Oral administration of 5-hydroxytryptophan aggravated periodontitis-induced alveolar bone loss in rats

archives of oral biology 60 (2015) 789–798

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Oral administration of 5-hydroxytryptophan aggravated periodontitis-induced alveolar bone loss in rats Xianxian Li a,b, Xiangnan Wu a,b, Yuanyuan Ma c,d, Zhichao Hao a,b, Shenyuan Chen e,f, Taozi Fu a,b, Helin Chen a,b, Hang Wang a,b,* a

State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, PR China West China College of Stomatology, Sichuan University, Chengdu 610041, PR China c Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou 510055, PR China d Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, PR China e Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing 401147, PR China f The Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing 401147, PR China b

article info

abstract

Article history:

Objective: 5-Hydroxytryptophan (5-HTP) is the precursor of serotonin and 5-HTP has been

Accepted 31 January 2015

widely used as a dietary supplement to raise serotonin level. Serotonin has recently been discovered to be a novel and important player in bone metabolism. As peripheral serotonin

Keywords:

negatively regulates bone, the regular take of 5-HTP may affect the alveolar bone metabo-

5-Hydroxytryptophan

lism and therefore influence the alveolar bone loss induced by periodontitis. The aim of this

Serotonin

study was to investigate the effect of 5-HTP on alveolar bone destruction in periodontitis.

Periodontitis

Design: Male Sprague-Dawley rats were randomly divided into the following four groups: (1)

Alveolar bone loss

the control group (without ligature); (2) the 5-HTP group (5-HTP at 25 mg/kg/day without

Osteoclast

ligature); (3) the L group (ligature + saline placebo); and (4) the L + 5-HTP group (ligature + 5-

Osteocyte

HTP at 25 mg/kg/day). Serum serotonin levels were determined by ELISA. The alveolar bones were evaluated with micro-computed tomography and histology. Tartrate-resistant acid phosphatase staining was used to assess osteoclastogenesis. The receptor activator of NFkB ligand (RANKL) and osteoprotegerin (OPG) expression in the periodontium as well as the interleukin-6 positive osteocytes were analysed immunohistochemically. Results: 5-HTP significantly increased serum serotonin levels. In rats with experimental periodontitis, 5-HTP increased alveolar bone resorption and worsened the micro-structural destruction of the alveolar bone. 5-HTP also stimulated osteoclastogenesis and increased RANKL/OPG ratio and the number of IL-6 positive osteocytes. However, 5-HTP treatment alone did not cause alveolar bone loss in healthy rats. Conclusion: The present study showed that 5-HTP aggravated alveolar bone loss, deteriorated alveolar bone micro-structure in the presence of periodontitis, which suggests 5-HTP administration may increase the severity of periodontitis. # 2015 Elsevier Ltd. All rights reserved.

* Corresponding author at: 14 Third Section, Renmin Nan Road, Chengdu 610041, PR China. Tel.: +86 28 85501441; fax: +86 28 85582167. E-mail address: [email protected] (H. Wang). http://dx.doi.org/10.1016/j.archoralbio.2015.01.015 0003–9969/# 2015 Elsevier Ltd. All rights reserved.

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1.

archives of oral biology 60 (2015) 789–798

Introduction

Periodontitis-induced alveolar bone loss is the most prevalent form of bone pathology1 and a major cause of tooth loss in adults. Although bacterial infections are the primary trigger of periodontitis, alveolar bone loss is mainly caused by the uncoupling of the normally balanced remodelling processes of bone resorption and bone formation.2 Systematic and local factors that modulate bone remodelling can greatly influence the occurrence and severity of alveolar bone loss. 5-Hydroxytryptophan (5-HTP) is the precursor of serotonin (i.e., 5-HT). 5-HT has been known as a neurotransmitter that regulates mood and behaviour in the central nervous system for decades. Recently, growing evidence has shed light on the important role of 5-HT in the regulation of bone metabolism,3 although debate about this issue still exists.4 Peripheral 5-HT down regulates osteoblast proliferation and mediates the effect of low-density lipoprotein receptorrelated protein 5 (LRP5).5,6 In contrast, 5-HT up regulates osteoclast formation and deficient osteoclastogenesis has been observed in 5-HT-depleted mice.7,8 In our previous in vitro study, 5-HT was found to activate ERK pathway and stimulate the secretion of interlukin-6 (i.e., IL-6),9 which is an important inflammatory cytokine in periodontitis and a multifunctional regulator of bone remodelling,10 by osteocytes. The pharmacologic inhibition of peripheral 5-HT genesis prevents the bone loss caused by ovariectomy.5,11 In contrast, the widely used SSRI antidepressants (i.e., inhibitors of 5-HT transporters) which elevate 5-HT level, have been found to decrease bone mineral density and increase the risk of bone fracture.12–15 Because alveolar bone is a part of the skeletal system, if 5HT is confirmed to regulate bone metabolism, it might also influence the progress of alveolar bone loss in periodontitis. Notably, a gene polymorphism for the 5-HT transporter was recently been found associated with aggressive periodontitis.16 However, we still have a very limited understanding of the role of 5-HT in periodontitis. Two recent studies investigated the effects of SSRIs on periodontitis and produced conflicting results. One study by Branco de Almeida et al.17 suggested a therapeutic effect of fluoxetine, whereas the other study by Carvalho et al.18 reported a deteriorative effect of venlafaxine on periodontal bone loss. As the precursor of 5-HT, 5-HTP has been widely used clinically as a 5-HT supplement for decades.19 The aromatic amino acid decarboxylase (AAAD) exclusively and freely converts 5-HTP to 5-HT without any biochemical feedback inhibition.20 If 5-HT can regulate bone and influence the alveolar bone loss induced by periodontitis, it is reasonable to assume the regular administration of 5HTP would have the same effect. This potential effect is particularly important because of the large number of people who take 5-HTP as a dietary supplement to improve depression, obesity or headaches. In the present study, we tested the hypothesis that the use of 5-HTP would increase circulating 5-HT levels and thus influence the progression of alveolar bone loss in a rat model of experimental periodontitis.

2.

Materials and methods

2.1.

Animals

Forty male Sprague-Dawley rats between the ages of 6 and 8 weeks with body weights that ranged from 180 to 220 g were purchased from the Laboratory Animal Centre of Sichuan University. The rats were housed in standard conditions (12-h light/dark cycle and temperature 22–25 8C) with free access to food and water. All of the protocols described in this study were approved by the Institutional Ethics Committee of the State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University.

2.2. Experimental groups and the induction of experimental periodontitis (EPD) The animals were randomly assigned into the following four experimental groups (n = 10 animals/group) after 7 days of acclimatization: (1) the control group (without ligature); (2) the 5-HTP group (5-HTP at 25 mg/kg/day without ligature); (3) the L group (ligature + saline placebo); and (4) the L + 5-HTP group (ligature + 5-HTP at 25 mg/kg/day). The 5-HTP was dissolved in saline solution. All treatments (saline or 5-HTP) were given orally (via gavage) 1 h before ligature attachment and daily during the experimental periods. To induce periodontitis, rats were general anesthetized and a ligature was put in a subgingival position around the cervixes of the right maxillary first molars. The upper molars were chosen as periodontitis can be induced by ligature more rapidly in the upper molar than the lower molar.21 The rats were killed under general anaesthesia 14 days after the attachment of the ligature. The maxillae were collected and fixed in 10% neutral formalin for 24 h and then submitted to micro-CT.

2.3.

Serum 5-HT determination

Serum was harvested from blood collected from the tail vein on days 7 and 14. Serum 5-HT was measured immediately after the blood collection with an ELISA kit (CUSABIO BIOTECH, Wuhan, China) according to manufacturer’s instructions. Briefly, a monoclonal antibody specific to 5-HT has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin-labelled 5-HT and unlabeled 5-HT (standards and samples) with the pre-coated antibody specific to 5-HT. After incubation (37 8C, 1 h), the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated (37 8C, 30 min) before washing. The amount of bound HRP conjugate is reverse proportional to the concentration of 5-HT in the sample. The substrate solution is added and incubated (avoid of light, 37 8C, 20 min). After adding the stop solution, run the microplate reader and conduct measurement at 450 nm immediately. The sensitivity of the ELISA kit for serotonin was 0.4 ng/ml.

2.4.

Micro-CT analysis

After fixation, the maxillae were transferred to 70% ethanol for micro-CT scanning (VivaCT 40, Scanco Medical, Basserdorf,

archives of oral biology 60 (2015) 789–798

Switzerland). Three-dimensional images were reconstructed to quantify the extent of bone destruction. The horizontal alveolar bone loss (ABL) measurements were performed along the long axis of the buccal roots of all of the right maxillary molars. The distances from the cement–enamel junctions to the alveolar bone crest were measured, and the total alveolar bone loss was determined by taking the sum of the measurements (i.e., three measurements for the first molar and two measurements each for the second and third molars) and subtracting the values obtained from the left maxilla (i.e., the non-ligature side) in millimetres.22 The roots were digitally removed and the alveolar bone around the right maxillary first molar was selected as the region of interest (ROI).23 The following micro-architecture parameters were analyzed in the ROIs: bone volume ratio (BV/TV); trabecular number (Tb.N); trabecular thickness (Tb.Th); trabecular separation (Tb.Sp); and bone surface density (BS/BV).

2.5.

Histological analyses

After the micro-CT, the right hemimaxillae were immersed in 10% EDTA solution for 30 days (solution were changed daily) at room temperature for decalcification. Next, the specimens were embedded in paraffin and were cut along the mesialdistal axis to produce 5 mm-thick serial sagittal sections of the right maxillary first, second, and third molars. The sections were then stained with haematoxylin and eosin (H&E) and evaluated using a microscope (Nikon 80i, Nikon Ltd., Japan).

2.6.

Osteoclastogenesis analysis

To identify the osteoclasts, three sections of each specimen were stained for tartrate-resistant acid phosphatase (TRAP) using a TRAP stain kit (387A, SIGMA, St. Louis, MO, USA) according to the instructions in the product manual. Osteoclasts were identified as TRAP+ multinuclei cells (>3 nuclei). In each section, five fields near the bone crest region were randomly selected at 400 magnification, and the TRAP+ cells in these fields were counted by two blinded observers.

2.7.

Immunohistochemical analysis

Immunolabeling was carried out using the Histostain-Plus Kit (Zymed, USA) as recommended by the manufacturer. Briefly, after deparaffinization and rehydration, slides were incubated in 3.0% H2O2 for 10 min to reduce endogenous peroxidase activity. Pepsin solution (Invitrogen, USA) was used for antigen retrieval. Subsequently, samples were incubated in serum blocking reagent for 30 min at 37 8C to saturate non-specific binding sites. Anti-OPG (bs-0431R, Bioss, China), anti-RANKL (bs-0747R, Bioss, China) and anti-IL-6 (ab6672, Abcam, USA) were diluted at 1:100 and applied to tissue sections, which were incubated overnight in humidified chambers at 4 8C. Sections were then incubated with biotin-labelled secondary antibody (30 min, 37 8C) followed by the avidin–biotin–peroxidase complex. Finally, sections were developed with 0.05% 3, 3-diaminobenzidine tetrachloride (DAB) and counterstained with Mayer’s haematoxylin. The negative control was carried out with the same steps as described above except replacing the primary antibody with phosphate buffer solution (PBS).

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The staining images were taken using a microscope with an attached digital camera (Nikon 80i, Nikon Ltd., Japan).

2.8.

Statistical analyses

The data were analysed with one-way ANOVAs. When a significant difference was found, multiple comparisons between the groups were performed using the S–N–K method. p values <0.05 were considered significant. All calculations were performed using GraphPad Prism 5 software (GraphPad, Inc., San Diego, CA, USA).

3.

Results

3.1.

Serum 5-HT concentration

ELISA tests were used to determine if feeding with 5-HTP, the precursor of 5-HT, increased the serum 5-HT levels in the periphery. As shown in Fig. 1, 25 mg/kg daily gavages of 5-HTP significantly increased serum 5-HT concentrations at both times evaluated (D7 and D14 p < 0.05). Therefore, 5-HTP gavage effectively elevated circulating 5-HT. However, no significant difference was found between the two time points in any group (p > 0.05), and the ligatures did not affect the circulating 5-HT levels (p > 0.05).

3.2.

Alveolar bone loss

The reconstructed 3-D images of the right sides of the maxillae are shown in Fig. 2a. No resorption of the alveolar bone was observed in the control group or the 5-HTP treatment alone group. Bone resorption with root exposure was observed in the L group. The animals that were subjected to EPD and received 5-HTP gavages (L + 5-HTP group) suffered more severe bone loss and root exposure than did the animals that received EPD alone (L group). The distances between the cemento–enamel junction and the alveolar bone crest were measured along the axes of the buccal roots. As shown in Fig. 2b, the EPD induced by the ligatures resulted in alveolar bone loss (control vs. L; p < 0.05) whereas 5-HTP treatment alone did not cause alveolar bone loss (control vs. 5-HTP; p > 0.05). However, HTP significantly increased the alveolar bone loss when the ligatures were used (L vs. L + 5-HTP p < 0.05). Together, these results indicate that

Fig. 1 – Serum serotonin levels determined by ELISA: 5-HTP significantly elevated the serum serotonin levels on both day 7 and 14.

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Fig. 2 – 5-HTP increases alveolar loss induced by periodontitis. (a) Buccal view micro-CT 3-D images of right maxillae from each group at day 14. (b) Linear measurements of the alveolar bone loss (ABL) in each group. The L group exhibited significant alveolar bone loss compared to the control group, and the L + 5-HTP group displayed the greatest alveolar bone loss. No significant difference was found between the 5-HTP group and the control group. (c–g) The micro-structure parameters of the bone surrounding the roots of first maxillary molar. (c) The fractions of bone volume vs. the total volume. The L + 5-HTP group exhibited the lowest BVF volume, and there were no significant differences between the other groups. (d) Trabecular numbers. The Tb.n of the L + 5-HTP group was significantly lower than that of the L group. However, no significant differences were found among the other groups. (e) The trabecular thicknesses. The smallest Tb.ths were observed in the L + 5-HTP group, no significant differences were found among the other groups. (f) Trabecular separations. The ligatures increased the trabecular separation (L group vs. control group), and the L + 5-HTP group exhibited significantly greater trabecular separation than did the L group. (g) The fractions of bone surface vs. the bone volume. L + 5HTP group exhibited the highest BS/BVs, and no significant differences were observed among the other groups. *p < 0.05.

5-HTP significantly aggravated the alveolar bone loss that was induced by EPD.

3.3.

Micro-architecture of the alveolar bone

To further explore the alveolar bone quality, we performed bone histomorphometry. As shown in Fig. 2c–g, there were no significant differences in any of the parameters between the 5-HTP group and the control group. Compared to the L group, the L + 5-HTP group was significantly worse (p < 0.05) in terms of all of the micro-architecture parameters that were evaluated. The L + 5-HTP group exhibited the lowest BVF value and associated Tb,n and Tb,th values and exhibited the highest BS/BV and Tb.sp values. These data are suggestive of deteriorations in the micro-architectures of the alveolar bones in the animals that received 5-HTP when EPD was induced.

3.4.

Histological analyses

As shown in Fig. 3, the H&E-stained maxillary bones revealed healthy periodontal tissue in which the junctional epithelium

is attached to the cement–enamel junction, and the alveolar bone crest was intact in the control and 5-HTP groups. In the L group, EPD alone decreased the height and the width of the alveolar bone, and apical migration of the junctional epithelium was observed. However, the animals in the L + 5-HTP group suffered the most severe periodontitis. In agreement with the measurements from the 3-D images, the L + 5-HTP group exhibited more extensive destruction of the alveolar bone than did the L group.

3.5.

Osteoclastogenesis

As shown in Fig. 4, a few osteoclasts were observed in the healthy alveolar bone of the control and 5-HT groups. No statistically significant difference was found between the osteoclast numbers (OCNs) of the control and 5-HT groups, which suggests that 5-HTP treatment alone did not increase osteoclastogenesis. The OCN was increased in the L group compared to the control group. In the L + 5-HTP group, the OCN was significantly greater than that in the ligature alone group (L group, p < 0.05). Furthermore, as seen in Fig. 4d, the

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Fig. 3 – Histological aspects of the periodontia of the interproximal regions between the first and second molars in each group. (a) Control group: normal periodontium. (b) 5-HTP group: normal periodontium. (c) L group: experimental periodontitis was established and included alveolar bone loss and migration of the junctional epithelium. (d) L + 5-HTP group: increased periodontial destruction and severe alveolar bone loss were observed. The sections were stained with H&E (magnification 100T).

sizes of osteoclasts were also greatly increased. Together, these results indicate that 5-HTP stimulated osteoclastogenesis in conditions of experimental periodontitis.

3.6.

RANKL and OPG and expression

The periodontium of EPD-induced rats (L group) showed markedly increased immunostaining for RANKL compared to the periodontium of the control group and 5-HT group. 5-HTP administration slightly increased the amount of immunostaining observed for RANKL in the periodontium of the EPD-induced rats (Fig. 5). On contrary, as shown in Fig. 6, the immunostaining for OPG was strong in the periodontium of the control group and 5-HT group (Fig. 6a and b). The OPG stain decreased when periodontitis occurred. The weakest OPG immunostaining was observed in the L + 5-HTP group.

3.7.

Osteocyte secretion of IL-6

We previously found that 5-HT stimulates IL-6 secretion from osteocyte-like MLO-Y4 cells.9 To test whether the 5-HTP administration-induced elevation of 5-HT would stimulate the secretion of IL-6 from osteocytes; we performed out immunohistochemical staining for IL-6. Fig. 7 shows that almost no IL-6-positive osteocytes were observed in the control group. In the 5-HTP group and the L group, small numbers of osteocytes were IL-6-positive. In contrast, a large increase of IL-6+ osteocyte numbers was observed in the L + 5-HTP group.

4.

Discussion

In this study, we evaluated horizontal alveolar bone loss both morphologically and histologically. The results were consistent with each other and indicate a clear association between 5-HTP and increased periodontitis-related alveolar bone loss. The 3-D intrabony micro-structures of the alveolar bones were also evaluated. The results revealed that 5-HTP not only reduced alveolar bone crest height but also deteriorated the micro-structure of the bone in the periodontitis model. Together, these results demonstrated that 5-HTP significantly aggravated the alveolar bone destruction induced by periodontitis. Recently, 5-HT was demonstrated to be an important regulator of bone metabolism. Chabbi-Achengli et al.8 demonstrated decreased osteoclast formation in 5-HT-depleted mice and that the impaired osteoclastogenesis can be rescued by 5-HTP administration. A study by Laporta et al.24 indicated that oral intake of 5-HTP during the transition from pregnancy to lactation increases bone resorption markers and osteoclast numbers in the femur. In our study, 5-HTP treatment significantly increased osteoclast number and size in the alveolar bone when periodontitis was induced (L group vs. L + 5-HTP group, Fig. 4). This finding indicates a role for 5-HT in osteoclastogenesis related to inflammatory bone loss. However, 5-HTP treatment alone failed to stimulate osteoclastogenesis (control group vs. 5-HTP group, Fig. 4) or change the alveolar bone heights or structures in the healthy periodontal tissue (control group vs. 5-HTP group, Fig. 2). The

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Fig. 4 – Osteoclastogenesis. TRAP-positive osteoclasts are stained red. In the L + 5-HTP group, the number and sizes of the osteoclasts were significantly increased. (a,a0 ) control group. (b,b0 ) 5-HTP group. (c,c0 ) L group. (d,d0 ) L + 5HTP group. (e) The osteoclast number/field for each group.*p < 0.05 (a,b,c,d magnification 100T. a0 ,b0 ,c0 ,d0 magnification 400T).

finding that 5-HTP only affected inflammatory alveolar bone could be explained by the modulation of local 5-HT level by platelets. Under physiological condition, 95% of circulating 5-HT was up-taken and stored in platelets.25 However, under inflammation, the stored 5-HT was released due to platelet activation.26 Thus, 5-HTP may exert a bigger influence on local 5-HT concentration under inflammatory conditions. Besides, when periodontitis occurs, other inflammatory factors might be coordinated with increased 5-HT to cause enhanced bone resorption. In the study by Laporta et al.,24 5-HTP administration increased osteoclastogenesis in lactating rats in the absence of inflammation. This is likely due to the particularity of lactation period during which prolactin level are high and

endogenous 5-HT releases.27 The high prolactin level also has a strong effect on platelet activation.28,29 Besides, the increased PTHrP secretion27 during lactation may function together with the elevated 5-HT to cause the increased osteoclastogenesis. As 5-HT cannot pass the blood-brain barrier, the central and peripheral 5-HT pools act differently and independently. In the periphery, 5-HT acts as a hormone to inhibit bone formation and promote bone resorption. In contrast, in the central nervous system, 5-HT exerts a positive effect on bone via sympathetic tone.25 The influences of 5-HTP on bone metabolism result from the combined effects of central and peripheral 5-HT changes because 5-HTP can cross the blood–brain barrier, and AAAD exists both centrally and in the periphery. When 5-HTP is orally ingested, it is converted to 5-HT in the periphery, and the peripheral 5-HT cannot pass the blood-brain barrier. This peripheral process depletes substrate levels, and consequently, little 5-HTP reaches the brain.19 Indeed, several studies have suggested that when 5-HTP is used alone without inhibiting the peripheral conversion of 5-HTP to 5-HT, the improvements in depression caused by low central 5-HT levels are minimal.20 Thus, it is reasonable to assume the 5-HTP gavages used in the present study primarily influenced peripheral 5-HT levels. That the negative effect of elevated peripheral 5-HT exceeded the positive effect of increased central 5-HT results in a deleterious effect on alveolar bone in periodontitis. However, further study is needed to clarify the roles of central and peripheral 5-HT in periodontal bone loss. The RANKL–RANK–OPG system plays an important role in periodontitis associated alveolar bone loss. In the present study, 5-HTP up-regulated the RANKL/OPG ratio when periodontitis exists. The increased RANKL/OPG ratio is in accordance with our observed increased alveolar bone loss and osteoclastogenesis. We suppose that OPG/RANKL ratio change is involved in 5-HTP induced alveolar bone loss in periodontitis. We observed that the OPG decrease is more prominent than the RANKL increase. OPG is a strong inhibitor of the RANK–RANKL interaction and its avidity for binding to RANKL is 10 times higher than that of RANK.30 The decrease in OPG thus could largely increase the chance for RANK–RANKL binding to support osteoclast formation and bone resorption. However, the in vitro study by Gustafsson et al.31 indicated that 5-HT increased OPG while decreased RANKL expression in MC3T3-E1 cells. The discrepancy may due to the differences between in vitro and in vivo studies and the fact that 5-HT influences bone metabolism differently in healthy and diseased conditions.32 The finding that the 5-HTP administration increased the number of IL-6-positive osteocytes (Fig. 5) is consistent with the result from our previous study that 5-HT stimulates osteocytic IL-6 secretion in vitro.9 IL-6 has been demonstrated to induce osteoclastogenesis by stimulating RANKL expression though activating gp130-STAT3 pathway in osteoblast/stromal cells.25 Interleukin-6 gene polymorphisms have been shown to modify the risk for periodontitis.33–39 The secretion of IL-6 by osteocytes may affect bone as osteocytes can influence bone remodelling though soluble factors.40 Osteocytes secretion of IL-6 has been shown to

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Fig. 5 – Immunohistochemical staining for RANKL in the periodontium. (a) Control group. (b) 5-HTP group. (c) L group. (d) L + 5-HTP group. Magnification 100T.

Fig. 6 – Immunohistochemical staining for OPG in the periodontium. (a) Control group. (b) 5-HTP group. (c) L group. (d) L + 5HTP. Group magnification 100T

stimulate osteoclast precursor adhesion via ICAM-1.41 The increased osteocytes IL-6 secretion observed is consistent with the elevated osteoclastogenesis. We suggest that, in

addition to RANKL/OPG change, the increased IL-6 secretion from osteocytes may also participate in the 5-HTP-induced osteoclastogenesis in periodontitis.

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Fig. 7 – Immunohistochemical staining for IL-6 of the alveolar bone. (a) Control group: no positive osteocytes. (b) 5-HTP group and (c) L group: moderate numbers of IL-6 + osteocytes. (d) L + 5-HTP group: the majority osteocytes appeared as IL-6positive (magnification 100T).

The pathology of periodontitis is very complicated involving changes in immune system, periodontal soft tissue and alveolar bone. It has been reported that 5-HT can enhance T cell activation29,42 as well as the antigen presenting ability of dendritic cell.43,44 Thus 5-HTP may mediate the host immune responses in periodontitis. Besides, 5-HT has been shown to increase MMP-3 and MMP-13 expression in cardiac fibroblast.45 As MMP-3 and MMP-13 are crucial to periodontal break down, it is possible that 5-HTP administration may affect the periodontal fibroblast. Thus, we speculate that the increased alveolar bone destruction we observed in this study is a result from a combined effect of an indirect bone resorption stimulation from the immune or periodontal cells and a direct control of bone turnover by 5-HT. Further study is needed to investigate the immune and soft tissue changes in 5-HTP treated animal to explore the underlying mechanism by which 5-HTP increases alveolar bone loss induced by periodontitis.

5.

Competing interests None declared.

Ethical approval statement The protocols described in this paper were approved by the Institutional Ethics Committee of the State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University.

Acknowledgements This work was kindly supported financially by the National Natural Science Foundation of China (Nos. 81170941 and 81300856).

Conclusions references

In conclusion, our results showed that in the presence of ligature-induced periodontitis, 5-HTP aggravated alveolar bone loss, deteriorated alveolar bone micro-structure with increased osteoclast formation. These findings indicate that 5-HTP administration may increase the severity of periodontitis.

Funding This work was supported financially by the National Natural Science Foundation of China (Nos. 81170941 and 81300856).

1. Leone CW, Bokhadhoor H, Kuo D, Desta T, Yang J, Siqueira MF, et al. Immunization enhances inflammation and tissue destruction in response to Porphyromonas gingivalis. Infect Immun 2006;74(4):2286–92. 2. Ishikawa I. Host responses in periodontal diseases: a preview. Periodontology 2000 2007;43:9–13. 3. Kawai M, Rosen CJ. Minireview: a skeleton in serotonin’s closet? Endocrinology 2010;151(9):4103–8. 4. Cui Y, Niziolek PJ, MacDonald BT, Zylstra CR, Alenina N, Robinson DR, et al. Lrp5 functions in bone to regulate bone mass. Nat Med 2011;17(6):684–91.

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5. Yadav VK, Ryu JH, Suda N, Tanaka KF, Gingrich JA, Schutz G, et al. Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell 2008;135(5):825–37. 6. Warden SJ, Robling AG, Haney EM, Turner CH, Bliziotes MM. The emerging role of serotonin (5-hydroxytryptamine) in the skeleton and its mediation of the skeletal effects of low-density lipoprotein receptor-related protein 5 (LRP5). Bone 2010;46(1):4–12. 7. Battaglino R, Fu J, Spate U, Ersoy U, Joe M, Sedaghat L, et al. Serotonin regulates osteoclast differentiation through its transporter. J Bone Miner Res: Off J Am Soc Bone Miner Res 2004;19(9):1420–31. 8. Chabbi-Achengli Y, Coudert AE, Callebert J, Geoffroy V, Cote F, Collet C, et al. Decreased osteoclastogenesis in serotonindeficient mice. Proc Natl Acad Sci U S A 2012;109(7):2567–72. 9. Li X, Ma Y, Wu X, Hao Z, Yin J, Shen J, et al. Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT(2B) receptor and the ERK1/2 signalling pathway. Biochem Biophys Res Commun 2013;441(4):809–14. 10. Blanchard F, Duplomb L, Baud’huin M, Brounais B. The dual role of IL-6-type cytokines on bone remodeling and bone tumors. Cytokine Growth Factor Rev 2009;20(1):19–28. 11. Yadav VK, Balaji S, Suresh PS, Liu XS, Lu X, Li Z, et al. Pharmacological inhibition of gut-derived serotonin synthesis is a potential bone anabolic treatment for osteoporosis. Nat Med 2010;16(3):308–12. 12. Eom CS, Lee HK, Ye S, Park SM, Cho KH. Use of selective serotonin reuptake inhibitors and risk of fracture: a systematic review and meta-analysis. J Bone Miner Res: Off J Am Soc Bone Miner Res 2012;27(5):1186–95. 13. Couturier J, Sy A, Johnson N, Findlay S. Bone mineral density in adolescents with eating disorders exposed to selective serotonin reuptake inhibitors. Eat Disord 2013;21(3):238–48. 14. Haney EM, Warden SJ, Bliziotes MM. Effects of selective serotonin reuptake inhibitors on bone health in adults: time for recommendations about screening, prevention and management? Bone 2010;46(1):13–7. 15. Rizzoli R, Cooper C, Reginster JY, Abrahamsen B, Adachi JD, Brandi ML, et al. Antidepressant medications and osteoporosis. Bone 2012;51(3):606–13. 16. Costa JE, Gomes CC, Cota LO, Pataro AL, Silva JF, Gomez RS, et al. Polymorphism in the promoter region of the gene for 5-HTT in individuals with aggressive periodontitis. J Oral Sci 2008;50(2):193–8. 17. Branco-de-Almeida LS, Franco GC, Castro ML, Dos Santos JG, Anbinder AL, Cortelli SC, et al. Fluoxetine inhibits inflammatory response and bone loss in a rat model of ligature-induced periodontitis. J Periodontol 2012;83(5):664–71. 18. Carvalho RS, de Souza CM, Neves JC, Holanda-Pinto SA, Pinto LM, Brito GA, et al. Effect of venlafaxine on bone loss associated with ligature-induced periodontitis in Wistar rats. J Negat Results Biomed 2010;9:3. 19. Birdsall TC. 5-Hydroxytryptophan: a clinically-effective serotonin precursor. Altern Med Rev: J Clin Ther 1998;3(4): 271–80. 20. Hinz M, Stein A, Uncini T. 5-HTP efficacy and contraindications. Neuropsychiatr Dis Treat 2012;8:323–8. 21. Carvalho Rde S, de Souza CM, Neves JC, Holanda-Pinto SA, Pinto LM, Brito GA, et al. Vitamin E does not prevent bone loss and induced anxiety in rats with ligature-induced periodontitis. Arch Oral Biol 2013;58(1):50–8. 22. Leitao RF, Ribeiro RA, Chaves HV, Rocha FA, Lima V, Brito GA. Nitric oxide synthase inhibition prevents alveolar bone resorption in experimental periodontitis in rats. J Periodontol 2005;76(6):956–63. 23. Yang H, Wen Q, Xue J, Ding Y. Alveolar bone regeneration potential of a traditional Chinese medicine,

24.

25. 26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40. 41.

797

Bu-Shen-Gu-Chi-Wan, in experimental periodontitis. J Periodont Res 2014;49(3):382–9. Laporta J, Peters TL, Weaver SR, Merriman KE, Hernandez LL. Feeding 5-hydroxy-l-tryptophan during the transition from pregnancy to lactation increases calcium mobilization from bone in rats. Domest Animal Endocrinol 2013;44(4):176–84. Ducy P, Karsenty G. The two faces of serotonin in bone biology. J Cell Biol 2010;191(1):7–13. Kopp S, Alstergren P. Blood serotonin and joint pain in seropositive versus seronegative rheumatoid arthritis. Mediat Inflamm 2002;11(4):211–7. Hernandez LL, Gregerson KA, Horseman ND. Mammary gland serotonin regulates parathyroid hormone-related protein and other bone-related signals. Am J Physiol Endocrinol Metabol 2012;302(8):E1009–15. Urban A, Masopust J, Maly R, Hosak L, Kalnicka D. Prolactin as a factor for increased platelet aggregation. Neuroendocrinol Lett 2007;28(4):518–23. Wallaschofski H, Kobsar A, Sokolova O, Siegemund A, Stepan H, Faber R, et al. Differences in platelet activation by prolactin and leptin. Horm Metab Res Hormon- und Stoffwechselforschung/Hormones et metabolisme2004;36(7): 453–7. Shoji S, Tabuchi M, Miyazawa K, Yabumoto T, Tanaka M, Kadota M, et al. Bisphosphonate inhibits bone turnover in OPG(/) mice via a depressive effect on both osteoclasts and osteoblasts. Calcif Tissue Int 2010;87(2):181–92. Gustafsson BI, Thommesen L, Stunes AK, Tommeras K, Westbroek I, Waldum HL, et al. Serotonin and fluoxetine modulate bone cell function in vitro. J Cell Biochem 2006;98(1):139–51. Kode A, Mosialou I, Silva BC, Rached MT, Zhou B, Wang J, et al. FOXO1 orchestrates the bone-suppressing function of gut-derived serotonin. J Clin Invest 2012;122(10):3490–503. Costa AM, Guimaraes MC, de Souza ER, Nobrega OT, Bezerra AC. Interleukin-6 (G-174C) and tumour necrosis factor-alpha (G-308A) gene polymorphisms in geriatric patients with chronic periodontitis. Gerodontology 2010;27(1):70–5. Franch-Chillida F, Nibali L, Madden I, Donos N, Brett P. Association between interleukin-6 polymorphisms and periodontitis in Indian non-smokers. J Clin Periodontol 2010;37(2):137–44. Galicia JC, Tai H, Komatsu Y, Shimada Y, Ikezawa I, Yoshie H. Interleukin-6 receptor gene polymorphisms and periodontitis in a non-smoking Japanese population. J Clin Periodontol 2006;33(10):704–9. Holla LI, Fassmann A, Stejskalova A, Znojil V, Vanek J, Vacha J. Analysis of the interleukin-6 gene promoter polymorphisms in Czech patients with chronic periodontitis. J Periodontol 2004;75(1):30–6. Jansson H, Lyssenko V, Gustavsson A, Hamberg K, Soderfeldt B, Groop L, et al. Analysis of the interleukin-1 and interleukin-6 polymorphisms in patients with chronic periodontitis. A pilot study. Swed Dent J 2006;30(1):17–23. Nibali L, Tonetti MS, Ready D, Parkar M, Brett PM, Donos N, et al. Interleukin-6 polymorphisms are associated with pathogenic bacteria in subjects with periodontitis. J Periodontol 2008;79(4):677–83. Shao MY, Huang P, Cheng R, Hu T. Interleukin-6 polymorphisms modify the risk of periodontitis: a systematic review and meta-analysis. J Zhejiang Univ Sci B 2009;10(12):920–7. Bonewald LF. The amazing osteocyte. J Bone Miner Res: Off J Am Soc Bone Miner Res 2011;26(2):229–38. Cheung WY, Simmons CA, You L. Osteocyte apoptosis regulates osteoclast precursor adhesion via osteocytic IL-6 secretion and endothelial ICAM-1 expression. Bone 2012;50(1):104–10.

798

archives of oral biology 60 (2015) 789–798

42. Ahern GP. 5-HT and the immune system. Curr Opin Pharmacol 2011;11(1):29–33. 43. Li N, Ghia JE, Wang H, McClemens J, Cote F, Suehiro Y, et al. Serotonin activates dendritic cell function in the context of gut inflammation. Am J Pathol 2011;178(2):662–71. 44. Muller T, Durk T, Blumenthal B, Grimm M, Cicko S, Panther E, et al. 5-Hydroxytryptamine modulates migration,

cytokine and chemokine release and T-cell priming capacity of dendritic cells in vitro and in vivo. PLoS ONE 2009;4(7):e6453. 45. Yabanoglu S, Akkiki M, Seguelas MH, Mialet-Perez J, Parini A, Pizzinat N. Platelet derived serotonin drives the activation of rat cardiac fibroblasts by 5-HT2A receptors. J Mol Cell Cardiol 2009;46(4):518–25.