Expression of Notch signal in rat model of chronic periodontitis

1Journal of Hainan Medical University 2019; 25(6): 1-5Journal of Hainan Medical UniversityCorresponding author: Bao-Liang Wang, Department of Stomatology, Linyi PeopleHospital, Linyi, 276000, Shandong Province, China.Fund Project: 1. Project supported by the National Natural Science Foundation of Hainan Province; 2. Science develop Foundation of Hainan Medical University. Project No: 1. 20158318; 2. HY2014-009.1. IntroductionChronic periodontitis is a commonly occurring oral infectiousdisease in dental sustentacular tissues such as gingiva, parodontium and alveolar bones [1]. With the further research of its pathogenesis,gradually it is found that the progressive loss of periodontal support tissues in chronic periodontitis is not directly caused by microbial infections, but indirectly caused by the host in the immune response to infection microorganisms and their toxic products [2]. In the immune response, CD4+ T cells are a kind of important cells involved in [3]. They can differentiate into many subtypes underdifferent microenvironment. Th17 cells and regulatory T cells (Tregs)play an important role in promoting or inhibiting the inflammatory reaction [4]. The differentiation of Th subsets is regulated by multiple signal transduction pathways. Notch signaling pathway is currently recognized as an important signal transduction system regulating cell differentiation [5], including cell proliferation, differentiation and apoptosis [6].To establish a chronic periodontitis model of SD rats and detect the expression of Notch in peripheral blood mononuclear cells (PBMCs) of chronic periodontitis model was detected by real-time quantitative polymerase chain reaction (RT-PCR), intracellular staining and flow cytometry. It is hoped that we can find out the potential significance of Notch related molecules in the development of chronic periodontitis and provide a theoretical basis for seeking new methods of treating chronic periodontitis.Li Zhang et al./ Journal of Hainan Medical University 2019; 25(6): 1-522. Materials and methods2.1 Experimental animalsSPF-grade 6-8-week-old male SD rats, weighing about 20–24 g, were purchased from Shanghai Stryker Laboratory Animal Co.,Ltd.. The feeding environment was temperature range 20-25 ℃ and relative humidity range 40%-70%. Rats were maintained with free access to water and food (SPF solid feed) for six days prior to the experiment.2.2 Gingivalis cultureDissolved the Porphyromonas gingivalis lyophilized powderin 0.2 mL culture medium and inoculated on blood agar plate at 37 ℃instrict anaerobic for 7-10 d. Picked up a loop of gingivalis from the plate and seeded it into a fresh liquid TSA medium containing 5% defibrinated sheep’s blood. Strictly anaerobic at 37 ℃ and 100 r/min Shaker training 3-5 d. Using 1 mL pipette to absorb 0.5 mL bacterial liquid and slowly poured into a sterile tube containing4.5 mL physiological saline and mixed on a homogenizer to makea 1:10 sample homogenate. Take another 1 mL pipette, according to the above sequence of operations, 10-fold increasing sample homogenization was made. According to the estimation of the sample concentration, 2-3 samples were selected and 10-fold incremental dilution was used to extract 0.5 mL bacteria solution in sterile dishes, each dilution made two dishes. At the same time, 0.5 mL blank diluents were added to two aseptic plates as blank control. Poured 15-20 mL 46 ℃ -blood agar medium into a plate and turned the flat to make it mixed evenly. This process will be completed in 15 min. After the agar solidified, the plate was turned over and anaerobically cultured for 7-10 d, and all the colonies on the plate were counted.Main reagents: tryptone (Sinopharm Chemical Reagent Co., Ltd.); Porphyromonas gingivalis lyophilized powder, soy peptone (Hangzhou Microbial Reagent Co., Ltd.); sodium chloride (Sinopharm Chemical Reagent Co., Ltd.); agar powder, (Qingdao Haibo); sterile defibrinated sheep blood (Nanjing Mao Jie Microbial Technology Co., Ltd.).2.3 Experimental grouping and modelingTwenty-seven adult male SD rats were randomly divided into two groups: normal control group (group N, 12) and experiment group (group M, 15). In addition to normal feeding group N, group M: 10% high glucose diet + ligation of left maxillary second molar neck + gingivalis; rats in experiment group were anesthetized by 10% chloral hydrate, fixed, the assistant used a cloth towel forceps to hook up the rats’ tongues and jaws and open their mouths, exposed molars of both sides. First, the left maxillary second molar was separated from the root surface, and then the second molar neck was ligated with 6-0 mousse wire, and 10% sugar water was drinking from the day of the experiment. Eight weeks after operation, 100 µg of gingivalis fluid was injected into the sulcus once a week for 4 weeks. The concentration was 1ˑ108/mL. The rats in the normal control group were not treated. 12 weeks after model establishment, taken blood from the abdominal aorta and EDTA anticoagulant was used to separate the PBMC, and the left side was fixed with 4% formaldehyde in the upper jaw molar for preparation. By the end of the experiment, no animal died.2.4 Observation projectThe rats' behavior, status, signs, ingestion, water intake, stool, urine and body weight were observed. The gingival index, tooth mobility and periodontal pocket depth were recorded. Twelve weeks after experiment, the animals were sacrificed, their maxillas were separated and the molars segments were harvested. 100 mL/L formaldehyde fixed, mixed acid decalcification, gradient ethanol dehydration, paraffin embedding, slice thickness 5 μm, conventional HE staining, light microscopy.2.5 Notch protein expression(1) Mixed 2.5 mL anticoagulant and 2.5 mL sample diluents; (2) Took acentrifuge tube, added the same amount of separation fluid and sample, 2 500 r/min centrifugation 15 min. At this time, the liquid in the centrifuge tube was divided into 4 layers from the top to the bottom; (3) Collected the second layer cells into tubes containing 15 mL PBS, after fully mixed, centrifuged at 2 000 r/min for 10 min; (4) washed the precipitation twice with PBS, the cell sedimentation was collected,namely PBMC; (5) fixed the cells and washed with 3 mL stain buffer (pH 7.4 phosphate buffer + 2% fetal bovine serum + 0.1% stack Sodium nitrate) twice; (6) Resuspended the above cell precipitation with PBS to 105-108/mL; (7) Took the above suspension 4 mL, collected cells in each group, blank (without antibody), IgG control Group, notch antibody group (notch 2, notch 3, notch 4) were set up respectively. After one hour of incubation, the fluorescent secondary antibody was added and protected from light and incubated at 4 ℃ for 30 min. FCM was performed on the machine and notch 2/3/4 was detected on the FL-1A channel. 2.6 Total RNA extraction and reverse transcription of whole blood in ratsTook 3 mL anticoagulated whole blood, diluted with an equal volume of saline and separated mononuclear cells by 5 mL lymphocyte separation solution. Centrifuged at 1 000 g for 30 min, collected mononuclear cell layer. The solution was centrifuged at 350 g for 10 min to wash the cells twice (pH 7.2, PBS), and the cell sedimentation was collected. Total ribonucleic acid (RNA)Li Zhang et al./ Journal of Hainan Medical University 2019; 25(6): 1-53was extracted using RNA extraction kit. The complementary deoxyribonucleic acid (cDNA) was synthesized from 1 µg total RNA of each sample using the PrimeScriptTM RT Master Mix reverse transcriptase kit (Takara Bio, USA).2.7 Quantitative PCRThe expression of the related genes in the fluorescence quantitative PCR analysis was carried out by the reverse transcriptase cDNA template. Relative quantificationof transcript levels was performed by the 2-ΔΔCt method.Table 1.Fluorescence was performed using reverse transcribed cDNA as a template, as a specific primer for the four Notch receptors and the internal reference in2.8 Statistical methodsThe data was expressed as Mean ± sd. The software SPSS 17.0 was used for statistical analyses. The t-test was used for intergroup data comparison. P<0.05 denote statistical significance.3. Results3.1 Macroscopic observations group MDue to the influence of general anesthesia and local modeling, animals in Group M all showed appetite loss, loose fur disorder, dark yellow, weight loss and other manifestations. Group M: Swelling and congestion, ulcers, erosion, touch bleeding of the left maxillary first molar gingival tissue; alveolar bone absorption, visible root exposure, combined with epithelial proliferation to the root, deep periodontal pocket formation. At the same time, three rats were infected with different degrees of interspace infection due to the diffusion of endotoxin in the modeling process. Group N: normal appetite, weight gain, no abnormal spirit, normal periodontal tissue.3.2 Histological observationExperiment group: junctional epithelium root proliferation, mesh-like, deep periodontal pocket formation. Telangiectasia, hyperemia and infiltration of lymphocytes and leukocytes were seen in the connective tissue. The crest of the alveolar ridge and the deep alveolar bone were absorbed, showing obvious osteoclasts and bone resorption lacuna (Figure 1). Control group: no telangiectasia and hyperemia in the connective tissue; the periodontal ligament cells were arranged in a consistent manner, and the surface of the alveolar bone was smooth with no osteoclasts and bone resorption lacuna(Figure 2).(HE×100) (HE×200)Figure 1. Experiment group (M) histomorphology.(HE×100)(HE×200)Figure 2. Control group (N) histomorphology.3.3 Gingivalis culture resultsGram-negative bacilli, which was colonies when they grew in the frozen hemolytic medium (Figure 3).Figure 3. P. gingivalis microscopic examination.Li Zhang et al./ Journal of Hainan Medical University 2019; 25(6): 1-543.4 Changes of Notch signaling pathway protein expressionThe flow test results showed the Notch receptor protein expression in Table 2. The expression of Notch 1, Notch 3 and Notch 4 protein in the experimental group were significantly lower than that in the control group (P<0.01). but Notch 2 protein was significantly higher (P<0.05).Table 2.3.5 Notch receptor gene expression changesThe expression of Notch1, Notch2, Notch3 and Notch4 mRNA in the experiment group was significantly lower than that in the control group (P<0.01) (Table 3).Notch 2 mRNA were significantly higher (P<0.05).Table 3.Expression of Notch gene.4. DiscussionPeriodontitis is a kind of disease of connective tissue attachment loss and alveolar bone damage caused by bacteria of dental plaque biofilm[7] and their products. The main link in the process of periodontitis is the indirect immune response mediated by the bacterial infection of the host T cells[8]. The Notch signaling pathway is a pathway that controls cell fate through local cell interactions[9]. It consists of Notch receptors, ligands, and downstream signaling molecules and nuclear response factors. Experiments showed that Notch1 signal was activated during Th17 differentiation in mice and humans[10]. Blocking Notch signaling can significantly decrease the level of Th17-associated inflammatory cytokines[11], suggesting that Notch signaling plays a key role in Th17 differentiation. Using chromatin immunoprecipitation, study found that IL-17 and retinoic acid-related orphan receptor γt (RORγt) in Th17 cells are directly regulated by Notch signaling[12]. Blocking Notch signaling can decrease IL-17 expression and delay the progression of Th17-stimulated immune diseases in EAE animals, further demonstrated the important role of Notch signaling in Th17 cell differentiation[13]. On the other hand, in vitro experiments showed that parathyroid hormone-related peptide (PTHrP) can stimulate the expression of Notch signalingligand-Jagged 1 in periodontal ligament cells, participate in the regulation of RANKL-induced osteoclastogenesis and differentiation[14], while the bone formation factor can improve the Notch of periodontal ligament cells, the expression level of signal ligand DII-1[15,16]. Jagged 1 inhibits osteoclast differentiation by binding to Notch 1 receptor; whereas DII-1[17] and Notch 2 receptor binding promotes the process of osteoclast precursor cells into osteoclasts[18]. Compared with the Jagged/Notch 1 negative regulation, positive regulation effect of DII-1/Notch2 was the dominant effect[19,20].The success of modeling was preliminarily explained, and the expression of four Notch receptors in mouse BMC was detected by RT-PCR and flow cytometry respectively from the mRNA and protein levels. The results showed that the expression of Notch 1, Notch 3 and Notch 4 protein in peripheral blood of mice with chronic periodontitis was significantly lower than that of the control group (P<0.01); the expression of Notch 1, Notch 3 and Notch 4 mRNA of the experiment group was significantly lower than that of the control group (P<0.01). However, Notch 2 mRNA and percentage of cell expression were significantly higher(P<0.05). The results show that Notch gene in chronic periodontitis model mouse at mRNA and protein levels were obviously changed, suggesting that Notch gene may be involved in the pathogenesis of chronic periodontitis, and through the signal path that affect the differentiation function in vivo cells, immuno-inflammatory responses in the rats with chronic periodontitis are induced and different Notch gene different trends may have a different impact on the development of chronic periodontitis mechanism, which provides ideas for the further study of chronic periodontitis mechanism and to seek treatment at the molecular level. However, the specific functions and mutual influence mechanism of different Notch genes in the development of chronic periodontitis remains to be further studied.Conflict of interest statementWe declare that we have no conflict of interest.References[1] C osgarea R, Juncar R, Heumann C. 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