Curcumin Inhibits NTHi-Induced MUC5AC Mucin Overproduction in Otitis Media via Upregulation of MAPK Phosphatase MKP-1

Abstract

Otitis media (OM), characterized by the presence of mucus overproduction and excess inflammation in the middle ear, is the most common childhood infection. Nontypeable Haemophilus influenzae (NTHi) pathogen is responsible for approximately one-third of episodes of bacteria-caused OM. Current treatments for bacterial OM rely on the systemic use of antibiotics, which often leads to the emergence of multidrug resistant bacterial strains. Therefore there is an urgent need for developing alternative therapies strategies for controlling mucus overproduction in OM. MUC5AC mucin has been shown to play a critical role in the pathogenesis of OM. Here we show that curcumin derived from Curcuma longa plant is a potent inhibitor of NTHi-induced MUC5AC mucin expression in middle ear epithelial cells. Curcumin inhibited MUC5AC expression by suppressing activation of p38 MAPK by upregulating MAPK phosphatase MKP-1. Thus, our study identified curcumin as a potential therapeutic for inhibiting mucin overproduction in OM by upregulating MKP-1, a known negative regulator of inflammation.

Figure 1

Curcumin suppresses NTHi-induced MUC5AC expression in middle ear epithelial cells in vitro and in vivo. (a) HMEECs were pretreated with 10, 20, or 50 μM curcumin for 1 h, followed by NTHi for 5 h, and MUC5AC mRNA expression was measured. (b) HMEECs were pretreated with 20 μM curcumin for 1 h, followed by NTHi strain 12, 2627, or 9274 for 5 h, and MUC5AC mRNA expression was measured. (c) HMEECs were transfected with MUC5AC promoter-driven luciferase vector. Cells were pretreated with 20 μM curcumin for 1 h, followed by stimulation with NTHi for 5 h. MUC5AC promoter-driven luciferase activity was measured. (d) HMEECs were pretreated with 20 μM curcumin for 1 h, followed by stimulation with NTHi for 12 h, and MUC5AC protein level in cell culture supernatants was measured by ELISA. (e) HMEECs were pretreated with 20 μM curcumin for 1 h, followed by NTHi stimulation for 12 h. MUC5AC protein (Alexa 488) was visualized by immunofluorescence staining. DAPI, nuclear stain. Original magnification: 400x. Scale bar, 20 μm. (f, g) Mice were given 50 mg/kg (i.p) curcumin 1 h prior to transtympanic inoculation of NTHi. (f) MUC5AC mRNA expression in dissected middle ear was measured after 6 h NTHi inoculation. (g) MUC5AC protein (Alexa 488) expression in dissected middle ear was visualized by immunofluorescence assay after 9 h NTHi inoculation. DAPI, nuclear stain. Original magnification: 400x. Scale bar, 20 μm. Data are mean ± s.d. (n = 3). (a) ^∗p < 0.05, ANOVA (Tukey's post hoc). (b)–(d), (f) ^∗p < 0.05, t-test. Data are representative of three or more independent experiments.

Figure 2

Curcumin suppresses NTHi-induced MUC5AC expression via inhibition of p38 MAPK. (a) HMEECs were transfected with Mock, MKK3-CA, or MKK6-CA plasmids. Cells were treated with 20 μM curcumin for 1 h, and MUC5AC mRNA expression was measured. (b) HMEECs were pretreated with 20 μM curcumin and 10 μM SB203580 for 1 h, followed by stimulation with NTHi for 5 h, and MUC5AC mRNA expression was measured. (c) HMEECs were transfected with Mock, p38α-DN, or p38β-DN plasmids. Cells were pretreated with curcumin (20 μM) for 1 h, followed by NTHi stimulation for 5 h and MUC5AC mRNA expression was measured. (d) HMEECs were transfected with wild-type MUC5AC promoter, distal AP-1 mutant MUC5AC promoter, or proximal AP-1 mutant MUC5AC promoter containing luciferase vectors. Cells were pretreated with 20 μM curcumin for 1 h, followed by NTHi stimulation for 5 h. MUC5AC promoter-driven luciferase activity was measured. Data are mean s.d. (n = 3). (a) ^∗p < 0.05, t-test. (b)–(d) ^∗p < 0.05, ANOVA (Tukey's post hoc). n.s., not significant. Data are representative of three or more independent experiments.

Figure 3

Curcumin inhibits NTHi-induced MUC5AC expression via upregulation of MKP-1 phosphatase. (a) HMEECs were pretreated with curcumin (20 μM) for 1 h; followed by NTHi stimulation for 1 h. MKP-1 protein (Alexa 488) was visualized by immunofluorescence staining. DAPI, nuclear stain. Original magnification: 400x. Scale bar, 20 μm. (b) HMEECs were treated with 20 μM curcumin, 5 μg/ml Actinomycin D (ACT-D), or both for 1 h, followed by stimulation with NTHi for 1 h, and MKP-1 mRNA expression was measured. (c, d) HMEECs were transfected with (c) Mock or myc-MKP-1, (d) Mock or MKP-1 shRNA. Cells were stimulated with NTHi for 5 h, and MUC5AC mRNA expression was measured. MKP-1 knockdown was confirmed by Q-PCR. (e) HMEECs were transfected with Mock or MKP-1 shRNA plasmid. Cells were pretreated with 20 μM curcumin for 1 h, followed by NTHi stimulation for 5 h, and MUC5AC mRNA expression was measured. (b, e) ^∗p < 0.05, ANOVA (Tukey's post hoc). (c, d) ^∗p < 0.05, t-test. n.s., not significant. Data are representative of three or more independent experiments.

Figure 4

Postinfection administration of curcumin inhibits MUC5AC expression in middle ear epithelial cells in vitro and in vivo. (a) HMEECs were pretreated with curcumin (20 μM) 1 h prior to NTHi stimulation or posttreated with curcumin (20 μM) 1 h after NTHi stimulation. MUC5AC mRNA expression was measured after 5 h NTHi stimulation. (b) HMEECs were posttreated with 20 μM curcumin 1 h after NTHi stimulation. 12 h after NTHi stimulation, MUC5AC protein levels in cell culture supernatants were measured by ELISA. (c, d) Mice were given 50 mg/kg (i.p) curcumin 1 h prior to transtympanic inoculation of NTHi or were given 50 mg/kg (i.p) curcumin 1 h after NTHi inoculation. (c) MUC5AC mRNA expression in dissected middle ear was measured after 6 h NTHi inoculation. (d) MUC5AC protein (Alexa 488) expression in dissected middle ear was visualized by immunofluorescence assay after 9 h NTHi inoculation. DAPI, nuclear stain. Original magnification: 400x. Scale bar, 20 μm. (e) Schematic representation illustrating that curcumin suppresses NTHi-induced MUC5AC expression via MKP-1-dependent inhibition of p38 MAPK. (a, c) ^∗p < 0.05, ANOVA (Tukey's post hoc). (b) ^∗p < 0.05, t-test. n.s., not significant. Data are representative of three or more independent experiments.