Substance P aggravates periodontitis by upregulating HIF-1 α and RANKL / OPG ratio

Background Both substance P (SP) and hypoxia-inducible factor 1 alpha (HIF-1α) get involved in inflammation and angiogenesis. But the interrelation between SP and HIF-1α in rat periodontitis is still little known. Methods Ligation‐induced rat periodontitis was established to observe the expression of SP and HIF-1α by immunohistochemistry. Gingival fibroblasts and bone marrow macrophages (BMMs) were respectively cultured and stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). 10 nM SP with or without 1µg/ml LPS was added to elaborate the relationship between SP and HIF-1α in gingival fibroblasts. The effect of SP on osteoclastogenesis was tested by TRAP staining. Western blotting was applied to investigate the expressions of HIF-1α, osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL). Results The expression levels of HIF-1α and SP were higher in periodontitis than normal tissues. SP could upregulate the level of HIF-1α and RANKL/OPG ratio in LPS-stimulated gingival fibroblasts. SP with or without LPS also facilitated RANKL induced osteoclastogenesis. Conclusion Substance P aggravates periodontitis by increasing HIF-1α and RANKL / OPG ratio.


Abstract
Background Both substance P (SP) and hypoxia-inducible factor 1 alpha (HIF-1α) get involved in inflammation and angiogenesis. But the interrelation between SP and HIF-1α in rat periodontitis is still little known.
Methods Ligation-induced rat periodontitis was established to observe the expression of SP and HIF-1α by immunohistochemistry. Gingival fibroblasts and bone marrow macrophages (BMMs) were respectively cultured and stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). 10 nM SP with or without 1µg/ml LPS was added to elaborate the relationship between SP and HIF-1α in gingival fibroblasts.
The effect of SP on osteoclastogenesis was tested by TRAP staining. Western blotting was applied to investigate the expressions of HIF-1α, osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL).
Results The expression levels of HIF-1α and SP were higher in periodontitis than normal tissues. SP could upregulate the level of HIF-1α and RANKL/OPG ratio in LPS-stimulated gingival fibroblasts. SP with or without LPS also facilitated RANKL induced osteoclastogenesis.
Conclusion Substance P aggravates periodontitis by increasing HIF-1α and RANKL / OPG ratio.

Background
Periodontitis is a chronic inflammatory disease with periodontal bone destruction and However, the interrelation between SP and HIF-1α in rat periodontitis is still little known.
In this study, recombinant substance P protein was applied to observed the expression of HIF-1α, osteoprotegerin (OPG) and receptor activator of NF-kB ligand (RANKL) to elaborate the relationship between SP and HIF-1α in LPS-stimulatedgingival fibroblasts.

Animals
Twelve male Wistar rats (220-260 g, Laboratory Animal Center, Shandong University) were maintained on a routine diet to acclimate for 1 week before the experiment. The rats were assigned to ligation (L) group and normal (N) group at random. Protocols of the study met approval from Ethics in the Care and Use of Laboratory Animals Committee of the School of stomatology, Shandong University.

Rat experimental periodontitis model
Rats in the L group were under general anesthesia for the experimental periodontitis model. A 4-0 silk suture and an orthodontic ligature wire was placed around the cervical region of the right first lower molars and then ligated firmly. After 6 weeks, all rats in the two groups were euthanized with a lethal dose (150 mg/kg) of sodium thiopental. The gingiva and alveolar bone tissues were collected and fixed in 4% paraformaldehyde for 48 hours. Then specimens were dehydrated, cleared and finally embedded in paraffin. Serial sections (5-μm thick) were obtained for hematoxylin-eosin staining (HE) staining and SP

TRAP staining
The cells were fixed with 4% paraformaldehyde, and then stained for TRAP using a commercially available kit (Joy Tech Bio. Co., Zhejiang, China). Osteoclasts were identified as TRAP-positive multi-nucleated cells containing three or more nuclei.

Statistical analysis
All data values were expressed as mean ± standard error of mean. T tests were conducted with GraphPad Prism 5 software. A value of P < 0.05 was considered statistically significant.

Rat experimental periodontitis model
6 weeks after ligation, obvious gingival recession of the first molars was observed.
Gingival inflammatory infiltration and obvious alveolar bone loss between the first and second molars were observed by hematoxylin-eosin staining (Figure 1).

Expressions of HIF-1α and SP in rat ligation-induced experimental periodontitis.
After the immunohistochemistry staining was performed in the gingiva of ligation-induced experimental periodontitis, positive expression of HIF-1α was observed. The positive staining region was localized in inflammatory infiltrating cells (Figure 2A, B and C).
Comparing with normal gingiva, gingiva of periodontitis expressed higher HIF-1α according to western blotting (*P<0.05) ( Figure 2D). In addition, we observed the positive staining of SP was the same with HIF-1α (Figure 3).

SP promoted RANKL-induced osteoclast differentiation in BMMs and upregulated
In the RANKL+10 nM SP ( Figure 5B2) and RANKL+50 nM SP groups ( Figure 5B3), more TRAP positive osteoclasts were detected than the RANKL group ( Figure 5B1). Both 10 nM and 50 nM SP were able to upregulate osteoclast differentiation induced by RANKL ( Figure   5C).
SP upregulated RANKL/OPG ratio in gingival fibroblasts tested by western blotting. The RANKL/OPG ratio was markedly increased in the LPS+SP group, compared to the LPS and SP groups (*P<0.05) (Figure 6). In neutrophils, it has been demonstrated that HIF-1α was able to increase the activity of NF-κB and inhibit neutrophil apoptosis through this pathway [14,15]. In this study, increased expressions of HIF-1α and SP were observed in rat ligation-induced experimental periodontitis.

Discussion
Our previous study has showed upregulating the expression of HIF-1α changes the metabolic pathway of human periodontal ligament cells. Both SP and HIF-1α get involved in inflammation and angiogenesis. But the interrelation between SP and HIF-1α in rat periodontitis is still little known. According to relevant research, 1µg/ml LPS and 10 nM SP [15,16] were applied in this study. We observed both 1µg/ml LPS and 10 nM SP could obviously induce TNF-α expression. 10 nM SP with or without 1µg/ml LPS both could upregulate the level of HIF-1α in gingival fibroblasts.
Substance P signaling regulates the functions of both osteoblast and osteoclast [17]. It has been clarified that SP inhibits osteoblastic cells differentiation and may be related to bone metabolism in periodontal diseases under conditions of stress [9,16]. Osteoclasts can express SP receptor, which will bind to SP and then induce osteoclastogenesis. If a certain quantity of SP was produced constantly, it will result in bone resorption [18,19]. Our results showed RANKL/OPG ratio was markedly increased in the LPS group, SP group, and LPS+SP group (P<0.05), compared with normal group. SP might upregulate RANKL/OPG ratio to enhance bone resorption, which was consistent with the result of Lee et al [20].
There are some other mechanisms through which SP plays a role in periodontitis, such as inducing interelukin-6 (IL-6) synthesis in monocytes, stimulating histamine release from mast cells, regulating the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) and so on [21]. Our study may provide a new mechanism for SP modulating periodontitis.