Photobiomodulation reduces inflammation but does not influence the hypoxia-inducible factor-1α in pulp tissue of rats after bleaching

Abstract Objectives: To evaluate the influence of photobiomodulation with infrared laser (IRL) in the rat pulp tissue after bleaching, considering the immunolabeling of interleukin (IL)-23 and hypoxia-inducible factor (HIF)-1α. Methodology: The right and left molars of forty rats were divided into groups: Control – with placebo gel and Bleached – with 35% hydrogen peroxide (H2O2). Half of the rats received one IRL application on both sides, establishing a split-mouth design, which resulted in 4 groups with 20 hemi-maxillae each: Control, Bleach, IRL, and Bleached-IRL. Rats (n=10) from each group were euthanized, at 2- and 30-days mark, and the pulp tissue was evaluated using inflammation and immunolabeling scores. Wilcoxon and Mann-Whitney statistical tests were performed (p<0.05). Results: At the 2-days mark, the Bleached group had severe inflammation and necrosis in the occlusal thirds of the pulp, and moderate to severe inflammation in cervical third, whereas the Bleached-IRL had mild to moderate inflammation (p<0.05). At the 30-days mark, there was no inflammation, but tertiary dentine formation in the bleached groups. Regarding IL-23, severe immunolabeling was observed in the Bleached group (p<0.05) at the 2-days mark; at the 30-days mark, there was a reduction in immunolabeling, in which the Bleached group had moderate and the Bleached-IRL group had mild immunolabeling (p>0.05). HIF-1α was more evident at the 2-days mark in the Bleached group, without significant difference with the Bleached-IRL (p>0.05). The difference was observed between the bleached and control groups, without immunolabeling (p<0.05); at the 30-days mark, the Bleached group had reduction in HIF-1α immunolabeling, while the Bleached-IRL had an increase; the difference remained between the bleached and the controls groups (p<0.05) Conclusion: Photobiomodulation using IRL minimized the inflammation and IL-23 immunolabeling in the pulp tissue of rats after dental bleaching, but did not influence significantly the HIF-1α immunolabeling.


Introduction
During dental bleaching, the oxidation of organic structures of the dental tissue occurs through the action of reactive oxygen species (ROS) released from hydrogen peroxide (H 2 O 2 ). 1 Due to the ability of ROS to penetrate through mineralized dental tissues, it can reach the dental pulp 1 and cause damage such as severe inflammation, necrosis, deposition of mineralized tissue, and pulp aging. 2 Clinically, the response to pulp damage is presented as an intense tooth sensitivity reported by patients immediately after the bleaching. 3 Some protocols have described methods to minimize the effects of bleaching on vital teeth, such as variations in the H 2 O 2 concentration and duration of the bleaching gel application. [4][5][6] Photobiomodulation therapy (PBM) was also evaluated 7-9 due its biostimulant effects, as well as its analgesic and anti-inflammatory actions. 10,11 Studies have shown that PBM stimulates adenosine triphosphate and protein synthesis, both capable of acting in tissue repair. 12 Moreover, PBM increases the expression of enzymes that minimize oxidative stress in wounded rats after irradiation. 10 Previously, it was observed that some cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-17, participate in the pulp tissue inflammatory process after bleaching. 5 An increased concentration of H 2 O 2 is accompanied by prolonged activation of CD5-positive cells (a receptor present in lymphocytelike cells) even 30 days after the bleaching, when the pulp tissue is already organized. 5 Some therapies can reduce the expression of these cytokines after bleaching. 13,14 Other important cytokine previously observed in inflamed pulp tissue, the IL-23, 15 could indicate if PBM would be able to act in this tissue. The combination IL-23 and IL-17 plays a critical role in inflammatory processes, 15,16 and IL-17 was present in the pulp inflammation after dental bleaching. 5,13 Thus, IL-23 may also be present in the pulp tissue of bleached teeth. PBM reduces IL-23 expression in epidermal treatment, 17 evidencing that the laser is capable of regulating this interleukin.
ROS have also been associated with hypoxic microenvironments. 18 The molecular response to hypoxia requires rapid action mechanisms that act within partial pressures of oxygen (O 2 ), leading to the activation of transcription factors as an attempt to regulate the O 2 supply and energy metabolism of the tissue. 18 The transcription factor of hypoxia-inducible factor (HIF)-1α is a critical mediator of these adaptive responses and is considered the main regulator in the transition of cellular response and development of hypoxia, which induces the therapeutic properties of mesenchymal stem cells. 19 With its interaction with genes related to angiogenesis, HIF-1α is characterized by stimulating collagen synthesis with consequent regulation of the cell cycle, favoring the survival and improvement of natural cell resistance. 20 Recent studies have shown that HIF-1α promotes the expression of IL-1β and TNF-α in dental pulp cells, suggesting that HIF-1α is involved in the progress of inflammation in dental pulp. 19 Angiogenesis is the formation of new capillaries and PBM has an important effect on this process. 21,22 Transient hyperemia in the pulp was observed after PBM application in teeth during orthodontic movement and PBM was related to accelerated pulp tissue repair. 21 It was also observed that angiogenesis on dorsal wound of rats was more intense after treatment with PBM. 22 Although vascular permeability is increased in pulp tissue of bleached rat teeth, 23 the angiogenesis process in these teeth has not yet been evaluated.
Thus, the aim of this study is to evaluate the influence of PBM on pulp inflammation of bleached teeth through the analysis of the inflammatory infiltrate and the immunolabeling of pro-inflammatory cytokine IL-23. Furthermore, this study evaluated the presence of HIF-1α in the pulp tissue of these teeth, and the influence of PBM on the immunolabeling of this transcription factor. The null hypothesis adopted was that PBM does not influence the inflammatory process and the HIF-1α immunolabeling in the pulp tissue of bleached teeth.   (Table 1). 2 The analysis was performed by a single calibrated operator in a blinded manner.

Immunohistochemical analyses
Other histological sections of groups were obtained for immunohistochemical assessments with an indirect immunoperoxidase technique 13 for IL-23 and HIF-1α.
The histological sections were deparaffinized in xylene and hydrated in a decreasing ethanol series.   Table 1 shows the scores . 2 Data were collected and analyzed by a single-calibrated and blinded operator.

Statistical analyses
The Wilcoxon signed-rank and the Mann-Whitney test were used for statistical comparisons of pulp damage, inflammatory response, and immunohistochemistry at the significance level of 5%.     had an extensive deposition of tertiary dentine ( Figure   2; Table 2). Figure 3 shows the representative images of the immunohistochemical analyses and Table 2  Regarding the immunohistochemical analysis of HIF-1α, the immunolabeling was more evident at the 2-days mark in the Bleached group, with a severe immunolabeling in most specimens, whereas the Bleached-IRL group had moderate immunolabeling; however, there was no significant difference between the groups (p>0.05). The difference was observed between the bleached groups and their respective controls, which had no immunolabeling (p<0.05). At the 30-days mark, the Bleached group had a reduction in HIF-1α immunolabeling, whereas the Bleached-IRL group had an increase in immunolabeling from moderate to severe; however, the difference remained only between the bleached groups and their controls (p>0.05) (Figure 3; Table 2).

Discussion
This study evaluated, in vivo, the influence of PBM applied on the inflammatory process following dental bleaching, using the immunolabeling of IL-23 cytokine and the angiogenesis marker (HIF-1α). We observed a significant reduction in the inflammatory infiltrate and in the presence of necrosis in the group that received PBM compared with the group that did not receive it. We also observed a reduction from severe IL-23 immunolabeling in the Bleached group to moderate immunolabeling in the Bleached-IRL group.
However, the immunolabeling of HIF-1α did not differ significantly between the bleached groups, regardless of the application of PBM. Thus, the null hypothesis that PBM does not influence the inflammation in the pulp of bleached teeth was rejected, but the null hypothesis that PBM does not influence the HIF-1α immunolabeling in pulp after bleaching was accepted.
A previous study compared the action of red laser (RL) and IRL on the pulp tissue of rat molars after bleaching and observed similar effects of reduced inflammatory infiltrate with a single application of IRL as well as with three applications of RL. 14 Our study used only a single application of the IRL, due to reduction of inflammation on the dental pulp and shorter clinical time. Another study that evaluated the potential of PBM in minimizing the damage caused by dental bleaching to pulp cells showed that the further reduction in the cytotoxicity of the bleaching gel was obtained using IRL compared to RL 7 , corroborating our results.
Studies have shown that the beneficial effects of PBM are related to its ability to stimulate cell proliferation, which was observed in pulp cells after stimulation. 25,26 Induction of gene expression of these cells and stimulation of dentine production was also observed. 26 In pulpotomy, PBM stimulated the repair and promoted the healing of the pulp tissue, 27 and presented anti-inflammatory potential. 28 Moreover, PBM favored the reduction of the exudative phase of the inflammatory process and promoted an increase in vascularization and collagen synthesis. 29 These studies showed the beneficial effects of PBM for the repair of pulp tissue, which corroborates our results.
Previously, an increased expression of proinflammatory cytokines was observed in the pulp tissue of bleached teeth. 5,13 The production of IL-23 cytokine is related to the formation of determined cell types, such as antigen-presenting cells, monocytes, activated dendritic cells, and macrophages. 30  A previous study, which evaluated the action of PBM in the treatment of cutaneous inflammation, showed that laser application suppressed the expression of  Our study showed that the IRL was able to This may be due to the induction of angiogenesis that PBM can provide to the tissue. 22 PBM also promoted bone regeneration, with an increase in related gene expression, including HIF-1α; according to the authors, cells exposed to laser radiation undergo a sustained increase in ROS production through the alteration of the mitochondrial membrane potential, which further amplifies the induction of HIF-1α. 38 Thus, it was expected that PBM would lead to a significant increase in HIF-1α immunolabeling in our study.
We observed that the pulp tissue of bleached teeth had a higher immunolabeling for HIF-1α. It is known that cells exposed to hypoxia have higher levels of HIF-1α than normal cells, 39 which corroborates the results found in this study, since H 2 O 2 promotes increased oxidative stress on the pulp tissue 1 , similar to tissues in cancerous environment. 37 However, the present data showed that PBM was not able to significantly change Considering the data observed in this and previous studies, 7,14 we can suggest that PBM represents satisfactory results in minimizing inflammatory processes in pulp tissue. However, PBM was not able to allow the recovery of a pulp tissue without changes, even 30 days after the bleaching, since there was a significant difference compared to its control group.
Moreover, this study highlights that the H 2 O 2 effects in pulp tissue are so intense that they can prevent the full action of therapies, such as the PBM. This reinforces the importance of using low concentrations of H 2 O 2 in the bleaching gel. 1,5 Conclusion The PBM, with IRL, minimized the inflammatory infiltrate and IL-23 immunolabeling in the pulp tissue of rats after dental bleaching, but did not significantly influence HIF-1α immunolabeling.