S100a9 is involved in the alleviation of OMT toward radiation-induced oral mucositis. All mice in the following experiments were housed in tailor-made cages. (A) The mRNA expression profile in tongue tissues from mice with or without OMT after head and neck irradiation by high throughput sequencing. (B) The expression levels of target genes which down-regulated following irradiation exposure and up-regulated after OMT were validated by qRT-PCR. Mean ± SEM. n = 12 per group. (C) Tissue sampling time points and scheme for sh-S100a9 injection combined with irradiation and OMT. (D) Mice that received sh-S100a9 plasmid were sacrificed 24 h after the retro-orbital sinus injection, and the luciferase activity in various organs (colon, small intestine and tongue) was detected by bioluminescent imaging. (E) The expression level of S100a9 was examined in tongue tissues by qRT-PCR. n = 12 per group. (F) Body weights were compared among three group mice after THI. Significant differences are shown relative to the “THI + OMT” group using two-way group ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.005). n = 18 per group. (G) The morphology of the tongue tissue was shown by H&E and immunohistochemistry staining among THI, THI + OMT and THI + OMT + sh-S100a9 group. Scale bar = 100 μm for first line, bar = 50 μm for second and third line. The solid diagonal line indicates the ulcer boundary, and dotted lines indicate the basement membrane. (H) The staining intensity of immunohistochemistry staining (CD45 and PCNA). (I) Photograph of mice in the three groups. The arrows point to the radiation-induced alopecia. (J-L) The expression levels of IL-1 (J), IL-6 (K) and TNFɑ (L) were examined in tongue tissues by quantitative PCR. Mean ± SEM. Significant differences are indicated: * P < 0.05, ** P < 0.01, *** P < 0.005 compared with THI + OMT; Student’s t-test, n = 8 per group. (M) The expression levels of S100A9 in peritumor tissues and tumor tissues of head and neck squamous carcinoma patients. (N) The overall survival rate of head and neck squamous carcinoma patients based on the expression of S100A9.