Yan-Hou-Qing formula attenuates ammonia-induced acute pharyngitis in rats via inhibition of NF-κB and COX-2

Abstract

Background: Yan Hou Qing (YHQ) is a Chinese medicinal formula designed to alleviate sore throat symptoms, but underlying mechanism of YHQ treatment for pharyngitis is poorly defined up to now.

Methods: In this study, the modulation of YHQ on pharyngitis is investigated in ammonia-induced acute pharyngitis rat models. After treatment with YHQ or dexamethasone respectively for five consecutive days, all rats were sacrificed for biomolecular and histopathologic study. Protein expressions of MAPKs, NF-κB, COX-2 and 5-LOX in pharyngitis tissue were evaluated by western blot analysis and the levels of TNF-α, IL-6, prostaglandin (PG) E₂, leukotrienes (LT)-B₄ and LT-D₄ in pharyngeal tissue were measured via ELISA assay. Evans blue (EB) dye exudation test was performed parallelly to assess the integrity of pharyngeal tissue.

Results: Compared with normal control group, EB dye exudation, and inflammatory cytokines in the model group were significantly increased, and the pharynx tissue was obviously infiltrated by inflammatory cells. YHQ treatment improved the inflammatory infiltrate in pharyngeal tissue, and reduced EB dye exudation in AP rat models. The up-regulated TNF-α and IL-6 in pharyngeal tissue of AP were significantly reduced by YHQ through inhibition of phosphorylation of p38, Erk and NF-κB. YHQ treatment also reversed the increased level of PGE₂ through down-regulation of COX-2.

Conclusions: YHQ formula attenuated the pharyngitis related symptoms via suppression of COX-2 and phosphorylation of p38, Erk and NF-κB (p65).

Keywords: Inflammation; NF-κB; COX-2; Pharyngitis; Yan Hou Qing.

Fig. 1

Representative HPLC-DAD chromatogram (at 279 nm) of the chemical standard of harpagoside (a), cinnamic acid (b) and YHQ formula (c)

Fig. 2

Schematic diagram of acute pharyngitis induction and YHQ treatment

Fig. 3

Modulation of YHQ on behaviors (a-f) and pharynx pathology (e-k) in ammonia-induced acute pharyngitis. Data are shown as the median with interquartile rage. Statistical analysis were conducted via one-way ANOVA followed by a Tukey’s Multiple Comparison test (^####p < 0.0001 vs. NC, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. AP)

Fig. 4

Effect of YHQ on ammonia-induced morphological damage of rat pharynx. a Evans Blue (EB) dye lesion due to morphological damage of rat pharynx; b Variations of EB dye in pharyngeal tissue. Data are shown as the mean ± SEM. Statistical analysis were conducted via one-way ANOVA followed by a Tukey’s Multiple Comparison test (^####p < 0.0001 vs. NC; ** p < 0.01, *** p < 0.001 vs. AP)

Fig. 5

The Effect of YHQ on ammonia-induced pharyngitis in rats. Pharynx tissue slices were fixed, embedded, and stained with HE. Microscope images were displayed at a magnification of 40 × (a) and 400 × (b)

Fig. 6

The effects of YHQ on TNF-α and IL-6 levels in pharynx tissue of rats. (a) Levels of TNF-α; (b) Levels of IL-6. Data are shown as the mean ± SEM. Statistical analysis were conducted via one-way ANOVA followed by a Tukey’s Multiple Comparison test (^##p < 0.01, ^###p < 0.001 vs. NC; *p < 0.05, ***p < 0.001 vs. AP)

Fig. 7

The effects of YHQ on LT-B₄ and LT-D₄ levels in pharynx tissue of rats. a Levels of LT-B4; b Levels of LT-D4. Data are shown as the mean ± SEM. Statistical analysis were conducted via one-way ANOVA followed by a Tukey’s Multiple Comparison test (^###p < 0.001 vs. NC; **p < 0.01, ***p < 0.001 vs. AP)

Fig. 8

The effects of YHQ on PGE2 levels and COX-2 expression in pharynx tissue of rats. Data are shown as the mean ± SEM. Statistical analysis were conducted via one-way ANOVA followed by a Tukey’s Multiple Comparison test (^###p < 0.001 vs NC; ***p < 0.001 vs. AP)

Fig. 9

Effects of YHQ on phosphorylated IKKβ (p- IKKβ), p-IκBα and p-NF-κB expression in pharynx tissue of rats. Data are shown as the mean ± SEM. Statistical analysis were conducted via one-way ANOVA followed by a Tukey’s Multiple Comparison test (^####p < 0.0001 vs. NC; ****p < 0.0001 vs. AP)

Fig. 10

The translocation of p65 to the nucleus was analyzed by confocal microscopy. Cells were immunostained using FITC for NF-κB p65 and DAPI for nucleus

Fig. 11

Effects of YHQ on phosphorylated p38 (p-p38), p-Erk and p-JNK expression in pharynx tissue of rats. Data are shown as the mean ± SEM. Statistical analysis were conducted via one-way ANOVA followed by a Tukey’s Multiple Comparison test (^####p < 0.0001 vs. NC; **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. AP)