Binding Protein A12, Receptor for Advanced Glycation Endproducts, and Nuclear Factor-Κ B Expression with Inflammation in Pulp Tissues from Tooth Caries
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Received: 27 November 2016 | Accepted: 22 June 2017 DOI: 10.1111/jicd.12290 ORIGINAL ARTICLE Endodontics Association of S100 calcium- binding protein A12, receptor for advanced glycation endproducts, and nuclear factor-κ B expression with inflammation in pulp tissues from tooth caries Mohammad M. Y. Khorasani1 | Pouria Andam-Shahsavari1 | Nahid Zainodini2 | Hossein Khoramdelazad3 | Reza Nosratabadi2,4 1Department of Endodontics, School of Dentistry, Rafsanjan University of Medical Abstract Sciences, Rafsanjan, Iran Aim: S100 calcium- binding protein A1 (S100A12) is a pro- inflammatory molecule 2 Immunology of Infectious Diseases Research which is secreted during inflammation and induces chemotaxis and the production of Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran pro- inflammatory cytokines via interaction with receptor for advanced glycation end- 3 Molecular Medicine Research products (RAGE) and subsequent, activation of nuclear factor-κ B (NF-κ B). The present Center, Rafsanjan University of Medical study was designed to determine the expression levels of S100A12, RAGE, and NF- B Sciences, Rafsanjan, Iran κ 4Department of Immunology, Faculty of in the inflamed pulp of carried teeth. Medicine, Rafsanjan University of Medical Methods: In the present study, mRNA from 50 inflamed pulp and 50 healthy pulp Sciences, Rafsanjan, Iran were used for expression studies using real- time polymerase chain reaction. The ex- Correspondence pression levels of S100A12, RAGE, and NF- κB were compared between inflamed and Dr Reza Nosratabadi, Immunology of Infectious Diseases Research Center, healthy tissues. Rafsanjan University of Medical Sciences, Results: The results revealed that the expression of S100A12, but not of RAGE or NF- Rafsanjan, Iran. Email: [email protected] κB, was significantly decreased in inflamed pulp when compared to healthy pulp. mRNA levels of RAGE were also increased in the inflamed pulp taken from men when Funding information Rafsanjan University of Medical Sciences, compared with women. Grant/Award Number: 9/991 Conclusion: The results suggest that S100A12 does not participate in the induction of inflammation in dental pulp. However, RAGE can participate in the inflammation in the pulp of males. KEYWORDS nuclear factor-κB, pulp, receptor for advanced glycation endproducts, S100 calcium-binding protein A1, tooth 1 | INTRODUCTION transcription factors in different cell systems.4 The treatment of im- mune related diseases can utilize specific antagonists of inflammatory One of the important complications of dental disease that can lead molecules, such as PRR.5 However, before such strategies can be ap- to several dental disorders is inflammation,1 and the main cause of plied to the management of dental caries and pulp inflammation, it is pulp inflammation is dental caries, which can lead to pulp necrosis.2 important to understand the mechanisms that lead to inflammation There are several inflammatory factors, such as pro- inflammatory cy- in the pulp. tokines, that play a role in the induction of inflammation in the pulp.3 Innate immune receptors, such as PRR, induce innate immune cells Several pathogen recognition receptors (PRR) and their correspond- via recognition of endogenous damage- associated molecular pattern ing intercellular signaling molecules can activate pro- inflammatory (DAMP) or exogenous pathogen- associated molecular pattern (PAMP) J Invest Clin Dent. 2017;e12290. wileyonlinelibrary.com/journal/jicd © 2017 John Wiley & Sons Australia, Ltd | 1 of 5 https://doi.org/10.1111/jicd.12290 2 of 5 | KHORASANI ET AL. ligands, and subsequently increase the expression of pro- inflammatory The current study was approved by the ethics committee of genes.6 Rafsanjan University of Medical Sciences, Rafsanjan, Iran (no. S100 calcium- binding protein A1 (S100A12) is an inflammatory IR.RUMS.REC.24), and the participants completed consent forms for calcium- binding protein that is produced and secreted by granulocytes, voluntary participation in the investigation. especially neutrophils.7 Secreted S100A12 has chemotactic effects, and can induce intracellular signaling via its corresponding receptor, 2.2 | Pulp preparation the receptor for advanced glycation endproducts (RAGE).8 Not surpris- ingly, S100A12 is a DAMP that is produced in several immune- related The teeth were split and arranged into two parts, longitudinally, for diseases. S100A12/RAGE binding leads to the activation of intracellu- pulp preparation. The procedures for the pulp preparation have been 9-11 lar signaling that subsequently results in the phosphorylation of Iκκ, the published previously. An excavator have been used for separation inhibitor of nuclear factor- κB (NF-κ B), which consequently causes Iκκ of pulp tissues. The separated tissues were kept in artificial CSF under 8 degradation and the activation of NF- κB. Therefore, S100A12, RAGE, sterile condition for using in further analysis. and NF- κB can be considered important candidates in the mechanism of inflammation in pulp. The aim of the present study was to evaluate 2.3 | RNA extraction, cDNA synthesis and real- time the expression levels of S100A12, RAGE, and NF- B in inflamed and κ polymerase chain reaction condition non- inflamed healthy pulp derived from carious and healthy teeth. For extraction of the whole RNA, the pulp tissues were made brit- tle by immersion in liquid nitrogen for 30 minutes and quickly placed 2 | MATERIALS AND METHODS in sterile RNase- free test tubes. Homogenized pulp were used to purify total mRNA. The full procedure for RNA extraction, cDNA 2.1 | Patients synthesis, and real- time polymerase chain reaction conditions are de- 12 In the present cross- sectional study, mRNA was extracted from 50 scribed elsewhere. β- actin was used as a housekeeping gene and the inflamed (tooth caries) and 50 healthy pulp from healthy teeth, and sequences of primers were as follow: S100A12 (forward): 5′- GGAGG assessed for S100A12, RAGE, and NF- κB expression levels. The vital- GAATTGTCAATATC- 3′, S100A12 (reverse): 5′- ATCTTGATTAGCATC ity of the pulp was evaluated using cooling, heating, and electric pulp CAGG- 3′; RAGE (forward): 5′- GACCCTGGAAGGAAGCAG- 3′, RAGE test tests. Participants treated with immune modulators or immune (reverse): 5′- CCCCTTACACTTCAGCACC- 3′; and β- actin (forward): suppressors, or those with allergic conditions, autoimmune or infec- 5′- GGCACCCAGCACAATGAAG- 3′, and β- actin (reverse): 5′- CCGA tious diseases, habit of smoking, addiction, or had undergone surgery TCCACACGGAGTACTTG- 3′. within the past 2 weeks or were pregnant were excluded from the study. The experimental groups included asymptomatic and decayed 2.4 | Statistical analysis teeth, in which the depth of decay was approximately 1- 2 mm, as as- sessed by radiography. Teeth with dental caries were extracted for Analyses were done using t tests and χ test to analyze age and sex, prosthetic and periodontal disease reasons. The control teeth were respectively. Mann- Whitney U test was used to analyze the expres- extracted for orthodontic reasons, and were obtained from healthy sion levels of S100A12, RAGE, and NF-κ B. The software used was individuals. The cases and controls were matched regarding age, sex, SPSS software package version 18 (SPSS, Chicago, IL, USA). Values and ethnic backgrounds (Table 1). Immediately after tooth extraction, were considered significant when P<0.05. the tips of the root (apical 3- 5 mm) were removed, and the teeth were gathered in artificial cerebrospinal fluid (CSF), which also included di- ethyl pyrocarbonate to inactivate RNase. Samples were kept at −20°C 3 | RESULTS until RNA extraction could be completed. The analysis showed that the two groups, from which pulp of teeth TABLE 1 Demographic data on patients with inflamed and with dental caries and the healthy pulp were extracted, were not dif- healthy pulp ferent in relation to age (P=0.271) and sex (P=1.0). Variables Case Control P- value Our results showed that the mean relative expression levels of S100A12 in the inflamed pulp was 0.0003 (range: 0.0000- 0.0007), Sex and in the healthy, non- inflamed pulp sample, it was 0.0016 (range: Female 25 25 0.579 0.0001- 0.0623). The statistical analysis showed that the differences Male 25 25 in expression between the groups was significant (P=0.038) (Figure 1). Age (years) The data analysis showed that the relative expression level of RAGE Mean 23.3 22.1 0.271 in the inflamed pulp was 0.0798 (range: 0.0133- 2.9795) compared to SD 0.58 0.91 the healthy, non- inflamed pulp, which was similar at 0.1337 (range: SD, Standard deviation. No significant differences were between groups 0.0293- 0.5549). These two groups showed no significant differences regarding age and sex. (P=0.912) (Figure 1). The mean relative expression of NF-κ B, in the KHORASANI ET AL. | 3 of 5 FIGURE 1 Relative mRNA expression levels of (A) S100A12; (B) RAGE and (C) NF-κB in inflamed and healthy pulps. The figure shows that expression levels of S100A12, but not RAGE and NF-κB, were significantly decreased in inflamed pulps in comparison to healthy pulp inflamed pulp was equal to 0.0890 (range: 0.0006- 1.3783), and in the FIGURE 2 Comparative mRNA expression levels of (A) S100A12; healthy, non- inflamed pulp, it was equal to 0.521 (0.0212- 0.1047). (B) RAGE and (C) NF-κB in the inflamed pulps of males versus Data are shown as the mean of the first quarter to the third quarter, females. The figures demonstrate that the expression levels of RAGE, and the statistical analysis showed that the difference between the but not S100A12 and NF-κB, were significantly increased in the inflamed pulps of males in comparison to females two groups was not significant (P=0.247) (Figure 1). The results of the present study also showed that, in inflamed pulp, the relative expression of RAGE (P=0.015), but not S100A12 (P=0.597) diseases and can exacerbate inflammation.13 The results of the current or NF- κB (P=0.112), was significantly increased in males when com- study demonstrated that mRNA levels of S100A12 were significantly pared with females (Figure 2).