Inflammation has synergistic effect with nicotine in periodontitis by up‐regulating the expression of α7 nAChR via phosphorylated GSK‐3β

Abstract Periodontitis is the leading cause of adult tooth loss, and those who smoke are at an increased risk of developing periodontitis. α7 nicotinic acetylcholine receptor (α7 nAChR) is proposed to mediate the potential synergistic effect of nicotine and inflammation in smoking‐related periodontitis. However, this has not been experimentally demonstrated. We isolated and cultured human periodontal ligament stem cells (PDLSCs) from healthy and inflamed tissues. PDLSCs were treated with either inflammatory factors or nicotine. We measured expression of genes that are associated with osteogenic differentiation and osteoclast formation using RT‐qPCR and Western blot analyses. Besides, immunohistochemical staining, micro‐CT analysis and tartaric acid phosphatase staining were used to measure α7 nAChR expression and function. Inflammation up‐regulated α7 nAChR expression in both periodontal ligament tissues and PDLSCs. The up‐regulated α7 nAChR contributed to the synergistic effect of nicotine and inflammation, leading to a decreased capability of osteogenic differentiation and increased capability of osteoclast formation‐induction of PDLSCs. Moreover, the inflammation‐induced up‐regulation of α7 nAChR was partially dependent on the level of phosphorylated GSK‐3β. This study provides experimental evidence for the pathological development of smoking‐related periodontitis and sheds new light on developing inflammation and α7 nAChR‐targeted therapeutics to treat and prevent the disease.

(α7 nAChR) is proposed to mediate the potential synergistic effect of nicotine and inflammation in smoking-related periodontitis. However, this has not been experimentally demonstrated. We isolated and cultured human periodontal ligament stem cells (PDLSCs) from healthy and inflamed tissues. PDLSCs were treated with either inflammatory factors or nicotine. We measured expression of genes that are associated with osteogenic differentiation and osteoclast formation using RT-qPCR and Western blot analyses. Besides, immunohistochemical staining, micro-CT analysis and tartaric acid phosphatase staining were used to measure α7 nAChR expression and function. Inflammation up-regulated α7 nAChR expression in both periodontal ligament tissues and PDLSCs. The up-regulated α7 nAChR contributed to the synergistic effect of nicotine and inflammation, leading to a decreased capability of osteogenic differentiation and increased capability of osteoclast formation-induction of PDLSCs. Moreover, the inflammation-induced up-regulation of α7 nAChR was partially dependent on the level of phosphorylated GSK-3β. This study provides experimental evidence for the pathological development of smoking-related periodontitis and sheds new light on developing inflammation and α7 nAChR-targeted therapeutics to treat and prevent the disease.

| INTRODUC TI ON
Periodontitis is a disease caused by bacterial infection. 1 At present, it is the leading cause of adult tooth loss. 2,3 Periodontitis not only increases the risk of oral disease, but also increases the risk of systemic diseases. 4,5 Therefore, the World Health Organization has called for increased attention and proposed integrated strategies to control and prevent periodontal inflammation globally. 6 Smoking is a significant risk factor for periodontitis. [7][8][9] Nicotine is one of the most toxic substances in cigarettes. 10,11 It has been revealed that α7 nicotinic acetylcholine receptor (α7 nAChR), the major nicotine receptor, is functionally expressed both in healthy tissue-derived periodontal ligament stem cells (H-PDLSCs) and in inflammatory tissue-derived PDLSCs (I-PDLSCs) . 12 More importantly, the destructive effect of nicotine on periodontal tissues is achieved mainly through its interaction with α7 nAChR. [13][14][15] It has been widely accepted that smoking promotes the destructive effect of inflammation in periodontitis. 10 However, it is also well recognized that smoking itself could not cause periodontitis independent of inflammation. Since very few studies have focused on the potential synergistic effect of nicotine and inflammation, a systematic investigation on this issue could provide a comprehensive explanation regarding the pathological mechanisms of smoking-related periodontitis.
It has been confirmed that there might be a bidirectional regulation between α7 nAChR and certain inflammatory factors like interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α). 16,17 Moreover, the inflammatory response of PDLSCs could activate multiple signalling pathways, such as those involving glycogen synthase kinase-3β (GSK-3β). 18 Furthermore, in the central nervous system, increased expression of phosphorylated GSK-3β can up-regulate the expression and promote the function of α7 nAChR, thus suppressing the progression of degenerative diseases. 19 Our previous study indicated that TNF-α significantly increases phosphorylated GSK-3β levels in bone marrow-derived mesenchymal stem cells and PDLSCs. 20 However, whether and how GSK-3β signalling is involved in the pathogenesis of smoking-related periodontitis has not been fully elucidated.
In this study, we investigated the changes of α7 nAChR expression in periodontal tissues and PDLSCs under inflammatory conditions and further clarified if α7 nAChR and inflammation synergistically affect osteogenesis and osteoclastogenesis of PDLSCs.
Our study provides the experimental basis to further investigate the mechanisms of smoking-related periodontitis and sheds new light on developing inflammation and α7 nAChR-targeted therapeutics to treat and prevent the disease. Written consents were obtained from all participants prior to conducting the study.

| Isolation and culture of human PDLSCs
Periodontal tissues were gently scraped from the middle third of the roots and cut into small pieces of approximately 1 mm 3 . These pieces were digested using type I collagenase (Sigma-Aldrich, Spruce) for 15 minutes and then resuspended in alpha minimum essential medium (α-MEM; HyClone) supplemented with 10% (v/v) foetal bovine serum (HyClone). Periodontal ligament tissues were seeded in a six-well plate (Corning) and cultured at 37°C in a humidified atmosphere with 95% air and 5% CO 2 . The medium was changed every 3 days. The limiting dilution technique was applied to obtain single colony-derived cell strains for further in vitro cultivation, as previously reported. 21

| Colony formation assay and toluidine blue staining
To assess the colony formation efficiency, a total of 1 × 10 3 cells at the third passage were seeded into 100-mm dishes (Corning). After 14 days of culture, cells were fixed using 4% paraformaldehyde (Henglee) and K E Y W O R D S bone metabolism, GSK-3β, nicotine, periodontal ligament stem cells, periodontitis, α7 nicotinic acetylcholine receptor stained using 1% toluidine blue (Hengyuan) for 20 minutes at room temperature. Aggregates of 50 or more cells were considered colonies.
The colony formation efficiency was calculated by dividing the total number of colonies by the total number of seeded cells.

| Flow cytometric analysis for surface markers
PDLSCs were labelled with antibodies against mesenchymal cell surface markers and analysed using flow cytometry, as described previously. 15 Briefly, to immunophenotype the PDLSCs, 1 × 10 5 cells at  After culturing for 4 weeks, the cells were fixed using 4% paraformaldehyde and stained with a fresh solution of Alizarin Red (Klamar) or Oil Red O (Sigma-Aldrich) for 20 minute at room temperature. The mineral nodules and lipid droplets were observed using a phase-contrast microscope (Olympus).
Quantitative PCR was performed in a volume of 20 μL using SYBR Premix Ex TaqTM II PCR kit (Takara). PCR conditions were selected according to the suggested protocol for the CFX Connect Real-Time PCR Detection System (Bio-Rad). β-actin was used as an internal reference gene, and the 2 −ΔΔCt method was used to calculate expression of target genes between the experimental group and control group.

| Western blot analysis
Western blot was performed according to a previously described protocol. 22 The primary antibodies used in this study included rabbit

| Immunohistochemical staining
Immunohistochemical staining was performed as previously described. 21 Paraffin serial sections (4 μm) were obtained, and immunohistochemical evaluations were performed using the fol-

| Lentiviral infection for gene knock-down
hPDLSCs were seeded in a 12-well plate (Corning). When the cells reached 50%-70% confluence, they were infected with lentiviral particles expressing α7 nAChR-specific shRNA or GSK-3β-specific shRNA (Santa Cruz) according to the manufacturer's protocol. Briefly, polybrene (Santa Cruz) was added to the α-MEM culture medium at a final concentration of 5 μg/mL. At 24 hours after incubation with the lentiviral particles, the cells were cultured with normal α-MEM and were routinely passaged. Puromycin dihydrochloride (Santa Cruz) at a concentration of 5 μg/mL was used to select stable clones with targeted knock-down of α7 nAChR or GSK-3β. Control lentiviral particles (Santa Cruz) were used to establish the negative control clones. All resistant clones were subcultured for further use. RT-qPCR and Western blot assays were conducted to analyse gene knock-down efficiency.

| Animal experiments
Nude mice (6-week-old, males) were purchased from Vital River Laboratory Animal Technology Company and maintained in the Animal Center of Air Force Medical University. hPDLSCs with indicated treatments were implanted subcutaneously into the nude mice according to previously described protocols. 21 Briefly, treated hPDLSCs were seeded in six-well plates at a density of 2 × 10 5

| Micro-CT scanning
The cell-HAP implants were analysed using a micro-CT device (Siemens AG). Scanning was performed at a 70 kV, 114 μA condition with an isotropic resolution of 10.5 μm. Sectional images of 2240 × 2240 pixels were obtained. A 1-mm section perpendicular to the long axis of the cylindrical scaffold material was taken in the middle, and three-dimensional images were reconstructed. Then, the ratios of the bone volume to the total volume and trabecular spacing were quantified. Images from one representative experiment are shown, and the quantitative analyses are summarized from three independent experiments.

| Tartaric acid phosphatase (TRAP) staining
The mouse monocyte/macrophage cell line RAW264.7 was purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco's modified eagle's medium (HyClone) supplemented with 10% FBS. Differently treated hPDLSCs were cocultured with RAW264.7 cells, and TRAP staining was performed as previously reported. 22 Briefly, hPDLSCs were seeded into 24-well plates (Corning; 1 × 10 5 cells/mL/well). After 12 hours, RAW264.7 cells (1 × 10 6 cells/mL/well) were directly added into the α-MEM culture medium containing 30 ng/mL human macrophage colony stimulatory factor (Wobai). After 14 days, the cells were subjected to TRAP staining using an acid phosphatase leucocyte kit (Sigma-Aldrich). Ten different visual fields in each group were randomly selected to calculate the number of TRAP-positive cells.

| Statistics
All experiments were performed in triplicate with hPDLSCs from at least three different patients. All results are presented as the mean ± standard deviation. A two-tailed unpaired Students' t test analysis was used to analyse data within two groups. A one-way analysis of variance followed by Tukey's post-test was used to analyse data within more than two groups. All data were analysed using SPSS software version 19.0 (IBM). A P-value less than .05 was considered statistically significant.

| Isolation and identification of H-PDLSCs and I-PDLSCs
After culturing the primary periodontal ligament tissues for 3-5 days, fibroblast-like cells were found on the edge of the seeded tissues ( Figure 1A). Single-cell clones ( Figure 1B), labelled as the first passage of PDLSCs, were selected and expanded by further culturing.
PDLSCs in the third to fifth passage ( Figure 1C) were used for further experiments.

| Effects of inflammation and nicotine on hPDLSC osteogenic differentiation and inducing osteoclast formation
In response to induction of osteogenic differentiation, 10 −9 mol/L nicotine did not statistically impact mRNA and protein expression of ALP, RUNX2, BSP and OCN (P > .05, Figure 2A and OCN in I-PDLSCs (P < .05, Figure 2B). Although nicotine did not synergistically decrease mRNA expression of BSP in I-PDLSCs (P > .05, Figure 2B), it significantly down-regulated BSP protein expression and other osteogenic differentiation-associated genes (P < .05, Figure 2E

| Periodontal ligament tissues and hPDLSCs derived from the inflammatory microenvironment have higher α7 nAChR expression
As shown in Figure 3A

| α7 nAChR knock-down or antagonizing alone does not affect the osteogenic differentiation and osteoclast formation-induction ability of hPDLSCs in response to inflammation
As shown in Figure S1A,B, qPCR indicated that the silencing efficiency of α7 nAChR was 56.6% and 73.0% in H-PDLSCs and I-PDLSCs while the α7 nAChR protein expression was reduced by 56.8% and 76.5% in H-PDLSCs and I-PDLSCs, respectively ( Figure S1C,D).
In response to osteogenic differentiation induction, α7 nAChR knock-down did not significantly alter ALP, RUNX2, BSP and OCN expression (P > .05, Figure 4A,C,D). Furthermore, treatment with α-BTX, a specific α7 nAChR antagonist, also did not significantly impact mRNA and protein expression of the osteogenic differentiation markers ( Figure 4A,C,D). Similarly, α7 nAChR inhibition in

| Silencing of α7 nAChR abrogates nicotineinduced impairment on osteogenic differentiation and enhancement on osteoclast formation-induction of hPDLSCs in the inflammatory microenvironment
The synergistic effect of nicotine and inflammation was demonstrated by significantly decreased expression of osteogenic differentiation indicator genes in H-PDLSCs, and this effect was reversed after silencing α7 nAChR expression (P < .05, Figure 5A,C,D).
Similarly, studies in I-PDLSCs showed that the synergistic effect of nicotine and inflammation could be significantly abrogated by silencing α7 nAChR expression (P < .05, Figure 5B,E,F).  Figure 4A-F, except cells were additionally treated with 10 −9 mol L −1 nicotine (nic). Cells were harvested to measure mRNA and protein levels of osteogenic differentiation-associated genes by RT-qPCR (A, B) and Western blot (C-F). G-J, Ceramic hydroxyapatite coated with differently treated H-PDLSCs (G) and I-PDLSCs (H) was subcutaneously implanted into the dorsal region of nude mice. At 8 wk after implantation, the ceramic hydroxyapatite was subjected to micro-CT analysis to examine structural changes. Quantitative analysis was conducted to determine bone volume/total volume and trabecular spacing (I, J). K-P, Changes of protein expression of ALP (K, L) and RUNX2 (M, N) on ceramic hydroxyapatite coated with differently treated H-PDLSCs (K, M) and I-PDLSCs (L, N) were determined by immunohistochemical staining. Bar graphs indicate semiquantitative analysis of staining for ALP and RUNX2 in H-PDLSCs (O) and I-PDLSCs (P). N = 3 for each group; *P < .05, **P < .01, ***P < .001; α7i, silencing the expression of α7 nAChR; if: inflammatory factors; nic: nicotine; osteo, osteogenic differentiation induction expression in H-PDLSCs (P < .05, Figure 6A). The synergistic effect of nicotine was attenuated by silencing α7 nAChR expression ( Figure 6A). Western blot assays showed similar trends for protein expression of these markers in H-PDLSCs (P < .05, Figure 6C,D).
In addition, knock-down of α7 nAChR in I-PDLSCs also abrogated the effect of nicotine on regulating mRNA ( Figure 6B) and protein ( Figure 6E,F) expression of RANKL and OPG.
As shown in Figure 6G,H, inflammation and nicotine synergistically promoted formation of TRAP + multinuclear osteoclast precursors in both H-PDLSCs and I-PDLSCs. Importantly, this effect was partially reversed by down-regulating α7 nAChR expression, which was further supported by comparing the numbers of TRAP + cells per visual field (P < .05, Figure 6I,J).

| Inflammation-induced up-regulated α7 nAChR expression in hPDLSCs is partially dependent on phosphorylated GSK-3β
As shown in Figure 7A,B, both H-PDL and I-PDL had comparable expression of GSK-3β (P > .05, Figure 7C). However, the expression level of phosphorylated GSK-3β (p-GSK-3β) in I-PDL was significantly higher compared to H-PDL (P < .05, Figure 7D

| D ISCUSS I ON
In this study, we isolated PDLSCs from healthy and inflamed peri-  Clinical observations have indicated that periodontitis patients who smoke consistently have more severe hard tissue resorption. 10 Under inflammatory conditions, the toxic effect of smoking was not due to a simple accumulation. Smoking, which alone does not lead to periodontal tissue resorption, can exacerbate periodontitis symptoms in response to local inflammation. 30 This clinical phenomenon suggests that toxic substances in cigarettes synergistically work to increase periodontitis. Our study demonstrated that the in vitro synergistic effect of nicotine and inflammation on osteogenic differentiation and osteoclast formation of PDLSCs was more potent than either stimulation with nicotine or inflammatory factors alone (Figure 2). This finding was in accordance with previous clinical observations.
Our previous work indicated that low concentrations of nicotine (10 −9 -10 −12 mol/L) had significantly decreased toxicity to human periodontal ligament cells. 14 Based on this, we chose to use nicotine at a concentration of 10 −9 mol/L in this study. Our results confirmed that nicotine at 10 −9 mol/L did not affect the osteoblastogenesis/ osteoclastogenesis balance of PDLSCs, but significantly exacerbated the destructive effect of inflammatory factors (Figure 2). To the best of our knowledge, this is the first report to demonstrate direct evidence of the in vitro synergistic effect of nicotine and inflammation in human PDLSCs.
α7 nAChR is a classical receptor that plays a role in the physiological regulation of neurons. 31 After identifying its expression in oral tissues, 32 α7 nAChR was considered a critical molecular regulatory target of oral diseases. 14 This study further suggests that α7 nAChR expression levels varied in periodontal tissues derived from different microenvironments ( Figure 3). These results are consistent with an experimental periodontitis animal model, 12 which also suggests an important role of α7 nAChR in periodontal inflammatory diseases.
In addition, the finding that inflammation up-regulates α7 nAChR expression is supported by results obtained in macrophages. 33 However, contradictory conclusions were reached from other investigations that focused on α7 nAChR expression in the nervous system. Inflammation in the central nervous system down-regulated α7 nAChR expression, which led to amyloid deposition and further mimicked the phenotype of central nervous degenerative diseases. 34 However, no clear mechanisms were clarified focusing on the different phenomena. 35 As a result, the mechanism by which inflammation up-regulates α7 nAChR expression in periodontal tissues and stem cells requires further investigation.
After confirming that nicotine and inflammation synergistically affected the balance of osteoblastogenesis and osteoclastogenesis in PDLSCs, we knocked down α7 nAChR expression in PDLSCs to further investigate the effects of loss of function. We showed that α7 nAChR knock-down partially reversed the synergistic effect of nicotine and inflammation (Figures 6 and 7). This finding further expands our understanding about the pathological mechanisms of smoking-related periodontitis. Considering the findings in the previous research, 12 α7 nAChR might play a more critical role in regulating the local microenvironment in periodontal tissues. That is, inflammatory factors up-regulate α7 nAChR expression in periodontal tissues.
Nicotine binding to α7 nAChR increased release of inflammatory factors. Thus, a positive feedback loop initiated by local inflammation can result in the imbalance of osteoblastogenesis and osteoclastogenesis of PDLSCs, exacerbating periodontitis in patients who smoke.
GSK-3β actively participates in inflammatory responses. It has already been demonstrated that inflammatory responses induced by lipopolysaccharides can suppress GSK-3β activity to regulate expression of inflammatory factors. 36 Functionally, suppressing GSK-3β can also regulate the nuclear factor-κB signalling pathway to affect inflammatory responses of monocytes and macrophages. 37 Our study also confirmed that in response to inflammation, GSK-3β activation in periodontal tissues was suppressed, as demonstrated by the up-regulated expression of phosphorylated GSK-3β ( Figure 7A-I).
In an inflammatory environment, phosphorylated GSK-3β regulates the expression of TNF-α through the wnt pathway to suppress osteogenic differentiation of PDLSCs. 38 Here, we further demonstrated that suppressing GSK-3β activity regulated the expression of α7 nAChR ( Figure 7J-O). Knock-down of GSK-3β partially reversed the effect of inflammatory factors on up-regulating α7 nAChR expression. This finding is in accordance with the results in degenerative diseases in the central nervous system. 19 Up-regulated expression of amyloid β can suppress expression of phosphorylated GSK-3β, leading to decreased expression and diminished function of α7 nAChR. 39,40 Thus, we speculated that in smoking-related periodontitis, decreasing phosphorylation of GSK-3β should be an effective therapy.
In summary, inflammation up-regulates α7 nAChR expression in human periodontal ligament tissues and PDLSCs. Up-regulated α7 nAChR expression significantly contributes to the synergistic effect of nicotine and inflammation, which exacerbates tissue destruction in smoking-related periodontitis patients. Increased α7 nAChR expression in response to inflammation is partially dependent on the phosphorylation of GSK-3β in PDLSCs. Our study provides an experimental basis for clarifying the synergistic effect of nicotine and inflammation in the pathological development of smoking-related periodontitis. In future work, detailed molecular mechanisms and research in smoking-related periodontitis animal models might be explored. Hospital. The funders of this work had no role in study design, data collection, data analysis, data interpretation, writing of the report, or decision to submit the article for publication.