The apoE-deficient rats showed higher plasma levels of total cholesterol, LDL cholesterol and VLDL cholesterol, and lower plasma levels of HDL cholesterol, as compared to control rats. Thus, the apoE-deficient rats exhibited dyslipidemia in this study. In addition, the periodontal tissues in the apoE-deficient rats exhibited higher expression levels of TLR2, TLR4, RANKL and TRAP than those in the control rats. TLR activates RANKL expression, resulting in osteoclast differentiation. It is possible that dyslipidemic conditions are associated with TLR-induced osteoclast differentiation in periodontal tissues.
Previous studies have demonstrated that OxLDL has direct effects on TLR2 and TLR4 expression[6, 7]. In this study, dyslipidemic conditions induced higher plasma levels of OxLDL than normal conditions. Increased plasma levels of OxLDL following dyslipidemic conditions may induce increased expression of TLR2 and TLR4. TLR 2 and 4 are critical receptors for oral bacterial LPS[8, 9]. Dyslipidemic conditions would alter the interaction between bacterial pathogens and the cellular membrane, by increasing plasma levels of OxLDL.
In this study, dyslipidemic conditions were associated with higher plasma levels of total cholesterol, LDL cholesterol and VLDL cholesterol, and lower plasma levels of HDL cholesterol when compared with normal conditions. In clinical studies, it has been shown associations between blood lipid parameters and periodontal condition[21–23]. Therefore, it is also possible that impaired plasma lipids had direct effects on TLR2 and TLR4 expression in the current model. However, we previously found that changes in osteoclast differentiation under hypercholesterolemic conditions did not depend on blood lipids[13, 24]. This suggests that impaired lipids do not play a crucial role in TLR2 and TLR4 expression in the periodontal tissue.
It has been reported that activation of TLR2 and TLR4 promotes expression of inflammatory cytokines[25, 26]. In the present study, dyslipidemic conditions induced gene expression of IL-1β, an inflammatory cytokine. Inflammatory cytokines promote osteoclast differentiation both directly and indirectly. These observations indicate that the increased osteoclast differentiation under dyslipidemic conditions is partly caused by induction of inflammatory cytokines. Furthermore, overproduction of inflammatory cytokines can stimulate inflammatory responses, and this would advance periodontal inflammation. In fact, inflammatory histological changes, such as increased number of polymorphonuclear leukocytes and root resorption, were also observed in the current dyslipidemia model.
In our previous study, hypercholesterolemic rats (age, 20 weeks) fed a high-cholesterol diet for 8 weeks showed increased numbers of RANKL-positive cells on the alveolar bone surface. In that study, the ratio of RANKL-positive cells to total cells (mean ± SD) was 0.15 ± 0.04 in the control group and 0.32 ± 0.10 in the hypercholesteromic group. In the present study using an apoE-deficient model (age, 16 weeks), the ratio of RANKL-positive cells to total cells (mean ± SD) was 0.18 ± 0.05 in the control group and 0.41 ± 0.07 in the dyslipidemic group. These results are in agreement with previous findings that impaired lipid metabolism induces RANKL expression.
Studies have shown positive association between dyslipidemia and periodontal disease[21, 29, 30]. In our previous animal study, feeding with a high-cholesterol diet increased serum lipid peroxidation and the number of TRAP-positive osteoclasts, with an increase in RANKL expression. In this study, we demonstrated that activation of TLR2 and TLR4 is involved in osteoclast differentiation on the surface of alveolar bone under dyslipidemic conditions. Therefore, suppression of TLR expression may be an effective method for preventing osteoclast differentiation under dyslipidemic conditions. However, further studies are necessary.
Our study has several limitations. As the bacterial flora in the gingival sulcus of rats differs from that in humans, we did not investigate how dyslipidemic conditions directly affect bacterial flora. More studies are thus necessary in order to clarify this issue. In addition, although osteoclast differentiation increased under dyslipidemic conditions, the linear distance between the cemento-enamel junction and alveolar bone crest did not change in our model. As the experimental period was only 7 days, long-term analysis is necessary.
In conclusion, dyslipidemia induces osteoclast differentiation on the alveolar bone surface by activation of TLR2 and TLR4 in the rat apolipoprotein E knockout model.