Analgesic Mechanisms of Steroid Ointment against Oral Ulcerative Mucositis in a Rat Model

Abstract

Despite the long history of use of steroid ointments for oral mucositis, the analgesic mechanism has not been fully elucidated. In this study, we examined the effects of triamcinolone acetonide (Tmc) on oral ulcerative mucositis-induced pain in conscious rats by our proprietary assay system. Based on evaluations of the physical properties and retention periods in the oral mucosa of human volunteers and rats, we selected TRAFUL^® ointment as a long-lasting base. In oral ulcerative mucositis model rats, TRAFUL^® with Tmc suppressed cyclooxygenase-dependent inflammatory responses with upregulations of glucocorticoid receptor-induced anti-inflammatory genes and inhibited spontaneous nociceptive behavior. When an ointment with a shorter residual period was used, the effects of Tmc were not elicited or were induced to a lesser extent. Importantly, TRAFUL^® with Tmc also improved oral ulcerative mucositis-induced mechanical allodynia, which has been reported to be independent of cyclooxygenase. Ca²⁺ imaging in dissociated trigeminal ganglion neurons showed that long-term preincubation with Tmc inhibited the hypertonic stimulation-induced Ca²⁺ response. These results suggest that the representative steroid Tmc suppresses oral ulcerative mucositis-induced pain by general anti-inflammatory actions and inhibits mechanical sensitivity in peripheral nerves. For drug delivery, long-lasting ointments such as TRAFUL^® are needed to sufficiently induce the therapeutic effects.

Keywords: drug delivery systems; glucocorticoids; orofacial pain; pain; stomatitis; triamcinolone acetonide; trigeminal ganglion.

Figure 1

Residual periods of ointments on the oral mucosa of healthy humans and rats and the effects of triamcinolone acetonide (Tmc) on the oral ulcerative mucositis score and blood glucose level. V: Vaseline, PB: Plastibase, TO: Traful and PRO: Traful PRO-quick. (A) Residual sensation of each ointment at four different mucosal areas on the back of the upper and lower lip. The ointments were randomly applied by the participant’s fingers (n = 14). ** and ⁺⁺, p < 0.01 for all time points, compared with V and PB, respectively, according to Tukey’s test following two-way ANOVA. (B) Residual weights of ointments on the oral mucosa of healthy humans 2 h after application. TO and PRO could be collected from 64% (n = 9/14) and 71% (n = 10/14) of the subjects. (C) Residual weights of ointments collected from the labial fornix region of rats (each ointment, n = 4). * and ⁺, p < 0.05 and ** and ⁺⁺, p < 0.01 compared with V and PB, respectively, and ^#, p < 0.05 compared with TO according to Tukey’s test following one-way ANOVA (f = 0.89). (D) Effects of TO and TO with Tmc on the oral ulcerative mucositis score (two-way ANOVA, f = 0.46, 0.74, 0.80, 0.82, 0.86 at days 2–6, respectively) in the oral ulcerative mucositis model. NT: non-treatment, TO: TO treatment without Tmc and TO + Tmc: TO treatment with Tmc; each group n = 6. (E) Effect of TO with Tmc on blood glucose levels in the oral ulcerative mucositis model (n = 5, one-way ANOVA, f = 0.25).

Figure 2

Anti-inflammatory effects of triamcinolone acetonide (Tmc) in Traful ointment (TO) and analgesic effect on spontaneous pain in the oral ulcerative mucositis model (OUM, 2 days after the acetic acid procedure). (A) Representative histological images of the oral mucosa in the naive rats and the oral ulcerative mucositis after local treatments with Tmc-containing ointments. NT: non-treatment, TO + Tmc: TO with Tmc and PB + Tmc: Plastibase with Tmc. Microphotographs in the lower panel are enlarged from square area in the microphotographs in the upper panel (scale bars = 50 µm). Oral ulcerative mucositis sections were collected from the OUM following 2 coats of ointments. Tissue sections from the other 2 rats in each group exhibited the same features. (B) Semi-quantitative analyses of inflammatory cell infiltration in the oral mucosa (n = 3 in each group) using a 4-step score (1: none, 2: mild, 3: moderate and 4: severe). ⁺⁺ p < 0.01 between naive and NT according to Student’s t-test (d = 8.00). Among OUM rats, ** p < 0.01, compared with NT according to Sidak’s test following one-way ANOVA (f = 0.79) (C) Representative toluidine blue-stained images of the oral mucosa in the naive and the OUM rats after local treatments with Tmc-containing ointments. (D) Quantitative analyses of mast cell counts in the oral mucosa (n = 3 in each group). ⁺⁺ p < 0.01 between naive and NT according to Student’s t-test (d = 7.49). Among OUM rats, ** p < 0.01, compared with NT according to Sidak’s test following one-way ANOVA (f = 0.85) (E) Prostaglandin E₂ (PGE₂) levels in the oral ulcerative mucositis in the NT and TO + Tmc rats (each n = 4). * p < 0.05, compared with NT according to Student’s t-test (d = 2.55). (F) Effects of ointments on spontaneous mouth rubbing. NT (pAA: before the acetic acid procedure in the same rats), TO: TO alone, TO + Tmc, TO + Dex: TO with dexamethasone and PB + Tmc; each group n = 5. ⁺ p < 0.05 between pAA and NT according to Student’s t-test (d = 1.45). Among OUM rats, * p < 0.05, compared with NT according to Sidak’s test following one-way ANOVA (f = 0.20).

Figure 3

Changes in gene expression following two ointment treatments. ⁺ p < 0.05 and ⁺⁺ p < 0.01 between the naive rats and the nontreated (NT) oral ulcerative mucositis model (OUM) rats according to Student’s t-test. Among OUM rats, * p < 0.05 and ** p < 0.01, compared with NT according to Sidak’s test following one-way ANOVA. TO: Traful ointment alone; TO + Tmc: TO with triamcinolone acetonide (Tmc); and PB + Tmc: Plastibase with Tmc. For each group, n = 5 (d = 2.91, 0.17, 6.80, 3.77, 1.19, 0.57. f = 0.87, 0.38, 0.74, 0.81, 0.77, and 0.72). (A) Cyclooxygenase-2 (COX-2). (B) Microsomal prostaglandin E synthase-1 (mPGES1). (C) Tumor necrosis factor-α (TNF-α). (D) Glucocorticoid-induced leucine zipper (GILZ). (E) Interleukin-1 receptor-associated kinases M (IRAKM). (F) Mitogen-activated protein kinase phosphatase 1 (MKP1).

Figure 4

Analgesic effect of triamcinolone acetonide (Tmc)-containing Traful ointment (TO) on oral ulcerative mucositis-induced mechanical allodynia and its cellular mechanism. (A) Effects of ointments on oral ulcerative mucositis-induced mechanical allodynia. NT: non-treated oral ulcerative mucositis model (OUM), (pAA: before the acetic acid procedure in the same rats), TO: TO alone, TO + Tmc: TO with Tmc and PB + Tmc: Plastibase with Tmc; each group n = 5. ⁺⁺ p < 0.01 between pAA and NT according to the Student’s t-test, d = 4.02. Among OUM rats, ** p < 0.01, compared with NT according to Sidak’s test following one-way ANOVA, f = 0.24. (B) Endothelin-1 (ET-1) levels in the oral ulcerative mucositis in the NT and TO + Tmc rats (each group n = 4, Student’s t-test, d = 0.25). Colony-forming units (CFUs) under aerobic (C) and anaerobic (D) conditions. Samples were collected from the oral ulcerative mucositis of the OUM rats two days after the acetic acid procedure. ⁺⁺ p < 0.01 between the naive and NT groups according to Mann–Whitney U tests, d = 1.60, 1.35, respectively. The OUM rats were compared by one-way ANOVA, f = 0.47 and 0.77 (each group n = 5). (E) Representative Ca²⁺ responses to the hypertonic solution (Hyper, +100 mOsm), the TRPA1 agonist allyl isothiocyanate (AITC, 1 mM), the TRPV1 agonist capsaicin (CPS, 1 µM), and KCl solution (HK, 50 mM) in dissociated trigeminal ganglion neurons after treatments with Tmc (long-term: 3 h of incubation prior to the recordings; transient: 2 min incubation prior to hypertonic stimulation). DMSO application did not show any responses. (F) Percent responder to Hyper, AITC, and CPS. In the control, 20.1% (n = 30/149), 35.6% (n = 53/149), and 63.1% (n = 94/149) of the neurons were hypersensitive, AITC sensitive, and CPS sensitive, respectively. In the long-term incubation, 9.8% (n = 14/143), 11.9% (n = 17/143), and 61.5% (n = 88/143) of the neurons were hypersensitive, AITC sensitive, and CPS sensitive, respectively. In transient incubation, 23.6% of the neurons were hypersensitive neurons (n = 30/127). * p < 0.05 and ** p < 0.01, compared with the control according to Fisher’s test. (G) Mean ∆ ratio changes in Ca²⁺ responses. * p < 0.05 compared with the control according to Student’s t-test.