Effect of Periodontal Treatment on Inflammasome Proteins in Periodontal Diseases

New insights into the first line of immune response to infection and stress signals at the cellular level have reported the participation of cytoplasmic nucleotide-binding domain-like receptors containing multi-protein complexes named inflammasomes. The inflammasomes play a key role in innate immunity by regulating maturation of proinflammatory cytokines of the interleukin 1beta and 18. The inflammasome depends on assembly of a sensor, for instance nod-like receptor family pyrin domaincontaining protein (NLRP), with an adaptor,apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), allowing recruitment and activation of an inflammatory caspase-1. Although different inflammasomes have been described, the NLRP3/ASC/caspase-1 multi-protein complex, known as NLRP3 inflammasome, has been most intensively studied.

Sample size was calculated based on a previous study concerning levels of inflammasome complex proteins in periodontal diseases.To maintain estimates at an optimal level of precision,minimize impact of exclusions and dropouts, and establish significant differences in results at a 95% confidence level, alfa value=0.05, and 83% power.Therefore, the study sample included a total of 45 patients( 15 periodontally healthy, 15 gingivitis, 15 sStage III Grade C Periodontitis). The whole mouth clinical periodontal examination included measurement of probing depth (PPD), clinical attachment level (CAL), presence of bleeding on probing (BOP), gingival index (GI), and plaque index (PI) at 6 sites per tooth, except the third molars. The presence and type of the alveolar bone loss were assessed on the digital panoramic radiograph in each participant, which was supplemented with periapical radiographs if necessary. Periodontal status of each patient was evaluated by a single calibrated periodontists with a manual probe. The diagnosis of periodontitis or periodontally health was determined according to the 2017 World Workshop on Classification of Periodontal and Peri-Implant Diseases and Conditions. Periodontally healthy individuals (n=15) in the control group had no sites with PD >3 mm and CAL >2 mm and also no radiographic evidence of alveolar bone loss. BOP was <10% in the whole mouth. Healthy group also exhibited no history of periodontitis. Gingivitis patients had PD≤3 mm, intact periodontium, no destruction in alveolar bone radiographically and BOP>10%. Stage III grade C patient had clinical attachment loss (CAL)≥ 5, radiographic bone loss (RBL) extending to the middle third of root and beyond, ≤ 4 tooth loss due to periodontitis, percentage of bone loss by age is >1.

Treatment The recruited periodontitis and gingivitis patients received conventional quadrant scaling and root planning (SRP). SRP was performed by the same clinician using ultrasonic inserts and manual periodontal curettes. Re-evaluations were performed at 1 and 3 months following the completion of the SRP. No periodontal intervention was carried out in the periodontally healthy controls.

Saliva and Serum Sampling A total of 5 mL of unstimulated whole saliva was collected by passive drool method between 9:00 and 10:00 am. The participants were advised to avoid food consumption for three hours before sample collection. The participants were seated upright and saliva was collected over a period of 5 minutes with instructions to pool saliva in the floor of the mouth and passively drool it into a sterile glass beaker. The saliva samples were centrifuged at 5000 rpm for 10 minutes at room temperature, and supernatants were collected and stored at -80°C. A total of 5 mL of blood was collected from the antecubital fossa by venepuncture method. Serum was isolated from the blood by centrifuging at 3000 rpm for 20 minutes followed by its rapid transfer to a sterile polypropylene tube and storage at -80°C.

Biomarker Immunoassays: Serum and salivary samples of IL-1beta, IL-18, NLRP3, ASC and Caspase-1 were measured by ELISA using commercial kits.

Statistical Analysis All statistical analyses were carried out with the standard statistical software package. For the intra-group comparisons, if the data were not normally disturbed, Friedman test and the Dunn test with the Bonferroni correction were used to analyze the change between baseline and 1 month and 3 months after treatment. For inter-group comparisons, Mann-Whitney U test for normally and non-normally disturbed data. The Spearman's rank correlation test was used to detect the correlations of biochemical parameters with clinical parameters and each others in diseased group before and after treatment. All tests were performed at significance level of P <0.05.