Probiotic Streptococcus salivarius K12 Alleviates Radiation-Induced Oral Mucositis in Mice

Abstract

Background: Oral mucositis is the most common oral complication of cancer patients receiving radiotherapy and/or chemotherapy, leading to poor quality of life. Limitations of the current interventions on radiation-induced oral mucositis (RIOM) urge the development of novel therapeutics. Here, we evaluated the treatment outcome of probiotic Streptococcus salivarius K12 on RIOM mice, and oral microbiota that is associated with the progress of RIOM was further investigated.

Methods: An experimental RIOM mouse model was established, and S. salivarius K12 was applied to the mouse oral cavity daily. Histological analyses were performed to evaluate the severity of oral mucositis and the treatment outcome of S. salivarius K12. The oral microbiota of mice was further analyzed by 16S rRNA sequencing, microbial culture and qPCR.

Results: Irradiation induced conspicuous mucositis in the oral cavity of mice. S. salivarius K12 treatment was beneficial for the healing of RIOM, as reflected by reduced ulcer size, increased basal layer epithelial cellularity and mucosal thickness, and elevated epithelial proliferation and attenuated apoptosis. RIOM mice presented significant oral microbial dysbiosis, with an overgrowth of oral anaerobes. S. salivarius K12 treatment reconstituted the oral microbiota and decreased the abundance of oral anaerobes of RIOM mice. In addition, S. salivarius K12 treatment inhibited NI1060 in Pasteurella genus and downregulated the expression of nitrate reductase.

Conclusions: S. salivarius K12 treatment can alleviate RIOM and reconstituted the dysbiotic oral microbiota in mice. S. salivarius K12 may represent a promising adjuvant treatment to improve the quality of life of cancer patients receiving radiotherapy.

Keywords: Streptococcus salivarius K12; dysbiosis; oral mucositis; probiotics; radiotherapy.

Figure 1

Streptococcus salivarius K12 alleviates body weight loss and reduces tongue ulcer area in RIOM mice. (A) Average body weight loss. (B) Total body weight loss. (C) Toluidine blue staining of harvested tongues. The area of mucositis with (red arrow) and without ulcer (green arrow) was stained blue. Blue arrow: staining at the site of incision to remove tongue. (D) Quantitative analyses of mucositis area (mucositis+ulcer/whole surface area). (E) Quantitative analyses of ulcer area (ulcer/whole surface area). Data are presented as mean ± SD. ***P < 0.001. N = 5 per group.

Figure 2

Streptococcus salivarius K12 promotes RIOM healing in mice. (A) Representative images of H&E staining indicating the integrity of lingual mucosa (Scale bar, 100 µm). (B, C) Quantitative analysis of mucosal thickness of ventral tongues and dorsal tongues, respectively. (D, E) Basal layer epithelial cellularity of ventral tongues and dorsal tongues, respectively. Data are presented as mean ± SD. N=6 per group. One-way ANOVA test followed by Tukey’s test. ***P<0.001.

Figure 3

Streptococcus salivarius K12 promoted the proliferation of mouse tongue basal layer cells and reduced apoptosis of mouse tongue mucosal cells. (A) Representative microscopic images of mouse tongues PCNA staining (Scale bar, 100 µm). (B) Percentage of basal layer PCNA positive cells in mouse ventral tongues. (C) Percentage of basal layer PCNA positive cells in mouse dorsal tongues. (D) Representative microscopic images of TUNEL staining on mouse tongues (Scale bar, 30 µm). (E) Percentage of TUNEL-positive cells in mouse ventral tongues. (F) Percentage of TUNEL-positive cells in mouse dorsal tongues. Data are presented as mean ± SD. N=6 per group. One-way ANOVA test followed by Tukey’s test. ***P < 0.001.

Figure 4

Streptococcus salivarius K12 modulates oral microbiota in RIOM mice. (A) The alpha diversity of oral microbiota. (B) Principal coordinate analysis (PCoA) of oral microbiota based on Bray-Curtis distance. (C) Prevalent genus with significant difference in abundance. Values are presented as median, interquartile range, minimum, and maximum. Kruska-Wallis H test with post hoc tests applying the Dunn’s test for multiple comparisons. (D) qPCR quantification of NI1060 (mean ± SD). One-way ANOVA test followed by Tukey’s test. N=6 per group. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Streptococcus salivarius K12 suppresses the overgrowth of oral anaerobes in RIOM mice. (A) Total bacteria in oral swabs were quantified by qPCR normalized as 16S/18S rRNA. (B) Total cultivable oral anaerobes in oral swabs. (C) Relative expression levels of nitrate reductase napA gene of oral microbiota. Data are presented as mean ± SD. N=6 per group. One-way ANOVA test followed by Tukey’s test. ***P < 0.001.