Evaluation of insulin and inflammatory pathways in skeletal muscle tissue of adult rats, offspring of rats with true endodontic-periodontal lesions

Author(s)

Bianca Elvira Belardi

• Document type: Master's Dissertation
• Press: Araçatuba. 2022-04-20.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Odontologia. Araçatuba
• Defense date: 2022-04-01
• Advisor: Doris Hissako Matsushita

Abstract

Fetal programming suggests that adverse stimuli when applied during early fetal development can alter offspring metabolism, increasing the risk of disease in adulthood. Studies have shown that both maternal periodontal disease (PED) and apical periodontitis (AP) in rats promote insulin resistance (IR) in their adult offspring. However, studies investigated the effects of maternal endodontic-periodontal lesions (EPL) on offspring health are scarce and, in these cases, the impact could be even more. The pulp and periodontium are known to have a close bond, and are intimately connected by anatomical structures such as the apical foramen, accessory canals, and dentinal tubules, which may interfere with each other's health. True EPL is an inflammatory process that extends to the pulp and periodontal tissue simultaneously, each with its origin. In addition, these pathologies, evaluated in isolation, are associated with increased tumor necrosis factor-alpha (TNF-alpha) that can stimulate IkappaB kinase (IKK) and c-Jun N-terminal protein kinase (JNK), which promote phosphorylation of insulin receptor substrate 1 (IRS-1) in serine residues, resulting in attenuation of the insulin signal (IS), contributing to IR in adult offspring. In this context, it has become essential to investigate if true EPL can promote more insulin resistance in its adult offspring In view of this, the aim of this study will be to evaluate in female rats with EPL: 1) bone markers such as tartrate-resistant acid phosphatase (TRAP) and osteocalcin (OCN) at the site of maternal injuries. Furthermore, the oaims of this study were evaluated in adult rats, offspring of rats with EPL: 1) glycemia and insulinemia; 2) insulin pathways; 3) inflammatory pathways in the gastrocnemius muscle (MG). Therefore, the 28 Wistar rats (2 months old) were distributed in 4 groups: 1) control rats; 2) rats with a AP induced in the first right upper molar; 3) rats with an induced PED in the right upper second molar; 4) rats with EPL, in which PED was induced in the upper right second molar, and AP in the upper right first molar. The AP was induced by exposing the pulp tissue to the oral environment, using a carbon steel drill with a 0.1 mm sphere at the end. PED was induced by ligation with sterile silk thread. After 30 days of inducing oral inflammation, rats from all groups were placed for mating with healthy rats. After weaning, the mother rats was sacrificed and the hemimaxyls (on the right side) will be collected to perform the following experiment: 10 1) Evaluation of TRAP and OCN at the site of maternal injuries by the immunohistochemical technique. Statistical analysis was performed using the Mann–Whitney test and Student's t test (p<0.05). When the male offspring of all rats completed 75 days of age, the following experiments were performed: 1) Assessment of body weight and food intake; 2) glycemia, insulinemia and the HOMA-IR index; 3) total TNF-α content in MG; 4) IKKα/β and JNK phosphorylation status in MG; 5) pp185 (tyrosine) and IRS-1 (serine) phosphorylation status in MG by western blotting method. Statistical analysis was performed using variance analysis (One way for analysis of experiments in items 1, 2, 3 and 4; Two way for item 5, described above) followed by Tukey's test (p<0.05. Histological and immunohistochemical analyzes of the site of maternal lesions showed that: 1) there was an increase in the inflammatory infiltrate in the EPL group when compared to the PED group; 2) there was no difference in this parameter between the groups of mother rats with AP and EPL; 3) a higher pattern of immunostaining for OCN was observed in the EPL group when compared to the AP group; 4) no statistical differences were found in this parameter between the groups of mother rats with EPL and PED; 5) There was greater immunostaining for TRAP in the EPL group compared to the AP and PED groups. Furthermore, the analyzes performed on the offsprings showed that: 1) maternal PED and EPL promote low birth weight (LBW) in the offspring; 2) there was no LBW in the AP-o group; 3) there were no statistical differences in body mass and food intake among the groups evaluated during the 75-day period; 4) the AP-o, PED-o and EPL-o groups showed an increase in insulinemia and IR in relation to the CN-o group; 5) the comparison among the groups with oral inflammation showed that the EPL-o group had higher insulinemia and IR when compared to the other groups; 6) unaltered fasting glucose among the evaluated groups; 7) the offspring of rats with PED and EPL showed an increase of IKKα/β phosphorilation status in MG in relation to the offspring of CN and AP rats; 8) the PED and EPL groups showed an increase in TNF-α content in the MG in relation to the CN-o and AP-o groups; 9) the AP-o, PED-o and EPL-o groups showed reduced tyrosine phosphorylation of pp185 when compared to the CN-o group. Furthermore, in this same parameter, the EPL-o group showed a more accentuated reduction in relation to the other groups; 10) there was no significant difference in IRS-1 serine phosphorylation status among the evaluated groups. The results of the present study demonstrate that the association of AP and PED promotes more pronounced changes both locally and systemically in the adult offspring. Maintaining good maternal oral health is very important to prevent local diseases such as AP and PED that can promote systemic changes in adult offspring, impairing their general health. (AU)

• FAPESP's process: 19/27662-0 - Evaluation of insulin and inflammatory pathways in skeletal muscle tissue of adult rats, offspring of rats with true endodontic-periodontal lesions
• Grantee: Bianca Elvira Belardi
• Support Opportunities: Scholarships in Brazil - Master