Background
Chronic periodontitis is the most common oral disease and the leading cause of tooth loss in adults [
1]. Furthermore, it is a known risk factor for many systemic diseases [
2‐
4], such as cardiovascular disease, diabetes, and rheumatoid arthritis.
Periodontitis is primarily initiated by exaggerated host immune responses to periodontal pathogens, which lead to the breakdown of periodontal connective tissues and alveolar bone loss [
5]. Among numerous periodontal pathogens, the Gram-negative anaerobic bacterium
Porphyromonas gingivalis (
P. gingivalis) is one of the key pathogens in chronic periodontitis.
P. gingivalis releases copious amounts of virulence factors, including proteases and lipopolysaccharides, which interact with toll-like receptors or penetrate into periodontal tissues and trigger immune responses, resulting in periodontal tissue destruction. Therefore, eliminating periodontal pathogens, particularly
P. gingivalis, is essential for blocking the process of periodontitis [
5]. Local drug therapy is an effective supplementary means for the treatment of periodontitis, for it can reach areas that curettage instruments can’t. Local drug therapies have focused on chlorhexidine and antibiotics such as roxithromycin, spiramycin and metronidazole. Unfortunately, patients have shown poor compliance due to the adverse side effects of these drugs, such as bitter taste, pigmentation, diarrhoea and vomiting. The emergence of drug resistance and dysbacteriosis also limits their clinical application [
6,
7]. Therefore, it is urgent to develop novel drugs with few side effects to fight periodontal pathogens. Recently, antimicrobial peptides have become promising antimicrobial agents due to their rapid bactericidal activities and low risk of producing drug-resistant bacterial strains [
8]. However, the application of antimicrobial peptides has important drawbacks in vivo due to their high degradation characteristics in the presence of biological fluids [
9‐
12]. Although they demonstrate effective in vitro activities, many antimicrobial peptides have failed in vivo application tests.
P-113 (AKRHHGYKRKFH-NH
2) is a 12-amino-acid histidine-rich peptide derived from saliva protein histatin 5. Considering its easy degradability, we improved its chemical structure by replacing the histidine residues with bulky amino acid β-naphthylalanine and synthesized a novel antimicrobial peptide, Nal-P-113. This structural alteration enables Nal-P-113 to be resistant to high salt conditions, including PBS, saliva and bovine calf serum [
13,
14]. The antimicrobial and anti-biofilm effects of the novel antimicrobial peptide Nal-P-113 have been successfully confirmed in our previous in vitro study together with its nontoxic effect on rat gingival tissue. Nevertheless, to date, no study has focused on the impact of Nal-P-113 in inhibiting periodontitis in an animal model. In this study, we explored the effect of locally administered Nal-P-113 in a rat model of periodontitis by measuring alveolar bone loss, microbe colonization and immune-inflammatory modulation. We postulated that Nal-P-113 can prevent periodontal tissue destruction in experimental periodontitis models not only by controlling the amount of pathogens and total bacteria but also by modulating immune-inflammatory factors in gingiva, representing an auxiliary new approach for periodontitis therapy.
Methods
Synthesis, purification, analysis and dilution of antimicrobial peptide Nal-P-113
Nal-P-113, Ac-AKR-Nal-Nal-GYKRKF-Nal-NH
2, was synthesized from P-113 (AKRHHGYKRKFH-NH2) by replacing His 4, 5 and 12 with β-naphthylalanines based upon the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis protocol [
13,
15]. Electrospray mass spectrometry was used to identify the peptide, and high-performance liquid chromatography (HPLC) was used to assess its purity. The peptide concentration was determined with a UV spectrophotometer at 280 nm. Nal-P-113 was dissolved in sterile deionized water at concentrations of 6.25 μg/mL, 25 μg/mL, 100 μg/mL and 400 μg/mL.
Bacteria strains
The Gram-negative bacterium P. gingivalis strain W83 was obtained from the American Type Culture Collection (ATCC) and stored at the Department of Oral Biology, China Medical University. P. gingivalis strain W83 was cultured at 37 °C for 18 h in an anaerobic chamber with 85% N2, 10% H2, and 5% CO2 in brain heart infusion broth (Difco, Detroit, MI) supplemented with yeast extract (5 mg/mL), haemin (5 μg/mL) and vitamin K1 (0.2 μg/mL).
Ethical statement
The use of rats in this study complied with Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines. The experimental protocols were approved by the ethics committee of China Medical University.
Animals
Five-week-old, healthy, male Sprague Dawley (SD) rats weighing approximately 200–250 g were used. Rats were randomly assigned to one of the following groups (24 rats for each group): a, P. gingivalis W83 alone; P. gingivalis W83 with b, 6.25 μg/mL of Nal-P-113; c, 25 μg/mL of Nal-P-113; d, 100 μg/mL of Nal-P-113; e, 400 μg/mL of Nal-P-113; and f, control group without P. gingivalis W83 or Nal-P-113.
Induction of rat periodontitis models
All rats received azithromycin (10 mg/500 mL) for 4 days to reduce the original oral flora before the periodontitis model commenced in a controlled-temperature environment (22 ± 2 °C). This treatment was followed by a 7-day antibiotics-free period. At day 0, rats in all groups (a-f) were anesthetized with 10% chloral hydrate. A 0.2-mm wire was placed in the dentogingival area of both mandibular first molars. Rats in the experimental groups (a-e) received 1 × 109 CFU/mL (1.5 mL) of P. gingivalis W83 in the oral cavity and oesophagus when feeding twice per day (at 8:00 a.m. and 8:00 p.m.). Rats in group f, i.e., the control group, received 1.5 mL of BHI broth. Two hours later, Nal-P-113 (1 mL) was dripped into the rats’ periodontal pockets for 1 min in groups b to e. Sterile deionized water was dripped into the periodontal pockets in groups a and f. The above procedures continued for four weeks and were accompanied with inhalational anaesthesia with sevoflurane each time (Hengrui Pharmaceutical Co. Ltd., Shanghai, China).
Measurement of alveolar bone loss
Four weeks later, all rats (144/144) were healthy. After sacrifice by spinal dislocation, the rat mandibles were separated gently from muscle and soft tissue and exposed overnight to 8% hydrochloric acid. A stereomicroscope (SZX12, Olympus, Japan) equipped with a 12.5× objective was used for observing the morphology of each rat alveolar bone in every group. YC-2008 μ micro-measurement software (Aoka, Suzhou, China) was used for measuring the distance between the enamelo-cemental junction and alveolar bone crest along the axis of the teeth. For evaluating the average alveolar bone loss, three points were measured on the buccal and lingual parts along the axis of the teeth. The average bone loss was calculated for each tooth.
Bacterial colonization and distribution
Unilateral subgingival plaque and bacteria from the buccal and lingual mucosa were collected using sterile toothpicks and stored in 1× PBS at −80 °C.
P. gingivalis W83 and total bacteria loads were assessed using absolute quantitative real-time PCR. The primers used were as follows:
P. gingivalis W83: F: 5′-CATAGATATCACGAGGAACTCCGATT-3′ and R: 5′-AAACTGTTAGCAACTACCGATGTGG-3′. Universal: F: 5′-AACTGGA GGAAGGTGGGGA-3′ and R: 5′-ACGCCAACCTAGTGGAGGA-3′. Each reaction tube contained 20 μL of real-time PCR reaction mixture, including 10 μL of 2× SYBR® Premix Ex Taq
§, 0.4 μL of 50× ROX Reference Dye II, 0.8 μL of 10 μM primers, 0.4 μL of template DNA and 6.8 μL of ddH
2O. The amplification cycle conditions were 95 °C for 30 s, 40 cycles of 5 s each at 95 °C, and 34 s at 60 °C. The absolute quantities of the target bacteria were determined using standard curves for
P. gingivalis W83 and total bacteria. Briefly, the DNA concentrations of the target bacterial strains were serially diluted 10-fold using sterile water to construct standard curves and determine the exact numbers of DNA copies in the samples by calculating ct values. All experiments were performed in triplicate to ensure data accuracy. The data were analysed with ABI 7500 software version 2.0.5 (Applied Biosystems, Foster City, CA, USA) [
16].
Contralateral molars were fixed with 2.5% glutaraldehyde (BioChemika, Fluka), washed with PBS and subsequently dehydrated with ethanol. The processed samples were smeared onto a copper plate followed by gold sputtering, and images of the enamelo-cemental junction of the first molar were acquired by scanning electron microscopy (Inspect F50, FEI Company, USA) at 20,000× magnification. Six views were randomly selected from each animal.
Histopathologic evaluation of periodontal tissue
All mandibular periodontal tissues and tooth samples were divided in half: the left part was immediately cooled to −80 °C for western blotting, and the right part was fixed in 10% formaldehyde solution at 4 °C for one week. The samples were washed with distilled water and placed in 10% EDTA for decalcification for 40 days, rinsed in PBS buffer for 12 h, dehydrated, embedded and cut into 3–4 μm sections for immunohistochemical examination of IL-1β and TNF-α expression in periodontal tissues. Sections were obtained from the tissue that contained tooth, junctional epithelium and gingival tissues. Six views were selected from each sample for mean value calculations. Integrated optical density quantified using a MetaMorph Imaging Microscopic Image Analysis System (UIC, USA) was used to assess the expression of IL-1β and TNF-α. The results were recorded as means ± standard deviations (SD).
Western blot detection of gingival tissue
Gingival tissue at the buccal root bifurcation was used to examine the levels of IL-1β and TNF-α. A total of 20 mg gingival tissue sample was lysed on ice for 30 min with RIPA lysis buffer containing protease and phosphatase inhibitors (Sigma, St Louis, MO, USA). Supernatant proteins were obtained by centrifugation at 10,000×
g at 4 °C for 10 min. Total protein was measured with a BCA protein assay kit (Thermo Fisher Scientific, USA). Equal amounts (15 ng) of protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) at 100 V for 2 h and electrotransferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in TBST (20 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) containing non-fat dry milk for 2 h at room temperature and incubated overnight in a 1:800 dilution of anti-IL-1β and anti-TNF-α (Kaiji Biological Technology Co. NJ, China) for 2 h. After the membrane was washed three times, it was incubated with a 1:5000 dilution of goat anti-rabbit secondary antibody (Kaiji Biological Technology Co. NJ, China) for 1 h at room temperature and scanned using a fluorescence system with an anti-GAPDH antibody as an internal reference [
17]. A gel imaging analysis system (SYNGENE G: BOX ChemiXR5, USA) and Gel-Pro32 software (Media Cybernetic, Inc., USA) were used for densitometry. Statistical analysis was conducted after calculating integral value/internal reference integral value ratios.
Statistical analysis
All experiments were performed in triplicate for each condition. An ANOVA was used to compare the significance among groups (SPSS Inc., Chicago, IL, USA). Pearson linear correlation analysis was used to calculate the correlation between P. gingivalis, total bacteria DNA replication and the expression of IL-1β and TNF-α cytokines. The data are presented as the means ± standard deviation. A P-value <0.05 was considered statistically significant.
Discussions
Periodontitis is a complex disorder dependent on many factors, but inflammatory cell accumulation induced by microorganisms in periodontal tissues is considered the primary causative factor.
P. gingivalis, a member of the red complex, is considered one of the most important pathogenic factors of periodontitis, which produces several virulence factors, including lipopolysaccharide, capsules, fimbriae and a group of proteolytic enzymes that damage periodontal tissue [
19‐
21]. In addition,
P. gingivalis can trigger changes to the amount and composition of the oral commensal microbiota, leading to inflammatory periodontal bone loss [
22]. In this condition, the removal of periodontal pathogens, such as
P. gingivalis, is an effective method to block chronic periodontitis. Although periodontal pathogens are required for disease initiation, the extent and severity of periodontal damage depend on the nature of the host response to bacterial challenge. The development of a novel therapeutic strategy combining both the antibacterial activity and the host-modulatory effect of a new drug could increase the likelihood of successfully managing periodontitis [
23]. Some studies have shown that antimicrobial peptides, in addition to exhibiting a direct antimicrobial activity, possess a wide range of immunomodulatory properties [
24]. In animal models, researchers have verified that antimicrobial peptides are effective in oral
Candidiasis infection, lung infection and sinusitis [
25‐
27], but few studies have focused on periodontitis therapy in vivo. The present study evaluated, for the first time, the anti-inflammatory and antimicrobial impact of a novel antimicrobial peptide Nal-P-113 in preventing the progression of periodontitis in a concentration-dependent manner.
In the current investigation, morphometric analysis demonstrated that therapy with Nal-P-113 promoted a significant decrease in bone loss compared to high periodontal breakdown in the non-treated group. The enamelo-cemental junction area served as an observation locus to explore the bacterial distribution on the rat tooth surface. Scanning electron microscopy showed that bacteria adhering to the surfaces of teeth in a scattered distribution were impacted by high concentrations of Nal-P-113. Consistent with the morphological images, quantitative real-time PCR confirmed the results that treatment with 100 μg/mL or 400 μg/mL of Nal-P-113 reduced the amount of
P. gingivalis and total bacteria loads compared with those in the control group (
P < 0.05). A previous study showed that Nal-P-113 inhibited bacterial growth even caused bacterial death by permeabilizing and/or forming pores within cytoplasmic membranes in a concentration-dependent manner in vitro [
14]. The present study demonstrated the ability of the novel antimicrobial peptide Nal-P-113 to inhibit biofilm formation, local inflammatory response and alveolar bone loss in an experimental rat periodontitis model.
Bacteria are essential for the development of periodontitis but not sufficient to cause disease alone. For periodontitis to develop, a susceptible host is required [
28,
29]. Page et al. in 1999 reported that periodontal disease was characterized by high concentrations of matrix metalloproteinases (MMPs), cytokines, and prostaglandins in the periodontal tissue.23 Certain cytokines, including IL-1β and TNF-α, play a critical role in the pathogenesis of periodontitis. IL-1β and TNF-α are both pro-inflammatory cytokines involved in the induction of several other inflammatory mediators in periodontal inflammation, such as IL-6, chemokines, MMPs and prostaglandin E
2 [
30]. Furthermore, IL-1β and TNF-α can induce secondary mediators, resulting in inflammatory response expansion, connective tissue destruction and osteoclastic bone resorption [
31]. To explore the host involvement in the disease process, immunohistochemical techniques and western blotting were used to detect IL-1β and TNF-α expression in the periodontal tissue of rats. The results showed that Nal-P-113 reduced IL-1β and TNF-α levels in periodontal tissue in a concentration-dependent manner. Many scholars have also confirmed that other antimicrobial peptides were capable of modulating innate immunity [
32‐
34]. For example, Pingel et al. reported that human beta-defensin (hBD)-3 significantly decreased the secretion of IL-6, IL-10, GM-CSF and TNF-α by human myeloid dendritic cells stimulated with recombinant
Porphyromonas gingivalis hemagglutinin B [
34]. LL-37 induced production of pro-inflammatory cytokines IL-4, IL-5 and IL-1β from human mast cell [
35] or by activation of P2X7 receptor from monocytes [
36]. Some immune cells, such as macrophages or mast cells, can express G protein-coupled receptors and have a role in both innate immune responses and antimicrobial activation [
24]. But in our study, we didn’t find Nal-P-113 alone change IL-1β or TNF-α levels in macrophages (Additional file
1: Figure S1). Other immune cells, such as mast cells, monocytes and neutrophils interact with Nal-P-113 should be investigated in the future studies.
It is worth noting that the TNF-α level detected by western blotting was slightly increased when 25 μg/mL of antimicrobial peptide Nal-P-113 was used. One potential explanation might be that antimicrobial peptides themselves can induce a slight up-regulation of TNF-α expression [
37]. Our results showed that 25 μg/mL or 400 μg/mL Nal-P-113 increased TNF-α expression levels in human periodontal ligament stem cells, but not in immortalized human gingival epithelial cells or macrophages (Additional file
1: Figure S1). The specific mechanism is not clear, maybe it decreased the MyD88 and AKT levels to affect the NF-κB signaling pathway and modulated TNF-α response [
38]. To some extent, the host-defence mechanisms are sustained by an large network of anti- and pro-inflammatory mediators that may exert antagonistic and/or synergistic biological activities, further studies are required to better understand the role of Nal-P-113 in the modulation of immune-inflammatory responses.
Based on the correlation analysis, IL-1β and TNF-α levels in gingival tissue were positively correlated with P. gingivalis but not with the total amount of bacteria. Therefore, in the process of host-microbial interactions, P. gingivalis played a more important role in promoting IL-1β and TNF-α expression than other oral bacteria. We surmise that the antimicrobial peptide Nal-P-113 might interrupt pre-inflammatory cytokine expression by reducing P. gingivalis loads in the rat oral cavity.
Conclusions
Overall, the present investigation demonstrates that a novel antimicrobial peptide, Nal-P-113, not only overcomes the degradation susceptibility of antimicrobial peptides in vivo but also exerts potent preventive effects on rat periodontitis. This peptide inhibits P. gingivalis-induced local inflammatory response and alveolar bone loss by reducing the amount of bacteria directly and down-regulating pro-inflammatory molecules such as IL-1β and TNF-α. Though further studies are needed to determine the exact mechanism of host-microbial interactions in periodontitis treated with antimicrobial peptides, these agents have the potential to be used as a new preventive or therapeutic strategy for chronic periodontitis in the clinic.