TRPA1 triggers hyperalgesia and inflammation after tooth bleaching

Hyperalgesia has become a major problem restricting the clinical application of tooth bleaching. We hypothesized that transient receptor potential ankyrin 1 (TRPA1), a pain conduction tunnel, plays a role in tooth hyperalgesia and inflammation after bleaching. Dental pulp stem cells were seeded on the dentin side of the disc, which was cut from the premolar buccal tissue, with 15% (90 min) or 40% (3 × 15 min) bleaching gel applied on the enamel side, and treated with or without a TRPA1 inhibitor. The bleaching gel stimulated intracellular reactive oxygen species, Ca2+, ATP, and extracellular ATP in a dose-dependent manner, and increased the mRNA and protein levels of hyperalgesia (TRPA1 and PANX1) and inflammation (TNFα and IL6) factors. This increment was adversely affected by TRPA1 inhibitor. In animal study, the protein levels of TRPA1 (P = 0.0006), PANX1 (P < 0.0001), and proliferation factors [PCNA (P < 0.0001) and Caspase 3 (P = 0.0066)] increased significantly after treated rat incisors with 15% and 40% bleaching gels as detected by immunohistochemistry. These results show that TRPA1 plays a critical role in sensitivity and inflammation after tooth bleaching, providing a solid foundation for further research on reducing the complications of tooth bleaching.

Tooth bleaching is one of the most frequently performed clinical procedures in esthetic dentistry 1 , which involves lightening the stained tooth by the application of chemical agents, hydrogen peroxide (H 2 O 2 ), or carbamide peroxide, to oxidize the organic pigmentation in the tooth. Despite the high success rate of bleaching of discolored teeth, bleaching sensitivity (BS) 2 remains the most common clinical side effect related to the bleaching procedure. Studies have reported that the incidence rate of BS reached up to 67-87% in trials [3][4][5] , and usually persists for up to 4-7 days after bleaching treatment 1 .
The mechanisms underlying BS have not yet been elucidated completely. BS has been suggested to result from a reversible inflammatory process caused by H 2 O 2 in the dental pulp 6,7 . Several clinical trials have evaluated the use of drugs for the reduction of BS in a perioperative protocol, including anti-inflammatory and analgesic drugs, and desensitizing gels 8 . However, topical application of desensitizers before or after dental bleaching did not reduce the incidence or intensity of BS [8][9][10] . Thus, it is necessary to further understand the mechanisms of BS, which can facilitate the development of novel strategies with a good efficacy and safety profile to relieve BS. H 2 O 2 and its by-products can diffuse through the enamel and dentin, ultimately reaching the pulp to trigger the release of inflammatory mediators 11 , thereby decreasing cellularity and cellular metabolism, altering vascular permeability, and even causing tissue necrosis 7,11 . Increasing evidence suggests that H 2 O 2 -induced pain is associated with transient receptor potential ankyrin 1 (TRPA1) 12,13 . TRPA1 is a non-selective cation channel, which has been recognized as a polymodal nociceptor activated by various irritants and oxidative stimuli, including H 2 O 2 , and contributes to nociceptive and inflammatory pain generation 14,15 . An increased H 2 O 2 content may contribute to visceral hyperalgesia by activating TRPA1 16 . H 2 O 2 production and subsequent TRPA1 activation reportedly contribute to the painful and inflammatory responses during acute gout attacks 13 . Another study showed that intramuscular injection of H 2 O 2 produces nociceptive and aversive behaviors via the TRPA1 receptor, and these H 2 O 2 -induced behaviors were blocked by TRPA1 antagonists 17 . These findings indicate the crucial role of TRPA1 in regulating peroxide-induced pain hyperalgesia, although the mechanism is not yet fully understood. TRPA1 expression has also been reported in dental pulp cells and odontoblasts 18,19 , and is upregulated in inflamed or injured pulp 18 . The functionality of TRPA1 channels has been shown to be modulated by cariesinduced inflammation, indicating a potential mechanism for inflammatory hyperalgesia 20 21 . ATP is released from mechanically stimulated odontoblasts and pulp via pannexin-1 (PANX1) in response to TRP channel activation, which then upregulates the expression of P2X3 receptors on the trigeminal neurons, increasing the intercellular calcium ions of the nerve 21 . Among the large family of TRP channels, TRPA1 and TRPV1 are the best known for their principal role in key hyperalgesia mechanisms 22 . As a small molecule drug that inhibits TRPA1, HC030031 can be used as a tool to study the role of TRPA1 channels in pain 23 . However, there has been no study on the relationship between the expression and functional significance of TRPA1 in dental pulp during bleaching treatment. In this study, we aimed to elucidate the functional properties of pulpal afferents in the sensory transduction of dentinal pain. The null hypothesis was that bleaching gel does not excite or sensitize the pulpal nociceptors via regulating TRPA1.
TRPA1 induces the hyperalgesia pathway in DPSCs after bleaching. In the hyperalgesia pathway, ATP is released in large quantities in cells, and the amount of ATP flowing out of the cells by the PANX1 channel is also increased to act on nerve cells, causing pain after an increase in intracellular Ca 2+ . Thus, we measured intracellular ( Fig. 3A

Discussion
With the increasing use of tooth bleaching technology, its complications have also attracted considerable attention, particularly BS. Understanding the underlying mechanism is essential to reduce the incidence of BS and promote clinical application of this procedure. To this end, we conducted a series of experiments to explore the effect of inhibiting TRPA1, a key factor in hyperalgesia, on sensitivity to bleaching gel. Our results suggest that the bleaching gel evidently upregulated TRPA1 expression in DPSCs, thereby activating the hyperalgesia gene PANX1 and the inflammatory genes TNFα and IL6 in a concentration-dependent manner. Profound changes in the protein levels of TRPA1, PANX1, TNFα, IL6, and the proliferation-related factors PCNA and caspase 3 were found in bleaching gel-treated teeth. Therefore, we must reject our initial hypothesis. In the sensory pathway, a receptor perceives the stimulation and then delivers a neurotransmitter, which in turn stimulates the nerve. Therefore, the sensor pathway is composed of receptors, neurotransmitters released by receptors, and nerves. Shibukawa et al. 21 immunohistochemically explored the receptors, TRPA1 channels that perceive the stimulation and PANX1 channels that secrete ATP as a neurotransmitter in the pulp tissue, making use of specific antibodies targeting these channels and nerves during odontoblast differentiation. TRPA1 is considered to be a receptor for harmful cold temperatures 24 , and its mRNA expression has been reported to increase under the influence of inflammation in the mouse colon 25 . TRPA1 expression is upregulated in the trigeminal ganglion after injection of nerve growth factor 26 or due to pulpal inflammation 27 . Moreover, TRPA1 expression is elevated in the odontoblasts of carious teeth 20 . Although the mechanism of dentin hyperalgesia remains unclear, the significant activation of protein expression in many TRP channels in the pulp or DPSCs suggests that TRP channels, specifically TRPA1, have a vital sensory function and play an important role in the pathogenesis of dentin hyperalgesia 28 . TRP channels in DPSCs can be switched on by the corresponding stimuli, thus participating in sensing and responding to various stimuli to ultimately induce hyperalgesia. Therefore, TRPA1 could be crucial to the dentin hyperalgesia induced by mechanical, thermal, or chemical stimulation.
Supporting this hypothesis, we found that bleaching gel enhanced the oxidative stress of DPSCs, based on an increase in intracellular ROS levels. The stimulation receptor TRPA1 was activated, promoting the transport of calcium ions into cells, therefore sensitizing PANX1 to secrete ATP into the extracellular zone. As mentioned above, ATP acts as a neurotransmitter to stimulate neurons and produce pain. Tooth bleaching produced  29 . Based on the mechanism underlying inflammation, the inflammatory response and complications of pulpitis can be attenuated.  The cytoplasmic extension of odontoblasts and the dentin fluid acts as a physical barrier for the penetration of foreign substances, minimizing the damage to the dental pulp 30,31 . Therefore, an in vivo model is of great significance in studying the response of the dental pulp tissue to invaders. Studies on human teeth 32 , animal teeth 33 , and cell cultures 34 have shown that the pulp of bleached teeth exhibits dramatic changes on the first day after bleaching, manifesting as severe inflammation or necrosis. Another study 35 showed that the concentration of the bleaching gel affects the ability of H 2 O 2 to penetrate the pulp cavity and is therefore related to the extent of injury in the pulp. In the present study, we found apparent expression of TRPA1, PANX1, and proliferationrelated proteins in the pulp after bleaching, especially in odontoblasts, which was consistent with the in vitro results. However, anterior teeth, which are the teeth most commonly bleached in clinical contexts, are rarely obtained; consequently, the enamel/dental disk in this study was made from human premolars. This issue needs to be addressed in future research.
In conclusion, we demonstrated that tooth bleaching leads to the activation of TRPA1, which promotes the intracellular absorption of calcium. In turn, PANX1 is activated, which induces the secretion of ATP that acts as a neurotransmitter to ultimately induce pain (Fig. 6). In addition, TRPA1 upregulates TNFα and IL6, thus promoting the occurrence of inflammation. This study thus clarifies the role of TRPA1 in the development of hyperalgesia and inflammation after bleaching, laying a solid foundation for further research on reducing tooth bleaching complications and promoting its clinical application.   Supplementary Fig. S4. Data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-tests. www.nature.com/scientificreports/ passage were used in the subsequent experiments as described previously 36 and cultured on the dentin surface of the disc. Informed consent was obtained from all participants in this study, and this study was performed in line with the principle of Declaration of Helsinki.

Methods
Cell identification. Identification of stem cell surface markers. Trypsin (Beyotime) was used to detach the cells, which were then counted and aliquoted in eight Eppendorf tubes (1.5 mL, 1 × 10 5 cells/tube). One tube was used as a negative control group and only had phosphate buffered saline (PBS) containing 2% FBS added to it. Then, 100 μL of PBS containing flow cytometry antibody and 2% FBS was added to the other seven tubes, which   Enamel/dentin disc preparation. Complete premolars were obtained from orthodontic patients aged 18-24 years with their consent. The inclusion criteria were as follows: (1) age, 18-24 years; (2) premolars had to be removed owing to orthodontics; and (3) the premolars were healthy, complete, and untreated. The exclusion criteria were as follows: (1) patients with systemic diseases, such as infectious, autoimmune, and genetic diseases and (2) those with abnormal tooth structure, such as dental fluorosis. Dental crowns were obtained by horizontally sectioning the tooth at the cementoenamel junction using a diamond saw (MCTBIO; MCTBIO, Yongin, Gyeonggi, Korea). The lingual sections of the crowns were discarded to fabricate a buccal enamel-dentin disc (Fig. S1). The apical wall was then sealed using light-cured resin (Filtek P60; 3M ESPE, St Paul, Minnesota [MN], US) to produce a bottomless cylinder. The specimens were disinfected with 75% ethanol (Aladdin; Aladdin, Shanghai, China) and ultraviolet light irradiation for 1 h before the in vitro experiments 11 . After disinfection, grouping was performed by simple randomization; that is, samples were numbered in sequence and then divided into groups. www.nature.com/scientificreports/ Experimental design. Cells were implanted on the dentin side of the disc. After attachment, the enameldentin disc was overturned in the medium to expose the enamel surface above the liquid level with a 1 mm gap; the enamel surface was surrounded by a light cured resin barriers (Opalescence PF; Ultradent Products, South Jordan, Utah [UT], US) to prevent the bleach from spilling into the medium (Fig. S1). After that, approximately 0.5 mL of thickened bleaching gel was daubed on the enamel surface at 37 °C.  TNFα  GAA CCC CGA GTG ACA AGC CT  TAT CTC TCA GCT CCA CGC CAT   IL6  GCA ATA ACC ACC CCT GAC CCAA  GCT ACA TTT GCC GAA GAG CC   TRPA1  GCA TGT TTA TTC CCT CAC TAC CCC ACA CAA GGA CAC ATA CAT AGCCA   PANX1  ACT TGG TTT CCC CGC ATG GT  GAA CAA AGC GCT TCC CTC  Thirty-six Sprague-Dawley rats weighing 250-350 g were kept in a standard animal room at 22 ± 1 °C, with 55% ± 10% humidity and a standard light/dark schedule of food and water. The rats were assigned to one of the following three groups: (1) no bleaching gel (n = 12); (2) 15% bleaching gel for 90 min (n = 12); and (3) 40% bleaching gel for three 15-min periods (n = 12). Bleaching gel (0.01 mL) was painted onto the incisor surface according to the relevant manufacturer instructions. Then, the rats were reared according to the aforementioned standards. Following bleaching, after 0 (n = 3), 1 (n = 3), 3 (n = 3), and 7 (n = 3) days of standard feeding, the rats were sacrificed under deep anesthesia with pentobarbital (

Statistical analysis.
The experiments were repeated at least three times, and the data are presented as the mean ± SD. Statistical analyzes were performed using GraphPad Prism 6.0 (GraphPad Software; GraphPad Software, San Diego, California [CA], US) by TWO-way ANOVA test (immunohistochemistry) or unpaired Student's t-tests (other data). Graphs were plotted using GraphPad Prism 6.0. The level of statistical significance was set at P < 0.05.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.