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The impact of smoking on levels of chronic periodontitis-associated biomarkers
The impact of smoking on levels of chronic periodontitis-associated biomarkers Chun-Yan He, Xiu-Qiu Gao, Li-Peng Jiang PII: DOI: Reference:
S0014-4800(16)30160-5 doi: 10.1016/j.yexmp.2016.07.004 YEXMP 3947
To appear in:
Experimental and Molecular Pathology
Received date: Revised date: Accepted date:
2 March 2016 5 July 2016 19 July 2016
Please cite this article as: He, Chun-Yan, Gao, Xiu-Qiu, Jiang, Li-Peng, The impact of smoking on levels of chronic periodontitis-associated biomarkers, Experimental and Molecular Pathology (2016), doi: 10.1016/j.yexmp.2016.07.004
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ACCEPTED MANUSCRIPT The impact of smoking on levels of chronic periodontitis-associated biomarkers Running title: Smoking and chronic periodontitis
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Department of Prosthodontics, the Second Affiliated Hospital of Liaoning Medical University,
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Jinzhou 121000, P.R. China;
Department of Radiation Oncology, the First Affiliated Hospital of Liaoning Medical University,
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Chun-Yan He 1, Xiu-Qiu Gao 1, Li-Peng Jiang 2, *
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Jinzhou 121000, P.R. China.
* Correspondence to: Dr. Li-Peng Jiang, Department of Radiation Oncology, the First Affiliated
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Hospital of Liaoning Medical University, No. 2, the Five Section of Renmin Street, Guta District,
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Jinzhou 121000, P.R. China. E-mail: [email protected]
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Tel. /fax: +86-416-2655167
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ACCEPTED MANUSCRIPT Abstract Objective: To investigate the effect of smoking on the expression levels of matrix metalloproteinase (MMP)-1, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and the concentrations of
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TNF-α and IL-10 in patients with chronic periodontitis (ChP).
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Methods: This is an ex-vivo study. Our study consisted of 78 cases, all of which were diagnosed
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with ChP and were selected according to the inclusion and exclusion criteria. Among these 78 cases, 38 patients were classified into the smoking group (S-ChP group), and 40 patients in the
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non-smoking group (NS-ChP group). The clinical periodontal parameters of all patients were recorded, including the plaque index (PLI), probing depth (PD), loss of attachment (LA) and sulcus
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bleeding index (SBI). Serum was collected from forearm blood to establish a Porphyromonas gingivalis (Pg) internalizing KB cell model. Enzyme-linked immunosorbent assay (ELISA) was
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used to determine the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cell lysis solution
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as well as IL-10 and TNF- in the gingival crevicular fluid (GCF).
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Results: Fewer Pg internalizing KB cell colonies were observed in the NS-ChP group than in the S-ChP group (P < 0.01). When 400 μL serum was added, there were remarkable differences in the concentrations of MMP-1 and TIMP-1 secreted from the KB cells between the S-ChP and NS-ChP
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groups (MMP-1: t = -21.71, P < 0.01; TIMP-1: t = 64.35, P < 0.001). Additionally, when 800 μL serum was added, there were significant differences in the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cells between the S-ChP and NS-ChP groups (MMP-1: t = -81.89, P < 0.001; MMP-9: t = -15.67, P < 0.001; TIMP-1: t = 109.4, P < 0.001). The TNF-α levels were higher, but the IL-10 levels were lower in the GCF from the ChP patients in the S-ChP group than those in the NS-ChP group (both P < 0.001). Conclusion: The serum of S-ChP patients can enhance the concentrations of MMP-1 and MMP-9, but reduce TIMP-1 secreted from Pg internalizing KB cells. However, the concentration of TNF-α was increased and IL-10 was decreased. Abnormal concentrations of ChP-associated biomarkers may be conducive to the development and progression of ChP. 2
ACCEPTED MANUSCRIPT Keywords: Chronic periodontitis; IL-10; TNF-α; MMP-1; MMP-9; TIMP-1; Porphyromonas
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gingivalis; KB cells
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ACCEPTED MANUSCRIPT Introduction Periodontitis, a chronic inflammatory disease, is characterized by loss of periodontal ligament and
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alveolar bone, and can lead to tooth loss and affect individual's oral health-related quality of life (De
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Pablo, et al., 2009; Durham, et al., 2013). It is estimated that severe periodontitis affects 5~20% of
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any population, and mild/moderate periodontitis is a problem of majority of adults (Kassebaum, et al., 2014; Dye, 2012; Petersen and Ogawa, 2012). Periodontitis is more common among men than
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women and is more prevalent in developing areas than in developed areas, and the occurrence of this disease shows an upward with aging (Ababneh et al., 2012; Darveau, 2010). Chronic
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periodontitis (ChP) can be developed by modifiable risk factors, including cigarette smoking, alcohol consumption, poor diet, and psychological stress and depression (Reynolds, 2014; Filoche,
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et al., 2010). It has been reported that smoking is strongly involved in the initiation and progression of periodontitis disease and that smokers were found altered amount of gingival crevicular fluid
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(GCF) and its components (Mokeem, et al., 2014; Kubota et al., 2011). Smoking is also reported to have a potential negative effect on the immune defense system of the periodontal host, which
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inhibits neutrophil and macrophage function and affects defense against bacteria (Gomes et al., 2015; Fan et al., 2015).
Matrix metalloproteinases (MMPs) are zinc- and calcium-dependent proteolytic enzymes that are responsible for the degradation of all extracellular matrix proteins and basement membrane components (Marcaccini et al., 2010). MMPs can be produced by inflammatory cells caused by bacterial infection in response to cytokines secreted from these cells in periodontal patients; thus, their presence, quantities, and activities are recognized to be very important for the characterization of various states of periodontitis and the determination of active destruction (Kim et al., 2013). Endogenous tissue inhibitors, especially tissue inhibitors of metalloproteinases (TIMPs), are 4
ACCEPTED MANUSCRIPT significant factors in the control of MMPs (Mouzakiti et al., 2012). An imbalance between MMPs and TIMPs is found in pathological conditions, including periodontitis and correlates with tissue
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destruction (Verstappen and Von den Hoff, 2006; Kubota et al., 2008). Tumor necrosis factor alpha
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(TNF-α), as a pro-inflammatory cytokine, can lead to propagation of inflammation and contribute to
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release of a high level of inflammatory mediators, which result in destruction of tooth-supporting periodontal structures (Passoja, et al., 2010; Yousefimanesh, et al., 2013). As a vital
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anti-inflammatory cytokine that impedes inflammatory responses, IL-10 is also implicated in ChP (Zhang et al., 2014). Porphyromonas gingivalis (Pg) is a predominant periodontal pathogen that
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expresses multiple potential virulence factors involved in the pathogenesis of periodontitis (Amano,
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2007). The virulence factors enable Pg to invade the periodontal tissue and subsequently spread into
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the systemic circulation (Blasco-Baque et al., 2016). Pg can also invade host cells, including epithelial cells (Amano et al., 2014). Gingival epithelial cells are spontaneously exposed to bacterial
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attacks and function in preventing bacteria invasion into deeper tissues (Amano, 2007). In the present study, we detected the levels of MMP-1, MMP-9, TIMP-1 in the supernatants of Pg
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internalizing KB cells and the levels of TNF-α and IL-10 in the GCF, in order to explore the effect of smoking on ChP.
Materials and Methods Subjects A total of 78 patients diagnosed with ChP were recruited into our study from the First Affiliated Hospital of Liaoning Medical University between September 2012 and March 2015. The ChP was diagnosed in accordance with the recommended standards from the Center for Disease Control and Prevention, the American Academy of Periodontology, on the condition of (1) presence of 2 interproximal sites with 3 mm clinical attachment loss, not on the same tooth, and (2) 5
ACCEPTED MANUSCRIPT presence of 2 interproximal sites with 4 mm probing depth (PD) occurring at two or more different teeth, or presence of 5 mm PD of a site (Armitage, 1999). According to the standard of WHO definition of the smoking population, patients who smoked more than 10 cigarettes daily for
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longer than 2 years were considered smoking patients with ChP (S-ChP group). The patients who quit smoking for more than 10 years or had no smoking habits were considered non-smoking
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patients with ChP (NS-ChP group). Our study consisted of 38 cases of S-ChP and 40 cases of NS-ChP. The exclusion criteria for patient enrollment and experimental teeth were as follows: (1)
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patients with systemic disease; (2) patients taking oral antibiotics, analgesics or hormonal drugs in the past three months, and those who had received periodontal treatment for nearly half of the year;
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(3) patients with less than 20 remaining teeth, and experimental teeth with periapical disease (based on clinical symptoms, X-ray examination and diagnostic results) and occlusal trauma (based on
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functional mobility, occlusion widened periodontal ligament and other local incentives diagnosis).
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In addition, periodontal clinical parameters were obtained from each patient with ChP, including the
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plaque index (PLI), PD, loss of attachment (LA) and sulcus bleeding index (SBI). Two diagonal quadrants were randomly selected where a tooth with the highest PD was used as the experimental molar tooth and the area with the deepest PD was the GCF sample site. This study was approved by
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the Ethics Committee of the First Affiliated Hospital of Liaoning Medical University, and the subjects’ written consent was obtained according to the current Declaration of Helsinki. Clinical parameter measurement and sample collection The periodontal clinical indices of the ChP patients were recorded. The PLI was determined and divided into four grades on the basis of the Silness & Loe PLI methods. The scores were as follows: grade 0, no plaque in the gingival margin area; grade 1, thin plaque in the gingival margin of the tooth surface that was not easy to see with the naked eye but could be scraped using the tip side of a probe; grade 2, visible medium plaque in the gingival margin or interproximal surface; grade 3, a large amount of soft scale in the gingival margin and the adjacent surface or gingival sulcus. The PD was the distance from the gingival margin to the bottom of periodontal pocket or the 6
ACCEPTED MANUSCRIPT bottom of gingival sulcus. The LA was the difference between the PD and the distance from the enamelo-cemental junction to the gingival margin; if gingival recession was present, the PD and the gingival recession distance were summed. The SBI was judged based on the Mazza (1981) method.
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The SBI was divided into six grades according to the gingival bleeding degree by Florida
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periodontal probing. The grades were as follows: grade 0, patients with healthy gingival, no
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inflammation or bleeding symptoms; grade 1, patients with gingival color changed with inflammation but no bleeding from probing; grade 2, punctate hemorrhage after probing; grade 3,
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probe hemorrhage diffusion along the gingival margin; grade 4, full bleeding and gingival fluid overflow; grade 5, spontaneous bleeding.
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Two diagonal quadrants were randomly selected in the mouth of each study subject. The molars or premolars were selected as experimental teeth on the condition that: no filling body or
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caries were present at the tooth neck; PD > 3 mm; LA more than or equal to 1 mm; and varying
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degrees of destruction or resorption of alveolar bone evident by X-ray. The GCF samples were
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taken from the deepest site of the PD. Filter paper strips (2 mm 10 mm) were placed at the bottom of the periodontal pocket and kept in place for 30 s to adsorb and collect the GCF. Four sites were selected, including the buccal side, far from the buccal side, the lingual side and far from the lingual
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side. The four filter paper strips were placed into the same EP tube, followed by sealing, labelling, weighing and recording, and then reserved at 80℃ in a low-temperature refrigerator for further use. The reserved GCF sample was taken out from the refrigerator. After back to room temperature, the sample was shocked, eluted for 1 h and centrifuged (13000 r/min) for 10 min at 4℃. The supernatant was collected in clean EP tubes (each 50 μl). All the above steps were completed by a highly-qualified and professionally trained physician from our hospital. Bacterial culture Venous blood (5 mL) was collected from the forearms of all of the study subjects andcentrifuged to obtain serum samples, which were then reserved at -80℃. After PgATCC33277 recovery at room temperature, it was inoculated into brain heart infusion agar (BHIA) medium 7
ACCEPTED MANUSCRIPT containing 1% hemoglobin chloride, 5% sterile defibrinated sheep blood, and 0.1% vitamin K1 at 37℃ in an anaerobic environment for 5~7 days. Gram staining and biochemical identification were performed to confirm the purity of PgATCC33277, which was then cultured at 4°C for 24 h. After
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10 min of centrifugation at 5000 r/min, the bacteria were collected. The bacteria were washed with
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PBS and suspended in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Life Technologies,
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Darmstadt, Germany) without an antibiotic. Then, a UV spectrophotometer was used to measure the absorbance at 600 nm, and the bacterial suspension was adjusted to 1 × 108 CFU/mL. Fifty samples
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were obtained randomly after the enrichment of the bacterial liquid extraction, and Gram staining and polymerase chain reaction (PCR) were used to identify Pg using the upstream primer
5'-ACGTCATCCACACCTTCCTC-3'.
the
downstream
primer
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Cell culture
and
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5'-TGTAGATGACTGATGGTGAAAACC-3'
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The conventional KB cell line ATCCCCL17 was thawed from cryopreservation, inoculated in
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low-glucose DMEM and incubated in a humidified atmosphere of 5% CO2 at 37℃. Under the microscope, the cells presented a "paving stone" shape. When the cells were 80% confluent, they were digested with trypsin containing 2.5 g/L ethylene diamine tetra-acetic acid (EDTA) and
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inoculated in 6-well plates (each well 2.5 mL), with the cell density adjusted to 2 × 108/L using low-glucose DMEM medium containing 10% fetal bovine serum (FBS). Bacterial colony counting When the adherent KB cells were 80%~90% confluent in the 6-well plates, the number of cells was 2.5 × 106 cells/well. These KB cells were then washed three times with PBS and placed into low-glucose DMEM along with 2.5 × 108 CFU/mL Pg to ensure a multiplicity of infection of 100:1. To avoid interference from other factors, we used wells without Pg as a negative control group. Moreover, in order to imitate the conditions in the serum of S-ChP or NS-ChP patients and to investigate the number of KB cells that internalized Pg with different volumes of serum, 200, 400, or 800 μL serum from the S-ChP and NS-ChP patients were added to Pg prior to KB cell infection. 8
ACCEPTED MANUSCRIPT A group without serum added was used as the negative control group. The samples were incubated for 2 h in triplicate. Subsequently, 3 PBS washes were performed to remove Pg that was not adhered to the KB cells. Then, the KB cells were incubated in DMEM containing antibiotics for 1 h,
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followed by 3 additional PBS washes. The supernatant (500 μL) was absorbed after 12 h and were
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then stored at -80℃. KB cells without remaining DMEM were first washed 4 times, and then 1 mL
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sterile distilled water was added to each well for 90 min of cell disruption. Following dilution (1:1), the suspension (100 μL) was seeded into BHI medium supplemented with 1% freshly prepared
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sodium chloride containing hemoglobin, 5% sterile defibrinated sheep’s blood, and 0.1% vitamin K1 for 5-7 days, and then the bacterial colonies were counted. The KB cells for our study were
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provided by the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and the Pg was purchased from Takara
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Biotechnology Ltd (Dalian, China).
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Enzyme-linked immunosorbent assay (ELISA)
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ELISA was used to detect the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cell supernatant as well as TNF-α and IL-10 in the GCF. The absorbance (A) [the optical density (OD)] value was measured by a microplate reader, with detection wavelength of 450 nm and correction
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wavelength of 570 nm. With the absorbance (A) value as the vertical axis and the standard concentration as the abscissa, a standard curve was obtained. Consequently, the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cell supernatant and the TNF-α and IL-10 concentration in the GCF were calculated. Data analysis Data analysis was performed using the statistical package for the social sciences (SPSS) version 19.0 (SPSS Inc.; Chicago, IL, USA). Categorical data were verified by χ2 and measurement data are presented as the means ± standard deviation (SD), in which two groups of samples were compared with t tests. The data that did not conform to normality or homogeneity of variance were verified using a non-parametric rank-sum test. Multi-group comparisons were performed with 9
ACCEPTED MANUSCRIPT one-way analysis of variance (ANOVA) based on α = 0.05. All tests were two-sided, with P < 0.05 indicating a significant difference.
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Results
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General information
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The 38 S-ChP cases included 26 males and 12 females with a mean age of 46.5 ± 8.3 years old, and the 40 NS-ChP cases included 29 males and 11 females with a mean age of 46.7 ± 9.1 years old.
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There was no significant difference in gender, age or PD values between the S-ChP and NS-ChP groups (all P > 0.05). Smoking history, PLI and LA values in the S-ChP patients were significantly
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higher and SBI was lower than those in the NS-ChP patients (all P < 0.01) (Table 1). The effects of different volumes of serum on the number of Pg internalizing KB cell colonies
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The serum of patients with ChP was collected from the S-ChP and NS-ChP groups to observe
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the effects of different volumes of serum from these two groups on Pg internalizing KB cells
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colonies. The results indicated that significant fewer Pg internalizing KB cell colonies were present in different volumes of serum from the NS-ChP group than in serum from the S-ChP group (all P < 0.01). With the addition of 200 μL, 400 μL or 800 μL serum, there was no significant difference in
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the number of Pg internalizing KB cell colonies in the NS-ChP group (F = 1.773, P = 0.174), while there was an increase in Pg internalizing KB cell colonies in the S-ChP group (F = 108.1, P < 0.001) (Figure 1, Table 2)
The effects of different volumes of serum on the concentrations of MMP-1, MMP-9 and TIMP-1 in KB cells With the addition of 200 μL, 400 μL or 800 μL serum, the KB cells in the S-ChP group had increased concentrations of MMP-1 and MMP-9, but decreased concentrations of TIMP-1 (Table 3). When 200 μL serum was added, the difference between the S-ChP and NS-ChP groups in the concentrations of MMP-1, MMP-9 and TIMP-1 secreted from the KB cells was not statistically significant (all P > 0.05). When 400 μL serum was added, there were remarkable differences 10
ACCEPTED MANUSCRIPT between the S-ChP and NS-ChP groups in the concentrations of MMP-1 and TIMP-1 secreted from the KB cells ChP and NS-ChP groups (MMP-1: t = -21.71, P < 0.01; TIMP-1: t = 64.35, P < 0.001). Additionally, when 800 μL serum was added, there were significant differences between the S-ChP
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and NS-ChP groups in the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cells (MMP-1:
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t = -81.89, P < 0.001; MMP-9: t = -15.67, P < 0.001; TIMP-1: t = 109.4, P < 0.001). Comparing the
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mass concentrations of the factors secreted by KB cells with or without the addition of Pg in the S-ChP and NS-ChP groups, we found that in both of the two groups, the mass concentration of the
without the addition of Pg (both P < 0.05).
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factors secreted from KB cells following the addition of Pg was significantly higher than that
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Concentrations of TNF-α and IL-10 in the GCF
The TNF-α levels in the GCF samples from the patients with ChP in the S-ChP group were
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6.61 ± 0.74 ng/mL, which was higher than those in the NS-ChP group, 5.88 ± 0.56 ng/mL (t = -4.9,
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P <0.001). The IL-10 levels in the GCF in the S-ChP group were 4.28 ± 0.46 ng/mL, which was
Discussion
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lower than those in the NS-ChP group, 5.26 ± 0.95 ng/mL (t = 5.84, P < 0.001) (Figure 2).
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The most important finding of our study was the effect of smoking on ChP. We found that Pg internalized KB cell colonies in the NS-ChP group were less than that in the S-ChP group. It has been recognized that smoking plays a significant role in the protective elements of the immune system (Palmer et al., 2005). A study reported that polymorphonuclear lymphocytes in smokers showed decreased chemotaxis, which prevented the elimination of periodontal pathogens, causing an increase in the extent and severity of periodontal destruction (Matthews et al., 2011). Periodontal bacteria (Pg included) were reported to be critically dependent on an inflammatory environment, where they obtained nutrients from tissue-breakdown products (Hajishengallis, 2014). Smoking was reported to provide a supporting environment for the growth of periodontal microbes (Gupta et al., 2016). Tobacco smoking was demonstrated to have critical effects on the host, leading to an 11
ACCEPTED MANUSCRIPT increased response to bacterial plaque accumulation (Llambes et al., 2015). Therefore, we assumed that smoking is supportive for Pg by creating an environment that promotes the bacterial growth. In the inflammation of the periodontal tissue, the level of MMPs increased in all the bodily fluids,
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including the GCF, saliva, serum and plasma; the level of TIMPs decreased and MMPs increased,
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jointly resulting in uncontrolled tissue destruction (Gupta et al., 2016). The balance between
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activated MMPs and TIMPs was shown to regulate extracellular matrix remodeling, and tissue degradation occurred due to the disruption of this balance (Surlin et al., 2014). With an imbalance
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of MMP/TIMP activity, changes in the TIMP-1 level could be critical in regulating the destruction of periodontal tissues by affecting the MMP levels (Emingil et al., 2006).
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In our study, we used Pg internalizing KB cell colonies to observe the differences in S-ChP and NS-ChP cells incubated with different volumes of serum and found that there were greater
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numbers of Pg internalizing KB cell colonies in the S-ChP group than in the NS-ChP group, and the
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Pg internalizing KB cell colonies in the S-ChP group increased as the volume of serum from S-ChP
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patients increased. Similar to this result, it was reported by Cogo et al. that exposure to cotinine (an important constituent of tobacco) at a high concentration might enhance Pg invasion into epithelial cells (Cogo et al., 2009). Furthermore, according to Zeller et al., smoking can reduce IgG (a type of
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antibody that eliminates plaque bacteria) production in response to Pg infection, thus promoting Pg survival and potentially increasing vulnerability to periodontitis (Zeller et al., 2014). The studies above showed that smoking might increase the likelihood of infection with Pg, and consequently, smokers might suffer from increased susceptibility to periodontitis. Notably, as the serum volume increased, the concentrations of MMP-1 and MMP-9 secreted by KB cells also increased while TIMP-1 decreased in both the S-ChP and NS-ChP groups, but with greater concentration changes in the S-ChP group. In agreement with one of the results, MMP-1, which plays a role in extracellular matrix destruction and collagen degradation, was found to exhibit high concentrations during periodontal disease (Beklen et al., 2007). Moreover, it was found that MMP-1 mRNA levels in the skin of smokers were significantly increased compared to those of 12
ACCEPTED MANUSCRIPT non-smokers (Lahmann et al., 2001). MMP-9 plays a role in the functions of type IV, V, and XI collagens; denatured collagens; elastins; and proteoglycans, and is mainly released from osteoclasts and polymorphonuclear neutrophils in periodontal tissues (Ertugrul et al., 2013). In ChP, there is an
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imbalance between MMPs and their inhibitors (TIMPs), with elevated MMPs and low levels of
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TIMPs (Rai et al., 2008). Ozcaka et al. discovered significantly elevated levels of MMP-9 as well as
2011), which further supports the results of this study.
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reduced levels of TIMP-1 in smokers with periodontitis compared with non-smokers (Ozcaka et al.,
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Moreover, our study demonstrated that the S-ChP group had higher levels of TNF-α and lower levels of IL-10 in the GCF than those in the NS-ChP group. Recently, Zhu et al. indicated that
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periodontitis patients seemed to have an activated TNF-α response, as lipopolysaccharide stimulated macrophages derived from the periodontal tissues produce greater amounts of TNF-α
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compared with individuals without periodontitis, and circulating TNF-α levels were also associated
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with more severe periodontitis in a large geriatric population (Jiang et al., 2013). Additionally,
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Gumus et al. showed that the TNF-α levels in the saliva and sera of smokers with periodontitis were higher than those in non-smokers (Gumus et al., 2014). As a potent anti-inflammatory cytokine, IL-10 is able to negatively modulate the synthesis of pro-inflammatory cytokines such as TNF-α,
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and IL-10 is suppressed in periodontal diseases, leading to increasing alveolar bone degradation and decreasing bone formation (Zhang et al., 2014). In accordance with this study, Goutoudi et al. demonstrated that the IL-10 concentration in the GCF of periodontitis patients was significantly lower in diseased sites and in S-ChP (Goutoudi et al., 2004). Bacteria or their products in periodontal connective tissue may induce immune responses involving the production of interleukins and TNF, which play important roles in regulating inflammatory processes (Llambes et al., 2015). In summary, the sera of smokers with periodontitis can enhance Pg internalization by KB cells, promote the expression of MMP-1 and MMP-9 and reduce the expression of TIMP-1; the level of the inflammatory factor TNF-α in the GCF increased, while the anti-inflammatory cytokine IL-10 13
ACCEPTED MANUSCRIPT decreased, due to smoking. Therefore, smoking can increase Pg colonies and pathogenicity, and exacerbate ChP. This study confirmed previous studies and provided a reference value for studies of the effect of smoking on periodontitis. However, there is still limit in our study; we did not give
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detailed description of how the quadrant was randomized. This will give us chances for improving
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our future studies.
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ACCEPTED MANUSCRIPT Acknowledgements The authors wish to express their gratitude to reviewers for their critical comments.
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Conflicts of interest
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None.
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inhibitors in periodontitis-affected gingival tissue. J Periodontol. 79, 166-73. Kubota, M., et al., 2011. Effect of smoking on subgingival microflora of patients with periodontitis in Japan. BMC Oral Health. 11, 1. Lahmann, C., et al., 2001. Matrix metalloproteinase-1 and skin ageing in smokers. Lancet. 357, 935-6. Llambes, F., et al., 2015. Relationship between diabetes and periodontal infection. World J Diabetes. 6, 927-35. Marcaccini, A. M., et al., 2010. Gingival crevicular fluid levels of MMP-8, MMP-9, TIMP-2, and MPO decrease after periodontal therapy. J Clin Periodontol. 37, 180-90. Matthews, J. B., et al., 2011. Effect of nicotine, cotinine and cigarette smoke extract on the neutrophil respiratory burst. J Clin Periodontol. 38, 208-18. Mokeem SA, et al., 2014. Influence of smoking on clinical parameters and gingival crevicular fluid volume in patients with chronic periodontitis. Oral Health Dent Manag. 13, 469-73. Mouzakiti, E., et al., 2012. Expression of MMPs and TIMP-1 in smoker and nonsmoker chronic periodontitis patients before and after periodontal treatment. J Periodontal Res. 47, 532-42. Ozcaka, O., et al., 2011. Smoking and matrix metalloproteinases, neutrophil elastase and myeloperoxidase in chronic periodontitis. Oral Dis. 17, 68-76. Palmer, R. M., et al., 2005. Mechanisms of action of environmental factors--tobacco smoking. J Clin Periodontol. 32 Suppl 6, 180-95. Passoja A, et al., 2010. Serum levels of interleukin-10 and tumour necrosis factor-alpha in chronic periodontitis. J Clin Periodontol. 37, 881-7. Petersen PE, Ogawa H, 2012. The global burden of periodontal disease: Towards integration with chronic disease prevention and control. Periodontol 2000. 60, 15-39. Rai, B., et al., 2008. Biomarkers of periodontitis in oral fluids. J Oral Sci. 50, 53-6. Reynolds, M. A., 2014. Modifiable risk factors in periodontitis: at the intersection of aging and disease. Periodontol 2000. 64, 7-19. Surlin, P., et al., 2014. Matrix metalloproteinase -7, -8, -9 and -13 in gingival tissue of patients with type 1 diabetes and periodontitis. Rom J Morphol Embryol. 55, 1137-41. Verstappen, J., Von den Hoff, J. W., 2006. Tissue inhibitors of metalloproteinases (TIMPs): their biological functions and involvement in oral disease. J Dent Res. 85, 1074-84. Yousefimanesh H, et al., 2013. Evaluation of salivary tumor necrosis factor-alpha in patients with the chronic periodontitis: A case-control study. J Indian Soc Periodontol. 17, 737-40. Zeller, I., et al., 2014. Altered antigenic profiling and infectivity of Porphyromonas gingivalis in smokers and non-smokers with periodontitis. J Periodontol. 85, 837-44. Zhang, Q., et al., 2014. Interleukin-10 inhibits bone resorption: a potential therapeutic strategy in periodontitis and other bone loss diseases. Biomed Res Int. 2014, 284836.
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ACCEPTED MANUSCRIPT Legends Figure 1. The effect of serum volumes of S-ChP and NS-ChP patients on Pg internalizing KB cell colonies. Note: The comparisons of Pg internalizing KB cell colonies between the S-ChP and
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NS-ChP patients were verified by t test; the data in the figure represent the mean ± standard
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deviation (x ± s); S-ChP, smoking patients with chronic periodontitis; NS-ChP, non-smoking
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patients with chronic periodontitis; Pg, Porphyromonas gingivalis.
Figure 2. The levels of TNF-α and IL-10 in the gingival crevicular fluid, comparing the S-ChP and
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NS-ChP patients. Note: The comparisons of the levels of TNF-α and IL-10 in the gingival crevicular fluid between the S-ChP and NS-ChP patients were verified by t test; the data in the
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figure represent the mean ± standard deviation (x ± s); S-ChP, smoking patients with chronic
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periodontitis; NS-ChP, non-smoking patients with chronic periodontitis.
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ACCEPTED MANUSCRIPT Table 1. General information of the patients in the S-ChP and NS-ChP groups NS-ChP group S-ChP group
t, χ2
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Gender (M/F) 29/11 26/12 0.16 0.693 Age (years) 46.7 ± 9.1 46.5 ± 8.3 0.10 0.921 * Smoking history (years) 1.91 ± 0.64 15.3 ± 4.47 -18.29 < 0.001 * PLI 3.97 ± 0.28 4.24 ± 0.55 -2.72 0.009 * SBI 3.48 ± 1.08 2.94 ± 0.73 2.57 0.012 PD (mm) 5.98 ± 1.46 5.61 ± 1.14 1.24 0.217 * LA (mm) 2.56 ± 0.75 2.98 ± 1.03 -2.05 0.044 The categorical data were verified by χ2 and measurement data by t test between NS-ChP group and S-ChP group; M, male; F, female; PLI, plaque index; SBI, sulcus bleeding index; PD, probing depth; LA, loss of attachment; S-ChP, smoking patients with chronic periodontitis; NS-ChP, non-smoking patients with chronic periodontitis; * represents P < 0.05 when compared with the NS-ChP group.
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Table 2. The effects of different volumes of serum from the patients in the S-ChP and NS-ChP groups on Pg internalizing KB cell colonies Pg internalizing KB cell colonies (×104 CFU/mL) Group Number 200 μL 400 μL 800 μL * *# S-ChP group 38 14.1 ± 1.7 16.9 ± 2.0 25.3 ± 2.8*#& NS-ChP group 40 12.8 ± 1.3 12.1 ± 1.5 13.0 ± 1.6 P < 0.001 < 0.001 < 0.001 t -3.77 -11.95 -23.62 Pg, porphyromonas gingivalis; CFU, clonal formation unit; S-ChP, smoking patients with chronic periodontitis; NS-ChP, non-smoking patients with chronic periodontitis; * represents P < 0.05 when compared with NS-ChP group; # represents P < 0.05 when compared with the 200 μL serum; & represents P < 0.01 when compared with the 400 μL serum; t represents the comparisons of the Pg internalizing KB cell colonies with the same volume of serum in the S-ChP and NS-ChP groups.
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ACCEPTED MANUSCRIPT Table 3. The effect of different volumes of serum from the patients in the S-ChP and NS-ChP groups on the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cells
TIMP-1
200 μL
62.2 ± 6.4
NS-ChP group (with Pg)
59.4 ± 6.2
#
S-ChP group (without Pg)
19.1 ± 2.5
NS-ChP group (without Pg)
18.6 ± 1.9
400 μL #
76.3 ± 14.7
150.1 ± 16.9
71.5± 8.2
#
78.3 ± 11.7
82.7 ± 10.7
48. 7 ± 7.3
51.0 ± 6.9
39.7 ± 7.8 38.9 ± 7.3 #
800 μL *#
#
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S-ChP group (with Pg)
#
46. 3 ± 5.9
#
330.9 ± 15.5
*#
#
49.1 ± 6.1
#
7.06 ± 0.50*
#
S-ChP group (with Pg)
2.59 ± 0.43
NS-ChP group (with Pg)
2.53 ± 0.51
#
3.92 ± 0.27
4. 23± 0.41
#
5.23 ± 0.53
S-ChP group (without Pg)
1.41 ± 0.22
1.42 ± 0.27
1. 96 ± 0.33
2.41 ± 0.39
NS-ChP group (without Pg)
1.48 ± 0.17
1.37 ± 0.21
1. 86 ± 0.27
2.37 ± 0.32
#
#
#
69.6 ± 5.8*
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4.04 ± 0.38
#
4.41 ± 0.47
76.8 ± 7.9*
#
#
S-ChP group (with Pg)
86.7 ± 8.7
NS-ChP group (with Pg)
89.2 ± 15.5
87.3 ± 13.4
85.6 ± 9.4
#
79.4 ± 6.4
S-ChP group (without Pg)
73.4 ± 9.3
69.1 ± 13.7
55.7 ± 11.2
20.4 ± 6.7
NS-ChP group (without Pg)
72.8 ± 9.1
68.2 ± 12.8
56.3 ± 11.5
20.1 ± 6.1
81.7 ± 9.6
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MMP-9
0 μL
#
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MMP-1
Concentration
Group
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Cytokines
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#
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The comparisons of the concentration of MMP-1, MMP-9 and TIMP-1 secreted by the KB cells with different volumes of serum from patients with chronic periodontitis between the S-ChP and NS-ChP group were verified by t test, as well as the mass concentrations of the secretion factors of KB cells with or without the addition of Pg; MMP-1, matrix metalloprotease-1; MMP-9, matrix metalloprotease-2; TIMP-1, tissue inhibitor of metalloproteinase; S-ChP, smoking patients with chronic periodontitis; NS-ChP, non-smoking patients with chronic periodontitis; * represents P < 0.001 when compared with the NS-ChP group; # represents P < 0.05 when comparing with or without Pg in the same group.
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S0014-4800(16)30160-5 doi: 10.1016/j.yexmp.2016.07.004 YEXMP 3947
To appear in:
Experimental and Molecular Pathology
Received date: Revised date: Accepted date:
2 March 2016 5 July 2016 19 July 2016
Please cite this article as: He, Chun-Yan, Gao, Xiu-Qiu, Jiang, Li-Peng, The impact of smoking on levels of chronic periodontitis-associated biomarkers, Experimental and Molecular Pathology (2016), doi: 10.1016/j.yexmp.2016.07.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT The impact of smoking on levels of chronic periodontitis-associated biomarkers Running title: Smoking and chronic periodontitis
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Department of Prosthodontics, the Second Affiliated Hospital of Liaoning Medical University,
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Jinzhou 121000, P.R. China;
Department of Radiation Oncology, the First Affiliated Hospital of Liaoning Medical University,
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Chun-Yan He 1, Xiu-Qiu Gao 1, Li-Peng Jiang 2, *
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Jinzhou 121000, P.R. China.
* Correspondence to: Dr. Li-Peng Jiang, Department of Radiation Oncology, the First Affiliated
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Hospital of Liaoning Medical University, No. 2, the Five Section of Renmin Street, Guta District,
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Jinzhou 121000, P.R. China. E-mail: [email protected]
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Tel. /fax: +86-416-2655167
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ACCEPTED MANUSCRIPT Abstract Objective: To investigate the effect of smoking on the expression levels of matrix metalloproteinase (MMP)-1, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and the concentrations of
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TNF-α and IL-10 in patients with chronic periodontitis (ChP).
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Methods: This is an ex-vivo study. Our study consisted of 78 cases, all of which were diagnosed
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with ChP and were selected according to the inclusion and exclusion criteria. Among these 78 cases, 38 patients were classified into the smoking group (S-ChP group), and 40 patients in the
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non-smoking group (NS-ChP group). The clinical periodontal parameters of all patients were recorded, including the plaque index (PLI), probing depth (PD), loss of attachment (LA) and sulcus
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bleeding index (SBI). Serum was collected from forearm blood to establish a Porphyromonas gingivalis (Pg) internalizing KB cell model. Enzyme-linked immunosorbent assay (ELISA) was
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used to determine the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cell lysis solution
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as well as IL-10 and TNF- in the gingival crevicular fluid (GCF).
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Results: Fewer Pg internalizing KB cell colonies were observed in the NS-ChP group than in the S-ChP group (P < 0.01). When 400 μL serum was added, there were remarkable differences in the concentrations of MMP-1 and TIMP-1 secreted from the KB cells between the S-ChP and NS-ChP
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groups (MMP-1: t = -21.71, P < 0.01; TIMP-1: t = 64.35, P < 0.001). Additionally, when 800 μL serum was added, there were significant differences in the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cells between the S-ChP and NS-ChP groups (MMP-1: t = -81.89, P < 0.001; MMP-9: t = -15.67, P < 0.001; TIMP-1: t = 109.4, P < 0.001). The TNF-α levels were higher, but the IL-10 levels were lower in the GCF from the ChP patients in the S-ChP group than those in the NS-ChP group (both P < 0.001). Conclusion: The serum of S-ChP patients can enhance the concentrations of MMP-1 and MMP-9, but reduce TIMP-1 secreted from Pg internalizing KB cells. However, the concentration of TNF-α was increased and IL-10 was decreased. Abnormal concentrations of ChP-associated biomarkers may be conducive to the development and progression of ChP. 2
ACCEPTED MANUSCRIPT Keywords: Chronic periodontitis; IL-10; TNF-α; MMP-1; MMP-9; TIMP-1; Porphyromonas
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gingivalis; KB cells
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ACCEPTED MANUSCRIPT Introduction Periodontitis, a chronic inflammatory disease, is characterized by loss of periodontal ligament and
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alveolar bone, and can lead to tooth loss and affect individual's oral health-related quality of life (De
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Pablo, et al., 2009; Durham, et al., 2013). It is estimated that severe periodontitis affects 5~20% of
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any population, and mild/moderate periodontitis is a problem of majority of adults (Kassebaum, et al., 2014; Dye, 2012; Petersen and Ogawa, 2012). Periodontitis is more common among men than
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women and is more prevalent in developing areas than in developed areas, and the occurrence of this disease shows an upward with aging (Ababneh et al., 2012; Darveau, 2010). Chronic
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periodontitis (ChP) can be developed by modifiable risk factors, including cigarette smoking, alcohol consumption, poor diet, and psychological stress and depression (Reynolds, 2014; Filoche,
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et al., 2010). It has been reported that smoking is strongly involved in the initiation and progression of periodontitis disease and that smokers were found altered amount of gingival crevicular fluid
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(GCF) and its components (Mokeem, et al., 2014; Kubota et al., 2011). Smoking is also reported to have a potential negative effect on the immune defense system of the periodontal host, which
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inhibits neutrophil and macrophage function and affects defense against bacteria (Gomes et al., 2015; Fan et al., 2015).
Matrix metalloproteinases (MMPs) are zinc- and calcium-dependent proteolytic enzymes that are responsible for the degradation of all extracellular matrix proteins and basement membrane components (Marcaccini et al., 2010). MMPs can be produced by inflammatory cells caused by bacterial infection in response to cytokines secreted from these cells in periodontal patients; thus, their presence, quantities, and activities are recognized to be very important for the characterization of various states of periodontitis and the determination of active destruction (Kim et al., 2013). Endogenous tissue inhibitors, especially tissue inhibitors of metalloproteinases (TIMPs), are 4
ACCEPTED MANUSCRIPT significant factors in the control of MMPs (Mouzakiti et al., 2012). An imbalance between MMPs and TIMPs is found in pathological conditions, including periodontitis and correlates with tissue
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destruction (Verstappen and Von den Hoff, 2006; Kubota et al., 2008). Tumor necrosis factor alpha
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(TNF-α), as a pro-inflammatory cytokine, can lead to propagation of inflammation and contribute to
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release of a high level of inflammatory mediators, which result in destruction of tooth-supporting periodontal structures (Passoja, et al., 2010; Yousefimanesh, et al., 2013). As a vital
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anti-inflammatory cytokine that impedes inflammatory responses, IL-10 is also implicated in ChP (Zhang et al., 2014). Porphyromonas gingivalis (Pg) is a predominant periodontal pathogen that
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expresses multiple potential virulence factors involved in the pathogenesis of periodontitis (Amano,
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2007). The virulence factors enable Pg to invade the periodontal tissue and subsequently spread into
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the systemic circulation (Blasco-Baque et al., 2016). Pg can also invade host cells, including epithelial cells (Amano et al., 2014). Gingival epithelial cells are spontaneously exposed to bacterial
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attacks and function in preventing bacteria invasion into deeper tissues (Amano, 2007). In the present study, we detected the levels of MMP-1, MMP-9, TIMP-1 in the supernatants of Pg
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internalizing KB cells and the levels of TNF-α and IL-10 in the GCF, in order to explore the effect of smoking on ChP.
Materials and Methods Subjects A total of 78 patients diagnosed with ChP were recruited into our study from the First Affiliated Hospital of Liaoning Medical University between September 2012 and March 2015. The ChP was diagnosed in accordance with the recommended standards from the Center for Disease Control and Prevention, the American Academy of Periodontology, on the condition of (1) presence of 2 interproximal sites with 3 mm clinical attachment loss, not on the same tooth, and (2) 5
ACCEPTED MANUSCRIPT presence of 2 interproximal sites with 4 mm probing depth (PD) occurring at two or more different teeth, or presence of 5 mm PD of a site (Armitage, 1999). According to the standard of WHO definition of the smoking population, patients who smoked more than 10 cigarettes daily for
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longer than 2 years were considered smoking patients with ChP (S-ChP group). The patients who quit smoking for more than 10 years or had no smoking habits were considered non-smoking
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patients with ChP (NS-ChP group). Our study consisted of 38 cases of S-ChP and 40 cases of NS-ChP. The exclusion criteria for patient enrollment and experimental teeth were as follows: (1)
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patients with systemic disease; (2) patients taking oral antibiotics, analgesics or hormonal drugs in the past three months, and those who had received periodontal treatment for nearly half of the year;
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(3) patients with less than 20 remaining teeth, and experimental teeth with periapical disease (based on clinical symptoms, X-ray examination and diagnostic results) and occlusal trauma (based on
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functional mobility, occlusion widened periodontal ligament and other local incentives diagnosis).
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In addition, periodontal clinical parameters were obtained from each patient with ChP, including the
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plaque index (PLI), PD, loss of attachment (LA) and sulcus bleeding index (SBI). Two diagonal quadrants were randomly selected where a tooth with the highest PD was used as the experimental molar tooth and the area with the deepest PD was the GCF sample site. This study was approved by
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the Ethics Committee of the First Affiliated Hospital of Liaoning Medical University, and the subjects’ written consent was obtained according to the current Declaration of Helsinki. Clinical parameter measurement and sample collection The periodontal clinical indices of the ChP patients were recorded. The PLI was determined and divided into four grades on the basis of the Silness & Loe PLI methods. The scores were as follows: grade 0, no plaque in the gingival margin area; grade 1, thin plaque in the gingival margin of the tooth surface that was not easy to see with the naked eye but could be scraped using the tip side of a probe; grade 2, visible medium plaque in the gingival margin or interproximal surface; grade 3, a large amount of soft scale in the gingival margin and the adjacent surface or gingival sulcus. The PD was the distance from the gingival margin to the bottom of periodontal pocket or the 6
ACCEPTED MANUSCRIPT bottom of gingival sulcus. The LA was the difference between the PD and the distance from the enamelo-cemental junction to the gingival margin; if gingival recession was present, the PD and the gingival recession distance were summed. The SBI was judged based on the Mazza (1981) method.
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The SBI was divided into six grades according to the gingival bleeding degree by Florida
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periodontal probing. The grades were as follows: grade 0, patients with healthy gingival, no
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inflammation or bleeding symptoms; grade 1, patients with gingival color changed with inflammation but no bleeding from probing; grade 2, punctate hemorrhage after probing; grade 3,
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probe hemorrhage diffusion along the gingival margin; grade 4, full bleeding and gingival fluid overflow; grade 5, spontaneous bleeding.
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Two diagonal quadrants were randomly selected in the mouth of each study subject. The molars or premolars were selected as experimental teeth on the condition that: no filling body or
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caries were present at the tooth neck; PD > 3 mm; LA more than or equal to 1 mm; and varying
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degrees of destruction or resorption of alveolar bone evident by X-ray. The GCF samples were
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taken from the deepest site of the PD. Filter paper strips (2 mm 10 mm) were placed at the bottom of the periodontal pocket and kept in place for 30 s to adsorb and collect the GCF. Four sites were selected, including the buccal side, far from the buccal side, the lingual side and far from the lingual
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side. The four filter paper strips were placed into the same EP tube, followed by sealing, labelling, weighing and recording, and then reserved at 80℃ in a low-temperature refrigerator for further use. The reserved GCF sample was taken out from the refrigerator. After back to room temperature, the sample was shocked, eluted for 1 h and centrifuged (13000 r/min) for 10 min at 4℃. The supernatant was collected in clean EP tubes (each 50 μl). All the above steps were completed by a highly-qualified and professionally trained physician from our hospital. Bacterial culture Venous blood (5 mL) was collected from the forearms of all of the study subjects andcentrifuged to obtain serum samples, which were then reserved at -80℃. After PgATCC33277 recovery at room temperature, it was inoculated into brain heart infusion agar (BHIA) medium 7
ACCEPTED MANUSCRIPT containing 1% hemoglobin chloride, 5% sterile defibrinated sheep blood, and 0.1% vitamin K1 at 37℃ in an anaerobic environment for 5~7 days. Gram staining and biochemical identification were performed to confirm the purity of PgATCC33277, which was then cultured at 4°C for 24 h. After
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10 min of centrifugation at 5000 r/min, the bacteria were collected. The bacteria were washed with
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PBS and suspended in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Life Technologies,
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Darmstadt, Germany) without an antibiotic. Then, a UV spectrophotometer was used to measure the absorbance at 600 nm, and the bacterial suspension was adjusted to 1 × 108 CFU/mL. Fifty samples
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were obtained randomly after the enrichment of the bacterial liquid extraction, and Gram staining and polymerase chain reaction (PCR) were used to identify Pg using the upstream primer
5'-ACGTCATCCACACCTTCCTC-3'.
the
downstream
primer
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and
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5'-TGTAGATGACTGATGGTGAAAACC-3'
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The conventional KB cell line ATCCCCL17 was thawed from cryopreservation, inoculated in
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low-glucose DMEM and incubated in a humidified atmosphere of 5% CO2 at 37℃. Under the microscope, the cells presented a "paving stone" shape. When the cells were 80% confluent, they were digested with trypsin containing 2.5 g/L ethylene diamine tetra-acetic acid (EDTA) and
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inoculated in 6-well plates (each well 2.5 mL), with the cell density adjusted to 2 × 108/L using low-glucose DMEM medium containing 10% fetal bovine serum (FBS). Bacterial colony counting When the adherent KB cells were 80%~90% confluent in the 6-well plates, the number of cells was 2.5 × 106 cells/well. These KB cells were then washed three times with PBS and placed into low-glucose DMEM along with 2.5 × 108 CFU/mL Pg to ensure a multiplicity of infection of 100:1. To avoid interference from other factors, we used wells without Pg as a negative control group. Moreover, in order to imitate the conditions in the serum of S-ChP or NS-ChP patients and to investigate the number of KB cells that internalized Pg with different volumes of serum, 200, 400, or 800 μL serum from the S-ChP and NS-ChP patients were added to Pg prior to KB cell infection. 8
ACCEPTED MANUSCRIPT A group without serum added was used as the negative control group. The samples were incubated for 2 h in triplicate. Subsequently, 3 PBS washes were performed to remove Pg that was not adhered to the KB cells. Then, the KB cells were incubated in DMEM containing antibiotics for 1 h,
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followed by 3 additional PBS washes. The supernatant (500 μL) was absorbed after 12 h and were
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then stored at -80℃. KB cells without remaining DMEM were first washed 4 times, and then 1 mL
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sterile distilled water was added to each well for 90 min of cell disruption. Following dilution (1:1), the suspension (100 μL) was seeded into BHI medium supplemented with 1% freshly prepared
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sodium chloride containing hemoglobin, 5% sterile defibrinated sheep’s blood, and 0.1% vitamin K1 for 5-7 days, and then the bacterial colonies were counted. The KB cells for our study were
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provided by the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and the Pg was purchased from Takara
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Enzyme-linked immunosorbent assay (ELISA)
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ELISA was used to detect the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cell supernatant as well as TNF-α and IL-10 in the GCF. The absorbance (A) [the optical density (OD)] value was measured by a microplate reader, with detection wavelength of 450 nm and correction
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wavelength of 570 nm. With the absorbance (A) value as the vertical axis and the standard concentration as the abscissa, a standard curve was obtained. Consequently, the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cell supernatant and the TNF-α and IL-10 concentration in the GCF were calculated. Data analysis Data analysis was performed using the statistical package for the social sciences (SPSS) version 19.0 (SPSS Inc.; Chicago, IL, USA). Categorical data were verified by χ2 and measurement data are presented as the means ± standard deviation (SD), in which two groups of samples were compared with t tests. The data that did not conform to normality or homogeneity of variance were verified using a non-parametric rank-sum test. Multi-group comparisons were performed with 9
ACCEPTED MANUSCRIPT one-way analysis of variance (ANOVA) based on α = 0.05. All tests were two-sided, with P < 0.05 indicating a significant difference.
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Results
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General information
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The 38 S-ChP cases included 26 males and 12 females with a mean age of 46.5 ± 8.3 years old, and the 40 NS-ChP cases included 29 males and 11 females with a mean age of 46.7 ± 9.1 years old.
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There was no significant difference in gender, age or PD values between the S-ChP and NS-ChP groups (all P > 0.05). Smoking history, PLI and LA values in the S-ChP patients were significantly
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higher and SBI was lower than those in the NS-ChP patients (all P < 0.01) (Table 1). The effects of different volumes of serum on the number of Pg internalizing KB cell colonies
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the effects of different volumes of serum from these two groups on Pg internalizing KB cells
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colonies. The results indicated that significant fewer Pg internalizing KB cell colonies were present in different volumes of serum from the NS-ChP group than in serum from the S-ChP group (all P < 0.01). With the addition of 200 μL, 400 μL or 800 μL serum, there was no significant difference in
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the number of Pg internalizing KB cell colonies in the NS-ChP group (F = 1.773, P = 0.174), while there was an increase in Pg internalizing KB cell colonies in the S-ChP group (F = 108.1, P < 0.001) (Figure 1, Table 2)
The effects of different volumes of serum on the concentrations of MMP-1, MMP-9 and TIMP-1 in KB cells With the addition of 200 μL, 400 μL or 800 μL serum, the KB cells in the S-ChP group had increased concentrations of MMP-1 and MMP-9, but decreased concentrations of TIMP-1 (Table 3). When 200 μL serum was added, the difference between the S-ChP and NS-ChP groups in the concentrations of MMP-1, MMP-9 and TIMP-1 secreted from the KB cells was not statistically significant (all P > 0.05). When 400 μL serum was added, there were remarkable differences 10
ACCEPTED MANUSCRIPT between the S-ChP and NS-ChP groups in the concentrations of MMP-1 and TIMP-1 secreted from the KB cells ChP and NS-ChP groups (MMP-1: t = -21.71, P < 0.01; TIMP-1: t = 64.35, P < 0.001). Additionally, when 800 μL serum was added, there were significant differences between the S-ChP
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and NS-ChP groups in the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cells (MMP-1:
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t = -81.89, P < 0.001; MMP-9: t = -15.67, P < 0.001; TIMP-1: t = 109.4, P < 0.001). Comparing the
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mass concentrations of the factors secreted by KB cells with or without the addition of Pg in the S-ChP and NS-ChP groups, we found that in both of the two groups, the mass concentration of the
without the addition of Pg (both P < 0.05).
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factors secreted from KB cells following the addition of Pg was significantly higher than that
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Concentrations of TNF-α and IL-10 in the GCF
The TNF-α levels in the GCF samples from the patients with ChP in the S-ChP group were
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6.61 ± 0.74 ng/mL, which was higher than those in the NS-ChP group, 5.88 ± 0.56 ng/mL (t = -4.9,
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Discussion
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lower than those in the NS-ChP group, 5.26 ± 0.95 ng/mL (t = 5.84, P < 0.001) (Figure 2).
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The most important finding of our study was the effect of smoking on ChP. We found that Pg internalized KB cell colonies in the NS-ChP group were less than that in the S-ChP group. It has been recognized that smoking plays a significant role in the protective elements of the immune system (Palmer et al., 2005). A study reported that polymorphonuclear lymphocytes in smokers showed decreased chemotaxis, which prevented the elimination of periodontal pathogens, causing an increase in the extent and severity of periodontal destruction (Matthews et al., 2011). Periodontal bacteria (Pg included) were reported to be critically dependent on an inflammatory environment, where they obtained nutrients from tissue-breakdown products (Hajishengallis, 2014). Smoking was reported to provide a supporting environment for the growth of periodontal microbes (Gupta et al., 2016). Tobacco smoking was demonstrated to have critical effects on the host, leading to an 11
ACCEPTED MANUSCRIPT increased response to bacterial plaque accumulation (Llambes et al., 2015). Therefore, we assumed that smoking is supportive for Pg by creating an environment that promotes the bacterial growth. In the inflammation of the periodontal tissue, the level of MMPs increased in all the bodily fluids,
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including the GCF, saliva, serum and plasma; the level of TIMPs decreased and MMPs increased,
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jointly resulting in uncontrolled tissue destruction (Gupta et al., 2016). The balance between
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activated MMPs and TIMPs was shown to regulate extracellular matrix remodeling, and tissue degradation occurred due to the disruption of this balance (Surlin et al., 2014). With an imbalance
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of MMP/TIMP activity, changes in the TIMP-1 level could be critical in regulating the destruction of periodontal tissues by affecting the MMP levels (Emingil et al., 2006).
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In our study, we used Pg internalizing KB cell colonies to observe the differences in S-ChP and NS-ChP cells incubated with different volumes of serum and found that there were greater
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numbers of Pg internalizing KB cell colonies in the S-ChP group than in the NS-ChP group, and the
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Pg internalizing KB cell colonies in the S-ChP group increased as the volume of serum from S-ChP
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patients increased. Similar to this result, it was reported by Cogo et al. that exposure to cotinine (an important constituent of tobacco) at a high concentration might enhance Pg invasion into epithelial cells (Cogo et al., 2009). Furthermore, according to Zeller et al., smoking can reduce IgG (a type of
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antibody that eliminates plaque bacteria) production in response to Pg infection, thus promoting Pg survival and potentially increasing vulnerability to periodontitis (Zeller et al., 2014). The studies above showed that smoking might increase the likelihood of infection with Pg, and consequently, smokers might suffer from increased susceptibility to periodontitis. Notably, as the serum volume increased, the concentrations of MMP-1 and MMP-9 secreted by KB cells also increased while TIMP-1 decreased in both the S-ChP and NS-ChP groups, but with greater concentration changes in the S-ChP group. In agreement with one of the results, MMP-1, which plays a role in extracellular matrix destruction and collagen degradation, was found to exhibit high concentrations during periodontal disease (Beklen et al., 2007). Moreover, it was found that MMP-1 mRNA levels in the skin of smokers were significantly increased compared to those of 12
ACCEPTED MANUSCRIPT non-smokers (Lahmann et al., 2001). MMP-9 plays a role in the functions of type IV, V, and XI collagens; denatured collagens; elastins; and proteoglycans, and is mainly released from osteoclasts and polymorphonuclear neutrophils in periodontal tissues (Ertugrul et al., 2013). In ChP, there is an
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imbalance between MMPs and their inhibitors (TIMPs), with elevated MMPs and low levels of
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TIMPs (Rai et al., 2008). Ozcaka et al. discovered significantly elevated levels of MMP-9 as well as
2011), which further supports the results of this study.
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reduced levels of TIMP-1 in smokers with periodontitis compared with non-smokers (Ozcaka et al.,
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Moreover, our study demonstrated that the S-ChP group had higher levels of TNF-α and lower levels of IL-10 in the GCF than those in the NS-ChP group. Recently, Zhu et al. indicated that
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periodontitis patients seemed to have an activated TNF-α response, as lipopolysaccharide stimulated macrophages derived from the periodontal tissues produce greater amounts of TNF-α
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compared with individuals without periodontitis, and circulating TNF-α levels were also associated
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with more severe periodontitis in a large geriatric population (Jiang et al., 2013). Additionally,
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Gumus et al. showed that the TNF-α levels in the saliva and sera of smokers with periodontitis were higher than those in non-smokers (Gumus et al., 2014). As a potent anti-inflammatory cytokine, IL-10 is able to negatively modulate the synthesis of pro-inflammatory cytokines such as TNF-α,
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and IL-10 is suppressed in periodontal diseases, leading to increasing alveolar bone degradation and decreasing bone formation (Zhang et al., 2014). In accordance with this study, Goutoudi et al. demonstrated that the IL-10 concentration in the GCF of periodontitis patients was significantly lower in diseased sites and in S-ChP (Goutoudi et al., 2004). Bacteria or their products in periodontal connective tissue may induce immune responses involving the production of interleukins and TNF, which play important roles in regulating inflammatory processes (Llambes et al., 2015). In summary, the sera of smokers with periodontitis can enhance Pg internalization by KB cells, promote the expression of MMP-1 and MMP-9 and reduce the expression of TIMP-1; the level of the inflammatory factor TNF-α in the GCF increased, while the anti-inflammatory cytokine IL-10 13
ACCEPTED MANUSCRIPT decreased, due to smoking. Therefore, smoking can increase Pg colonies and pathogenicity, and exacerbate ChP. This study confirmed previous studies and provided a reference value for studies of the effect of smoking on periodontitis. However, there is still limit in our study; we did not give
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detailed description of how the quadrant was randomized. This will give us chances for improving
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our future studies.
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ACCEPTED MANUSCRIPT Acknowledgements The authors wish to express their gratitude to reviewers for their critical comments.
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Conflicts of interest
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None.
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ACCEPTED MANUSCRIPT References
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Ababneh, K. T., et al., 2012. Prevalence and risk indicators of gingivitis and periodontitis in a multi-centre study in North Jordan: a cross sectional study. BMC Oral Health. 12, 1. Amano, A., 2007. Disruption of epithelial barrier and impairment of cellular function by Porphyromonas gingivalis. Front Biosci. 12, 3965-74. Amano, A., et al., 2014. Genetic characteristics and pathogenic mechanisms of periodontal pathogens. Adv Dent Res. 26, 15-22. Armitage GC, 1999. Development of a classification system for periodontal diseases and conditions. Ann Periodontol. 4, 1-6. Beklen, A., et al., 2007. MMPs, IL-1, and TNF are regulated by IL-17 in periodontitis. J Dent Res. 86, 347-51. Blasco-Baque, V., et al., 2016. Periodontitis induced by Porphyromonas gingivalis drives periodontal microbiota dysbiosis and insulin resistance via an impaired adaptive immune response. Gut. Cogo, K., et al., 2009. The effects of nicotine and cotinine on Porphyromonas gingivalis colonisation of epithelial cells. Arch Oral Biol. 54, 1061-7. Darveau, R. P., 2010. Periodontitis: a polymicrobial disruption of host homeostasis. Nat Rev Microbiol. 8, 481-90. De Pablo P., et al., 2009. Periodontitis in systemic rheumatic diseases. Nat Rev Rheumatol. 5, 218-24. Durham J., et al., 2013. Impact of periodontitis on oral health-related quality of life. J Dent. 41, 370-6. Dye BA, 2012. Global periodontal disease epidemiology. Periodontol 2000. 58, 10-25. Emingil, G., et al., 2006. Gingival crevicular fluid matrix metalloproteinase (MMP)-7, extracellular MMP inducer, and tissue inhibitor of MMP-1 levels in periodontal disease. J Periodontol. 77, 2040-50. Ertugrul, A. S., et al., 2013. MMP-1, MMP-9, and TIMP-1 levels in oral lichen planus patients with gingivitis or periodontitis. Arch Oral Biol. 58, 843-52. Fan, Y., et al., 2015. [The impact of smoking on human beta defensin 2,3 in gingival crevicular fluid and gingival tissue of patients with chronic periodontitis]. Shanghai Kou Qiang Yi Xue. 24, 735-8. Filoche SK, et al., 2010. Smoking, chronic periodontitis and smoking cessation support: Reviewing the role of dental professionals. N Z Dent J. 106, 74-7. Gomes, S. C., et al., 2015. Influence of supragingival biofilm control and smoking habit on Interleukin-1beta concentration. Braz Oral Res. 29, 1-8. Goutoudi, P., et al., 2004. Effect of periodontal therapy on crevicular fluid interleukin-1beta and interleukin-10 levels in chronic periodontitis. J Dent. 32, 511-20. Gumus, P., et al., 2014. Saliva and serum levels of B-cell activating factors and tumor necrosis factor-alpha in patients with periodontitis. J Periodontol. 85, 270-80. Gupta, N., et al., 2016. The effect of type 2 diabetes mellitus and smoking on periodontal parameters and salivary matrix metalloproteinase-8 levels. J Oral Sci. 58, 1-6. Hajishengallis, G., 2014. Immunomicrobial pathogenesis of periodontitis: keystones, pathobionts, and host response. Trends Immunol. 35, 3-11. Jiang, Z. L., et al., 2013. Study of TNF-alpha, IL-1beta and LPS levels in the gingival crevicular fluid of a rat model of diabetes mellitus and periodontitis. Dis Markers. 34, 295-304. Kassebaum NJ, et al., 2014. Global burden of severe periodontitis in 1990-2010: A systematic review and meta-regression. J Dent Res. 93, 1045-53. Kim, K. A., et al., 2013. Correlation of expression and activity of matrix metalloproteinase-9 and -2 in human gingival cells of periodontitis patients. J Periodontal Implant Sci. 43, 24-9. Kubota, T., et al., 2008. Altered gene expression levels of matrix metalloproteinases and their 16
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inhibitors in periodontitis-affected gingival tissue. J Periodontol. 79, 166-73. Kubota, M., et al., 2011. Effect of smoking on subgingival microflora of patients with periodontitis in Japan. BMC Oral Health. 11, 1. Lahmann, C., et al., 2001. Matrix metalloproteinase-1 and skin ageing in smokers. Lancet. 357, 935-6. Llambes, F., et al., 2015. Relationship between diabetes and periodontal infection. World J Diabetes. 6, 927-35. Marcaccini, A. M., et al., 2010. Gingival crevicular fluid levels of MMP-8, MMP-9, TIMP-2, and MPO decrease after periodontal therapy. J Clin Periodontol. 37, 180-90. Matthews, J. B., et al., 2011. Effect of nicotine, cotinine and cigarette smoke extract on the neutrophil respiratory burst. J Clin Periodontol. 38, 208-18. Mokeem SA, et al., 2014. Influence of smoking on clinical parameters and gingival crevicular fluid volume in patients with chronic periodontitis. Oral Health Dent Manag. 13, 469-73. Mouzakiti, E., et al., 2012. Expression of MMPs and TIMP-1 in smoker and nonsmoker chronic periodontitis patients before and after periodontal treatment. J Periodontal Res. 47, 532-42. Ozcaka, O., et al., 2011. Smoking and matrix metalloproteinases, neutrophil elastase and myeloperoxidase in chronic periodontitis. Oral Dis. 17, 68-76. Palmer, R. M., et al., 2005. Mechanisms of action of environmental factors--tobacco smoking. J Clin Periodontol. 32 Suppl 6, 180-95. Passoja A, et al., 2010. Serum levels of interleukin-10 and tumour necrosis factor-alpha in chronic periodontitis. J Clin Periodontol. 37, 881-7. Petersen PE, Ogawa H, 2012. The global burden of periodontal disease: Towards integration with chronic disease prevention and control. Periodontol 2000. 60, 15-39. Rai, B., et al., 2008. Biomarkers of periodontitis in oral fluids. J Oral Sci. 50, 53-6. Reynolds, M. A., 2014. Modifiable risk factors in periodontitis: at the intersection of aging and disease. Periodontol 2000. 64, 7-19. Surlin, P., et al., 2014. Matrix metalloproteinase -7, -8, -9 and -13 in gingival tissue of patients with type 1 diabetes and periodontitis. Rom J Morphol Embryol. 55, 1137-41. Verstappen, J., Von den Hoff, J. W., 2006. Tissue inhibitors of metalloproteinases (TIMPs): their biological functions and involvement in oral disease. J Dent Res. 85, 1074-84. Yousefimanesh H, et al., 2013. Evaluation of salivary tumor necrosis factor-alpha in patients with the chronic periodontitis: A case-control study. J Indian Soc Periodontol. 17, 737-40. Zeller, I., et al., 2014. Altered antigenic profiling and infectivity of Porphyromonas gingivalis in smokers and non-smokers with periodontitis. J Periodontol. 85, 837-44. Zhang, Q., et al., 2014. Interleukin-10 inhibits bone resorption: a potential therapeutic strategy in periodontitis and other bone loss diseases. Biomed Res Int. 2014, 284836.
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ACCEPTED MANUSCRIPT Legends Figure 1. The effect of serum volumes of S-ChP and NS-ChP patients on Pg internalizing KB cell colonies. Note: The comparisons of Pg internalizing KB cell colonies between the S-ChP and
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NS-ChP patients were verified by t test; the data in the figure represent the mean ± standard
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deviation (x ± s); S-ChP, smoking patients with chronic periodontitis; NS-ChP, non-smoking
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patients with chronic periodontitis; Pg, Porphyromonas gingivalis.
Figure 2. The levels of TNF-α and IL-10 in the gingival crevicular fluid, comparing the S-ChP and
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NS-ChP patients. Note: The comparisons of the levels of TNF-α and IL-10 in the gingival crevicular fluid between the S-ChP and NS-ChP patients were verified by t test; the data in the
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figure represent the mean ± standard deviation (x ± s); S-ChP, smoking patients with chronic
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periodontitis; NS-ChP, non-smoking patients with chronic periodontitis.
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Fig. 2
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ACCEPTED MANUSCRIPT Table 1. General information of the patients in the S-ChP and NS-ChP groups NS-ChP group S-ChP group
t, χ2
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Gender (M/F) 29/11 26/12 0.16 0.693 Age (years) 46.7 ± 9.1 46.5 ± 8.3 0.10 0.921 * Smoking history (years) 1.91 ± 0.64 15.3 ± 4.47 -18.29 < 0.001 * PLI 3.97 ± 0.28 4.24 ± 0.55 -2.72 0.009 * SBI 3.48 ± 1.08 2.94 ± 0.73 2.57 0.012 PD (mm) 5.98 ± 1.46 5.61 ± 1.14 1.24 0.217 * LA (mm) 2.56 ± 0.75 2.98 ± 1.03 -2.05 0.044 The categorical data were verified by χ2 and measurement data by t test between NS-ChP group and S-ChP group; M, male; F, female; PLI, plaque index; SBI, sulcus bleeding index; PD, probing depth; LA, loss of attachment; S-ChP, smoking patients with chronic periodontitis; NS-ChP, non-smoking patients with chronic periodontitis; * represents P < 0.05 when compared with the NS-ChP group.
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Table 2. The effects of different volumes of serum from the patients in the S-ChP and NS-ChP groups on Pg internalizing KB cell colonies Pg internalizing KB cell colonies (×104 CFU/mL) Group Number 200 μL 400 μL 800 μL * *# S-ChP group 38 14.1 ± 1.7 16.9 ± 2.0 25.3 ± 2.8*#& NS-ChP group 40 12.8 ± 1.3 12.1 ± 1.5 13.0 ± 1.6 P < 0.001 < 0.001 < 0.001 t -3.77 -11.95 -23.62 Pg, porphyromonas gingivalis; CFU, clonal formation unit; S-ChP, smoking patients with chronic periodontitis; NS-ChP, non-smoking patients with chronic periodontitis; * represents P < 0.05 when compared with NS-ChP group; # represents P < 0.05 when compared with the 200 μL serum; & represents P < 0.01 when compared with the 400 μL serum; t represents the comparisons of the Pg internalizing KB cell colonies with the same volume of serum in the S-ChP and NS-ChP groups.
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ACCEPTED MANUSCRIPT Table 3. The effect of different volumes of serum from the patients in the S-ChP and NS-ChP groups on the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cells
TIMP-1
200 μL
62.2 ± 6.4
NS-ChP group (with Pg)
59.4 ± 6.2
#
S-ChP group (without Pg)
19.1 ± 2.5
NS-ChP group (without Pg)
18.6 ± 1.9
400 μL #
76.3 ± 14.7
150.1 ± 16.9
71.5± 8.2
#
78.3 ± 11.7
82.7 ± 10.7
48. 7 ± 7.3
51.0 ± 6.9
39.7 ± 7.8 38.9 ± 7.3 #
800 μL *#
#
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S-ChP group (with Pg)
#
46. 3 ± 5.9
#
330.9 ± 15.5
*#
#
49.1 ± 6.1
#
7.06 ± 0.50*
#
S-ChP group (with Pg)
2.59 ± 0.43
NS-ChP group (with Pg)
2.53 ± 0.51
#
3.92 ± 0.27
4. 23± 0.41
#
5.23 ± 0.53
S-ChP group (without Pg)
1.41 ± 0.22
1.42 ± 0.27
1. 96 ± 0.33
2.41 ± 0.39
NS-ChP group (without Pg)
1.48 ± 0.17
1.37 ± 0.21
1. 86 ± 0.27
2.37 ± 0.32
#
#
#
69.6 ± 5.8*
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4.04 ± 0.38
#
4.41 ± 0.47
76.8 ± 7.9*
#
#
S-ChP group (with Pg)
86.7 ± 8.7
NS-ChP group (with Pg)
89.2 ± 15.5
87.3 ± 13.4
85.6 ± 9.4
#
79.4 ± 6.4
S-ChP group (without Pg)
73.4 ± 9.3
69.1 ± 13.7
55.7 ± 11.2
20.4 ± 6.7
NS-ChP group (without Pg)
72.8 ± 9.1
68.2 ± 12.8
56.3 ± 11.5
20.1 ± 6.1
81.7 ± 9.6
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MMP-9
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MMP-1
Concentration
Group
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Cytokines
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#
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The comparisons of the concentration of MMP-1, MMP-9 and TIMP-1 secreted by the KB cells with different volumes of serum from patients with chronic periodontitis between the S-ChP and NS-ChP group were verified by t test, as well as the mass concentrations of the secretion factors of KB cells with or without the addition of Pg; MMP-1, matrix metalloprotease-1; MMP-9, matrix metalloprotease-2; TIMP-1, tissue inhibitor of metalloproteinase; S-ChP, smoking patients with chronic periodontitis; NS-ChP, non-smoking patients with chronic periodontitis; * represents P < 0.001 when compared with the NS-ChP group; # represents P < 0.05 when comparing with or without Pg in the same group.
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