A Phase II Randomized Clinical Trial and Mechanistic Studies Using Improved Probiotics to Prevent Oral Mucositis Induced by Concurrent Radiotherapy and Chemotherapy in Nasopharyngeal Carcinoma

Abstract

Earlier evidence has proven that probiotic supplements can reduce concurrent chemoradiotherapy (CCRT)-induced oral mucositis (OM) in nasopharyngeal cancer (NPC). The incidence of severe OM (grade 3 or higher) was the primary endpoint in this study. We first enrolled 85 patients with locally advanced NPC who were undergoing CCRT. Of them, 77 patients were finally selected and randomized (1:1) to receive either a probiotic cocktail or placebo. To investigate the protective effects and the mechanism of probiotic cocktail treatment on OM induced by radiotherapy and chemotherapy, we randomly divided the rats into the control (C) group, the model (M) group, and the probiotic (P) group. After treatment, samples from the tongue, blood, and fecal and proximal colon tissues on various days (7th, 14th, and 21st days) were collected and tested for the inflammatory response, cell apoptosis, intestinal permeability, and intestinal microbial changes. We found that patients taking the probiotic cocktail showed significantly lower OM. The values of the incidence of 0, 1, 2, 3, and 4 grades of OM in the placebo group and in the probiotic cocktail group were reported to be 0, 14.7, 38.2, 32.4, and 14.7% and 13.9, 36.1, 25, 22.2, and 2.8%, respectively. Furthermore, patients in the probiotic cocktail group showed a decrease in the reduction rate of CD3+ T cells (75.5% vs. 81%, p < 0.01), CD4+ T cells (64.53% vs. 79.53%, p < 0.01), and CD8+ T cells (75.59 vs. 62.36%, p < 0.01) compared to the placebo group. In the rat model, the probiotic cocktail could ameliorate the severity of OM, decrease the inflammatory response, cause cell apoptosis and intestinal permeability, and restore the structure of gut microbiota to normalcy. In conclusion, the modified probiotic cocktail significantly reduces the severity of OM by enhancing the immune response of patients with NPC and modifying the structure of gut microbiota. Clinical Trial Registration: The Clinical Trial Registration should be the NCT03112837.

Keywords: intestinal microbiota; nasopharyngeal cancer; oral mucositis; probiotics; radiotherapy and chemotherapy.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Xia, Jiang, Li, Wei, Hong, Li, Feng, Wei, Xin and Chen.

Figures

Figure 1

Selection of probiotic strains. (A) The non-metric multidimensional scaling (NMDS) analysis of healthy people (HP) from employees of the Jiangxi Cancer Hospital, tumor patients (TP) from the Jiangxi Cancer Hospital, and non-cancer people (NT) (healthy residents at the cancer-free village). (B) The Venn diagram of the intestinal microbiota among HP, TP, and NT. (C) The relative abundance of the bacteria among groups HP, TP, and NT. (D) The relative abundance of bacteria among the HP, TP, and NT groups with quantitative real-time PCR (qRT-PCR). The data are presented as means ± SD, where ***p < 0.001 and **** p < 0.0001.

Figure 2

Patients included in the study.

Figure 3

The combination of probiotics reduced oral mucositis (OM) by improving the immunity of patients with nasopharyngeal cancer (NPC). (A) Mucosal index. (B) Reduction rate of CD3+T cells. (C) Reduction rate of CD4+T cells. (D) Reduction rate of CD8+T cells. (E) Reduction rate of lymphocyte. (F) Reduction rate of hemoglobin. (G) Weight loss. The data are presented as means ± SD, where **p < 0.01.

Figure 4

Effects of the combination of probiotics on the composition of bacterial communities in patients with NPC. (A) The relative abundance of bacteria among HP from the employees of the Jiangxi Cancer Hospital, before the treatment of radiotherapy plus chemotherapy plus a placebo (BRCP), after treatment with radiotherapy plus chemotherapy plus a placebo (ARCP), before the treatment of radiotherapy plus chemotherapy plus the probiotic combination (BRCPM), and after treatment with radiotherapy plus chemotherapy plus the probiotic combination (ARCPM). (B) The Venn diagram of the intestinal microbiota among HP, BRCP, ARCP, BRCPM, and ARCPM groups. (C) The NMDS analysis of groups HP, BRCP, ARCP, BRCPM, and ARCPM.

Figure 5

The probiotic cocktail relieved tongue tissue inflammatory response in rats with OM caused by radiotherapy and chemotherapy. (A) Mucosal index. (B) The expression of IL-6 in the mRNA level. (C) The expression level of TNF-α in the mRNA level. (D) The expression of IL-1β in the mRNA level. (E) H&E staining of the tongue tissue among the C, M, and T groups on the 7th, 14th, and 21st days. The data are presented as means ± SD, where *p < 0.05, **p < 0.01, and ##p < 0.01.

Figure 6

The combination of probiotics ameliorated the upregulation of TLR4/NF-κB, tongue tissue apoptosis and improved the expression of intestinal tight junction (TJ) in rats with OM caused by radiotherapy and chemotherapy. (A) Protein expression level of TLR4, P-NF-κB, and NF-κB. (B) Protein expression level of apoptosis-associated factors, Bcl-2 and Bax. (C) The expression level of intestinal TJ proteins, ZO-1 and claudin-1. The data are presented as means ± SD, where *p < 0.05, **p < 0.01, #p < 0.05, and ##P < 0.01.

Figure 7

Effects of probiotic cocktail on the composition of bacterial communities in rats with OM caused by radiotherapy and chemotherapy. (A) Shannon index of intestinal bacterial communities among C, M, and T groups at days 7, 14, and 21. (B) Simpson index of intestinal bacterial communities among C, M, and T groups on the 7th, 14th, and 21st days. (C) Principle coordinate analysis (PCoA) of intestinal bacterial communities among C, M, and T groups on the 7th, 14th, and 21st days. (D) Relative abundance of Bacteroidetes of intestinal bacterial communities at the phylum level among C, M, and T groups on the 7th, 14th, and 21st days. (E) Relative abundance of Firmicutes of intestinal bacterial communities at the phylum level among C, M, and T groups on the 7th, 14th, and 21st days. (F) Relative abundance of Bacteroidetes of intestinal bacterial communities at the genus level among C, M, and T groups on the 7th, 14th, and 21st days. (G) Relative abundance of Lachnospiraceae of intestinal bacterial communities at the genus level among C, M, and T groups on the 7th, 14th, and 21st days. (H) Relative abundance of Ruminococcus of intestinal bacterial communities at the genus level among the C, M, and T groups on the 7th, 14th, and 21st days.