Inhibition of carrageenan-induced dental inflammatory responses owing to decreased TRPV1 activity by Dexmedetomidine

Dexmedetomidine (Dex) is a highly selective agonist of the α2 adrenergic receptor and a common sedative; however, its anti-inflammatory effect has been studied. In this study, the inhibitory effect of Dex on inflammation in dental pulp cells was assessed. For this, the effect of Dex on inflammation induced by carrageenan (Car) in human dental pulp cells (hDPCs) was evaluated. Car incubation induced a robust inflammatory response in hDPCs as well as activation of PKA–STAT3 and PKC–nuclear factor kappa B (NF-κB) signaling pathways. Dex reduced the expression of inflammatory cytokines in a dose-dependent manner. Meanwhile, the phosphorylation of PKA, PKC, STAT3, and NF-κB as well as the nuclear accumulation of STAT3 and NF-κB were significantly increased in Dex-treated Car-induced hDPCs. Western blotting results also showed that the phosphorylation level of transient receptor potential cation channel subfamily V member 1 (TRPV1) was downregulated as a result of Dex treatment. Furthermore, we found that administration of the TRPV1 agonist capsaicin (Cap) reversed the effects of Dex on proinflammatory cytokines; however, the expression and activation of PKA–STAT3 and PKC–NF-κB signals were not altered owing to Cap administration. These results indicate that Dex plays a defensive role in dental pulp inflammation by regulating the TRPV1 channel and can be used as a potential target for human dental pulp inflammation intervention.


Introduction
Pulp exposure and injury leads to pulpitis and induces severe inflammation, frequently resulting in persistent pain and referred pain. Dental pulp inflammation is a common phenomenon, usually a sequela of dental caries or trauma [1]. Clinically, it could cause severe pain [2], and if not controlled, it may eventually lead to fatal systemic inflammatory disease [3]. The mechanism of acute pulpitis is complex and involves repetitive trauma, inflammation, bacterial invasion, stimulation of the afferent nerve, secondary hyperalgesia, and in rare cases, periodontitis. Without effective treatment, the outcome is always root canal treatment. Therefore, considering the immediate effects of pulpitis, the identification of a new therapeutic target is significantly important for treating pulpitis. However, several studies have focused on the effect of immune cells [4], such as macrophages, dendritic cells, and lymphocytes.
Human dental pulp cells (hDPCs) are the main cell types present in dental pulp and play multiple roles in host defense and regeneration [5][6][7]. HDPCs induced by proinflammatory mediators, including tumor necrosis factor alpha (TNF-α) and lipopolysaccharide (LPS), can locally secrete numerous cytokines to attract additional immune cells and initiate and regulate inflammation [8,9].
During inflammation, primary nociceptive neurons (nociceptors) are sensitized and the pain sensation (hyperalgesia) is increased. The direct effect of inflammatory mediators such as prostaglandins (PGI2 and PGE2) and sympathetic amines (epinephrine and dopamine) on their receptors in the nociceptor membrane can cause sensitization. Transient receptor potential cation channel subfamily V member 1 (TRPV1), a ligandgated ion channel, is involved in pain modulation [10]. The flavonoid eriodictyol (an antagonist of the TRPV1 channel) also plays a part by reducing nociceptive behavior [11]. In addition, the nociceptor is partially characterized by the expression of TRPV1 [12].
As a specific agonist of the α2 adrenergic receptor, dexmedetomidine (Dex) is commonly used for analgesia and sedation purposes in the operation room and intensive care unit [13,14]. Dex was recently reported to have a protective effect against inflammation that is triggered by endotoxin [15], spinal cord injury [16], sepsis [17], or lung injury [18]. In the present study, carrageenan (Car), an inflammation inducer [19], was used to induce pulp inflammation. STAT3 and NF-κB are common targets for IL-6-induced macrophages and carrageenan-induced mouse paw edema [20], while TRPV1 is crucial for proinflammatory STAT3 signaling [21]. Therefore, this study sought to study the protective effect of Dex on Car-induced pulp inflammation and determine the role of TRPV1, STAT3, and NF-κB on inflammation of hDPCs.

Expression of proinflammatory cytokines induced by car in hDPCs
To explore hDPC inflammation following Car treatment, the expression of proinflammatory cytokines in hDPCs was assessed. qPCR and ELISA test results revealed that messenger RNA (mRNA) and protein expressions of IL-1β, IL-6, and TNF-α in HDPCs after Car treatment were higher than those in the control group (P < 0.01) ( Fig. 1a and b).

Car-induced activation of PKA-STAT3 and PKC-nuclear factor kappa B (NF-κB) in induced hDPCs
Because the activation of the PKA-STAT3 and PKC-NF-κB pathways is crucial for inducing cytokine expression [22,23] the expression and phosphorylation of PKA, STAT3, PKC, and NF-κB after Car treatment were evaluated. qPCR results suggest that Car upregulates the mRNA expressions of STAT3 and NF-κB (P < 0.05), while the PKA and PKC expressions were not altered (Fig. 2a). Moreover, the WB results indicated that the levels of PKA, STAT3, PKC, and NF-κB phosphorylation were increased following Car treatment (Fig. 2B). Additionally, the nuclear localization of STAT3 and NF-κB was clearly increased as a result of Car incubation (Fig. 2c). Cellular fractionation was also performed to detect the nuclear (N) and cytoplasmic (C) distribution of STAT3 and NF-κB in hDPCs. The results showed that the "N" fraction of STAT3 and NF-kB in cells with Car treatment was increased, while the "C" fraction was reduced (Fig. 2d). These results suggest that Car Fig. 1 Car-triggered inflammation in hDPCs. hDPCs were treated with either 10 μM Car or PBS (as control) for 2 h. After lysis, the expressions of IL-1β, IL-6, and TNF-α were assessed by (a) qPCR and (b) ELISA. Data are expressed as means ± standard deviations. Comparisons between two groups were analyzed by t-test. **P < 0.01, ***P < 0.001

Dex ameliorated inflammatory responses triggered by car
Previous studies have shown that local Dex administration reverses inflammation induced by Car [24,25]. Therefore, ELISA was utilized to detect the production of proinflammatory cytokines under Car stimulation and various concentrations of Dex. IL-1β, IL-6, and TNF-α were downregulated in a dose-dependent manner at 2 h after Dex treatment in hDPCs (P < 0.05) ( Fig. 3a, b, and c). WB results showed that PKA, STAT3, PKC, and NF-κB phosphorylation was downregulated in hDPCs owing to stimulation using 5 μM Dex (P < 0.05) (Fig. 4a). Meanwhile, we found that STAT3 and NF-κB were redistributed in the cytoplasm following Dex treatment (Fig. 4b), suggesting that these two signaling pathways were blocked as a result of Dex treatment in hDPCs.

Dex desensitized TRPV1 channel in car-treated hDPCs
Previous studies have suggested that TRPV1 (a substrate of different protein kinases) is activated by multiple inflammatory mediators, such as PKC [26] and PKA [27]. Therefore, we attempted to examine the activation of TRPV1 during Car induction and Dex treatment. qPCR results showed that TRPV1 expression did not significantly change following Dex treatment (Fig. 5a); however, its activity, which was indicated by CGRP release, was clearly reduced after Dex treatment (Fig. 5b). Furthermore, WB indicated that TRPV1 phosphorylation was downregulated and confirmed that its expression did not change (Fig. 5c). The data suggested that TRPV1 was desensitized owing to Dex administration.

Effects of the TRPV1 agonist cap on inflammation in hDPCs
To further examine the influence of TRPV1 on Dexameliorated and Car-induced inflammation in hDPCs, cells were coadministered 5 μM of capsaicin (Cap), a TRPV1 agonist. We found that CGRP release was obviously increased after Cap treatment (P < 0.05) (Fig. 6a). In addition, TPRV1 phosphorylation was markedly increased after hDPCs were coadministered with Cap. Meanwhile, the activation of the PKA-STAT3 and PKC-NF-κB pathways was not altered, suggesting that TRPV1 was located downstream of these two pathways (Fig. 6b). In addition, the sensitization of TRPV1 resulted in the restoration of proinflammatory cytokine production ( Fig. 6c), suggesting that TRPV1 sensitization counteracts the effects of Dex on hDPC inflammation.

Discussion
Many molecules can be used to induce hDPC inflammation, including LPS, IL-6, IL-8, and TNF-α. More specifically, LPS can penetrate the dental pulp and induce (See figure on previous page.) Fig. 2 Car activated both PKA-STAT3 and PKC-NF-κB signaling in hDPCs. a Cell lysates were subjected to qPCR to assess the expressions of PKA, PKC, STAT3, and NF-κB. b WB was performed to probe with antibodies for phosphorylation and expression PKA, PKC, STAT3, and NF-κB in cell lysates. c IFA detected the cellular location of STAT3 and NF-κB in hDPCs. d Cell fractionation assay showed the location of STAT3 and NF-κB in nuclear and cytoplasmic fractions of hDPCs. e hDPCs were co-treated with either 100 μM Car and 10 μM H-89/Go6983 for 2 h. The expressions of IL-1β, IL-6, and TNF-α were assessed by ELISA. Data are expressed as means ± standard deviations. Comparisons between two groups were analyzed by t-test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group; # P < 0.05, ## P < 0.01 vs. Car group Data are expressed as means ± standard deviations. Comparisons between multiple groups were analyzed by one way ANOVA. **P < 0.01, ***P < 0.001 vs. Car group; ## P < 0.01, *** P < 0.001 vs. Control group inflammation and plays a key role in pulp infection [28]. Proinflammatory cytokines such as IL-6, IL-8, and TNFα are usually detected in inflamed pulp and are considered key biomarkers and mediators for the diagnosis of pulp inflammation [29][30][31]. In the present study, Car was used to induce the inflammation of hDPCs. A previous study revealed that the Car-induced acute inflammatory response can be examined to elucidate the inflammatory period related to phagocyte infiltration, excessive production of free radicals, and release of inflammatory mediators, e.g., TNF-α, cyclooxygenase-2, and inducible nitric oxide synthase [32]. An in vivo model  showed that Car-induced acute inflammatory response reached its peak at 2-3 h after injection, with the inflammation later disappearing within 24-74 h [33]. The inflammatory response induced by Car is considered to be a biphasic situation in which multiple mediators induce the generation of inflammation [34]. In this study, the robust generation of proinflammatory cytokines, i.e., IL-1β, IL-6, and TNF-α, and the phosphorylation and nuclear localization of STAT3 and NF-κB were observed after 100-μM Car treatment for 2 h. These findings indicated the successful induction of dental pulp inflammation by Car.
Existing evidence has revealed that TRPV1 is involved in the occurrence and development of immune-related diseases and is a therapeutic target that is easily blocked by small molecules. For instance, TRPV1 is currently considered to be an anticipated molecular target for the clinical evaluation of epilepsy therapy [35]. Its antagonists are employed for the treatment of pruritus, inflammation-related pain, tissue damage, ischemia, and other diseases [36,37], and its agonists have been used in a phase III clinical trial for the treatment of cluster headache, analgesia, and osteoarthritis [38,39]. Various inflammatory mediators can sensitize TRPV1, including neurotransmitters, cytokines and chemokines, lipids, peptides or small proteins, and growth factors. Many inflammatory mediators enhance the activity of TRPV1 [40]. TRPV1 is lowly expressed in pulpal sensory neurons relative to their expression in the pulpal trigeminal ganglia [41]. LPS treatment in dental trigeminal ganglia induced pulpitis by upregulating expression of the TRPV1 channel [42]. Previous studies have shown that local Dex administration reverses inflammation induced by Car [22,23]. In the present study, treatment using Dex led to impaired inflammation and deactivation of the TRPV1 channel. Further usage of the TRPV1 agonist Cap restored the generation of proinflammatory cytokines, suggesting that the TRPV1 channel was involved in the Car-triggered inflammation of DPCs.
Previous studies demonstrated that TRPV1 can be activated by inflammatory mediators and corresponding receptors, i.e., PKC [26] and PKA [27]. Both kinases can phosphorylate TRPV1 at different serine and threonine residues, resulting in TRPV1 sensitization. Alternatively, the calcium signaling pathway can be activated and TRPV1 phosphorylation can be induced by increased intracellular calcium in the TRPV1 channel, leading to downstream activation of STAT3 [21] and NF-κB and cytokine production [43]. STAT3, a member of the STAT protein family, is an important regulator in tumor Fig. 6 Cap treatment restored Car-induced inflammation in hDPCs. hDPCs were cotreated with Car (100 μM), Dex (5 μM), and/or Cap (5 μM) for 2 h. a The release of CGRP in cell culture medium was determined at 2 h after Car and/or Dex cotreatment by ELISA. b WB analyses were used to examine the phosphorylation and expression of PKA, PKC, STAT3, NF-κB, and TRPV1 in cell lysates. c ELISA was conducted to assess IL-1β, IL-6, and TNF-α expression. Data are expressed as means ± standard deviations. Comparisons between two groups were analyzed by t-test. **P < 0.01, ***P < 0.001 cells and plays a critical role in inflammation and tumorigenesis by regulating cell metabolism [44]. STAT3 protein exists in an inactive form in the cytoplasm and can be activated by associated kinases and phosphorylated at multiple phosphorylation sites [45]. The most common form of NF-κB is a heterodimer of p50 and p65/RelA proteins. Similarly, in the deactivated condition, NF-κB is present in an inactive form that is retained in the cytoplasm by the inhibitory protein IκB. Both STAT3 and NF-κB are transcription factors that are involved in immune responses during inflammation [22,23]. Previous studies have demonstrated that STAT3 [46,47] and NF-κB [22,[48][49][50][51] can be upregulated and activated in the inflammatory DPCs or dental pulp stem cells. In the present study, PKA-STAT3 and PKC-NF-κB signaling was activated by Car administration, while Dex treatment clearly reduced the phosphorylation and nuclear subcellular location of STAT3 and NF-κB, indicating that STAT3 and NF-κB activation might be attributed to Car-induced inflammation of DPCs. However, both signals were not altered after Cap administration, indicating that TRPV1 activation occurs downstream of these two signals.

Conclusions
In summary, our study demonstrated the intrinsic mechanism underlying dental pulp inflammation in hDPCs. Car can induce inflammation by sensitization of TRPV1 via the PKA-STAT3 and PKC-NF-κB pathways, which can be ameliorated by Dex. The present study shows that Dex can be used as a potential drug for gingivitis, which can induce anti-inflammatory potency in dental pulp cells.

Ethics
All experiments were approved by the Ethics Committee of Rizhao People's Hospital. All subjects signed informed consent forms.

Separation and culture of hDPCs
Healthy permanent premolars for orthodontic or impacted third molars were collected from subjects aged 18-26 years. As mentioned above, hDPCs were separated and cultured using a previously described enzymatic method [52]. The dental pulp tissue was separated, cut into small pieces, and digested at 37°C for 20 min with 3 mg/mL of type I collagenase (Gibco, USA). Next, the chopped pulp tissue was cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum, 100 U/mL of penicillin (Gibco, USA), and 100 mg/mL of streptomycin with 5% CO 2 . The medium was replaced every 3 days. After the cells achieved 80% confluence, they were separated by trypsin/ethylenediaminetetraacetic acid (Gibco, USA) and sub-cultured at a ratio of 1:2.

Dex incubation
Dex (1,179,333, Sigma, 350 μM) was frozen at − 20°C and diluted with DMEM/F-12 to a specified concentration if necessary. Before Dex treatment, cells were allowed to reach approximately 70-80% confluence. The cells were exposed to Dex at various concentrations (0, 0.1, 1, 5, or 10 μM) for 2 h to determine the optimal concentration. Cells were then divided into the following groups: control group, in which the cells were incubated without Dex treatment in a humidified environment at 37°C with 5% CO 2 ; Car group, in which the cells were incubated with 100 μM of Car for 2 h; and Dex/Car group, in which the cells were pretreated with 5 μM of Dex and 100 μM of Car for 2 h.
Immunofluorescence assay (IFA) hDPCs were inoculated in a 24-well plate and fixed with 4% polyformaldehyde (28,906, Thermofisher) for 15 min. hDPCs were permeated for 30 min with 0.1% Triton X-100, cultured at ambient temperature for 15 min with 10% goat serum, and treated at 4°C with the primary antibody overnight. hDPCs were washed with PBS three times and incubated at ambient temperature for 1 h with the secondary Cy3-labeled antibody in dark conditions. Then, the cells were stained with 4′,6-diamidino-2-phenylindole (D1306, Thermofisher) for 15 min. Images were obtained at 400× magnification using a fluorescent microscope.

Subcellular fractionation
Cells (1 × 10 6 ) were plated on 12-cm dishes and grown for 36 h. Then, the cells were harvested via scraping into 500 μL of cell lysis buffer containing 10 mM HEPES (pH 7.4), 10 mM NaCl, 1 mM KH 2 PO 4 , 5 mM NaHCO 3 , 1 mM CaCl 2 , 0.5 mM MgCl 2 , and 5 mM EDTA with complete protease inhibitor cocktail. Cells were allowed to swell for 5 min, followed by Dounce homogenization for 50-time strokes. The cells were then centrifuged at 5000 rpm for 5 min, generating a pellet containing nuclei and debris and a supernatant of cytosol and plasma. Pellets were resuspended in 1 mL of buffer containing 10 mM Tris (pH 7.5), 300 mM sucrose, 1 mM EDTA, and 0.1% NP40 with complete protease inhibitor cocktail and then pelleted, resuspended, and washed twice. The final pellets obtained were pure nuclei.

Enzyme-linked immunosorbent assay (ELISA)
According to the manufacturer's instructions, the concentrations of interleukin (IL)-1β (BMS224-2, Thermofisher), IL-6 (EH2IL6, Thermofisher), TNF-α (KHC3011, Thermofisher), and CGRP (ABIN1095216, Antibodiesonline) in the cell culture supernatants were analyzed using an ELISA kit. An automated microplate reader (SpectraMax® M5) was used for the measurement of the optical density (OD) at 450 nm. The concentrations of each sample were detected based on optical density and the concentration of the standard.

Statistical analysis
The results of our study are presented as means ± standard deviations. Comparisons between two groups or multiple groups were analyzed using one-way ANOVA or a two-tailed Student's t-test, respectively. A P-value of < 0.05 was considered to indicate a statistically significant difference.