Osteoprotegerin/receptor activator of nuclear factor-κB ligand are involved in periodontitis-promoted vascular calcification

Abstract

The present study explored the potential role of osteoprotegerin (OPG)/receptor activator of nuclear factor-κB (RANK)/receptor activator of nuclear factor-κB ligand (RANKL) in promoting vascular calcification by periodontitis. Thirty-six male Wistar rats were randomly assigned to four groups to establish animal models as follows: the sham group (group C), vascular calcification group (group VDN), periodontitis group (group CP), and test group (group CP+VDN). After eight weeks, all the rats were sacrificed. The periodontal and vascular calcification indices were detected. Quantitative polymerase chain reaction (qPCR), immunohistochemistry, western blot analysis, and enzyme-linked immunosorbent assay (ELISA) were used to quantify OPG/RANK/RANKL expression in vascular tissue and serum. Protein expression analyses revealed the expression of OPG and RANKL in the vascular tissues of the four groups. The expression of OPG in group C was the highest, which was similar to group CP+VDN, and the expression of OPG in groups CP and VDN were lower. However, the expression of RANKL was inversely correlated with OPG, and the ratio of RANKL/OPG was also higher in groups CP and VDN than that in groups C and CP+VDN. In conclusion, OPG/RANK/RANKL may play an essential role in the promotion of vascular calcification by periodontitis. However, the expression levels of OPG and RANKL were not simply superimposed when periodontitis and vascular calcification co-existed.

Keywords: RANK; RANKL; animal models; osteoprotegerin; periodontitis; vascular calcification.

Figure 1

(A) (a-h) Representative vascular immunohistochemistry. OPG expression was higher in group C than in any other group. RANKL expression was higher in the other three groups, while the CP+VDN group exhibited the highest expression (black arrows indicate positive cells; scale bar, 50 µm.). (B) (a) IOD/area value of OPG was calculated with Image-Pro Plus software. (b) IOD/area value of RANKL was calculated with Image-Pro Plus software. (c) Representative RANKL/OPG value. ^*P<0.05, compared with groups CP and VDN; ^▲P>0.05, compared with group C (non-significant difference). Groups: the sham group (group C), vascular calcification group (group VDN), periodontitis group (group CP), and test group (group CP+VDN). OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-κB ligand.

Figure 2

(A) Quantitative analysis of OPG and RANKL, using western blot analysis of the vascular tissue. (B) The relative levels of (a) OPG (b) RANKL and (c) RANKL/OPG value by western blot analysis. ^*P<0.05, compared with groups C and CP+VDN; ^▲P>0.05, compared with group C (non-significant difference); ^ΔP<0.05, compared with group CP. Groups: the sham group (group C), vascular calcification group (group VDN), periodontitis group (group CP), and test group (group CP+VDN). OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-κB ligand.

Figure 3

Expression of (A) OPG, (B) RANKL and (C) the ratio of RANKL/OPG by real-time qPCR. ^*P<0.05, compared with groups C and CP+VDN; ^▲P>0.05, compared with group C (non-significant difference); ^ΔP<0.05, compared with group CP. Groups: the sham group (group C), vascular calcification group (group VDN), periodontitis group (group CP), and test group (group CP+VDN). OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-κB ligand.

Figure 4

Expression of (A) OPG, (B) RANKL and (C) the RANKL/OPG ratio by ELISA. ^*P<0.05, compared with groups C and CP+VDN; ^▲P>0.05, compared with group C (non-significant difference); ^ΔP<0.05, compared with group CP. Groups: the sham group (group C), vascular calcification group (group VDN), periodontitis group (group CP), and test group (group CP+VDN). OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-κB ligand.