was observed with a TEM. (C) The in vitro cumulative release profiles of simvastatin from simvastatin-loaded nanomicelles using human serum albumin as a dissolution medium by a dynamic dialysis method. The simvastatin concentration was measured via HPLC at an ultraviolet absorbance of 238 nm. The results are the mean values of three independent measurements (±SD). Abbreviations: SD, standard deviation; SVG, simvastatin group; SVNG, simvastatin-loaded nanomicelles group; TEM, transmission electron microscope.

Figure 2

The effects of different concentrations of SVNs and SV on MG63 cells. Notes: When the dosing concentration was 0.25 and 0.025 μmol/L, the effects of SVNs and SV on the cellular proliferation activity (A) and ALP activity were determined (D). When the dosing concentration was 2.5 μmol/L, the effects of SVNs and SV on cell proliferation activity were determined (C), and massive cell death occurred in both the SVNG and the SVG as shown by an inverted microscope on the third day (B). The results are represented as the mean values of three independent measurements (±SD). ^*P<0.05 vs BCG; ^▲P<0.05 SVNG vs SVG (0.25 μmol/L); ^∆P<0.05 SVNG vs SVG (0.025 μmol/L); ^♦P<0.05 SVNG vs SVG (2.5 μmol/L). Abbreviations: BCG, blank control group; DFNG, drug-free nanomicelles group; SD, standard deviation; SV, simvastatin; SVG, simvastatin group; SVNs, simvastatin-loaded nanomicelles; SVNG, simvastatin-loaded nanomicelles group.

Figure 3

The effects of SVNs and SV on the ERK1/2 pathway. Notes: SVNs and SV can increase the expression of p-ERK1/2 in the early stage of the experiment (A). On 1 day (B), 7 days (C), and 14 days (D), both SVNs and SV exhibited a significant, sustained inhibitory effect on the expression of the p-ERK1/2. In addition, the expression of p-ERK1/2 in each experimental group was inhibited significantly after the MG63 cells were pretreated with the ERK1/2 pathway inhibitor PD98059 (50 μM) prior to drug intervention (B–D). The results are the mean values of three independent measurements (±SD). ^*P<0.05 vs BCG; ^#P<0.05 SVNG vs SVG; ^▲P<0.05 drug vs drug + PD98059. Abbreviations: BCG, blank control group; DFNG, drug-free nanomicelles group; p-ERK1/2, phosphorylated ERK1/2; SD, standard deviation; SV, simvastatin; SVG, simvastatin group; SVNs, simvastatin-loaded nanomicelles; SVNG, simvastatin-loaded nanomicelles group; t-ERK1/2, total ERK1/2.

Figure 4

The role of the ERK1/2 pathway in the regulation of cell proliferation by SVNs and SV. Notes: The results are the mean values of three independent measurements (±SD). ^*P<0.05 vs BCG; ^#P<0.05 SVNG vs SVG; ^▲P<0.05 drug vs drug + PD98059; ^♦P<0.05 vs BCG + PD98059; ^∆P<0.05 SVNG + PD98059 vs SVG + PD98059. Abbreviations: BCG, blank control group; DFNG, drug-free nanomicelles group; SD, standard deviation; SV, simvastatin; SVG, simvastatin group; SVNs, simvastatin-loaded nanomicelles; SVNG, simvastatin-loaded nanomicelles group.

Figure 5

The role of the ERK1/2 pathway in the regulation of the cell cycle by SVNs and SV. Notes: The results are the mean values of three independent measurements (±SD). ^*P<0.05 vs BCG; ^#P<0.05 SVNG vs SVG; ^▲P<0.05 drug vs drug + PD98059; ^♦P<0.05 vs BCG + PD98059; ^∆P<0.05 vs SVG + PD98059. Abbreviations: BCG, blank control group; DFNG, drug-free nanomicelles group; SD, standard deviation; SV, simvastatin; SVG, simvastatin group; SVNs, simvastatin-loaded nanomicelles; SVNG, simvastatin-loaded nanomicelles group.

Figure 6

The role of the ERK1/2 pathway in the regulation of cell apoptosis by SVNs and SV. The results are the mean values of three independent measurements (±SD). ^▲P<0.05 drug vs drug + PD98059; ^♦P<0.05 vs BCG + PD98059. Abbreviations: BCG, blank control group; DFNG, drug-free nanomicelles group; SD, standard deviation; SV, simvastatin; SVG, simvastatin group; SVNs, simvastatin-loaded nanomicelles; SVNG, simvastatin-loaded nanomicelles group.

Figure 7

The role of the ERK1/2 pathway in the regulation of osteogenic differentiation of cells by SVNs and SV. By inhibiting the ERK1/2 pathway, SVNs and SV increased the ALP activity (A) and the protein expression levels of OSX (B) and OC (C) in MG63 cells. The results are represented as the mean values of three independent measurements (±SD). ^*P<0.05 vs BCG; ^#P<0.05 SVNG vs SVG; ^▲P<0.05 drug vs drug + PD98059; ^♦P<0.05 vs BCG + PD98059; ^∆P<0.05 SVNG + PD98059 vs SVG + PD98059. Abbreviations: BCG, blank control group; DFNG, drug-free nanomicelles group; OC, osteocalcin; OSX, osterix; SD, standard deviation; SV, simvastatin; SVG, simvastatin group; SVNs, simvastatin-loaded nanomicelles; SVNG, simvastatin-loaded nanomicelles group.