Sequential Role of Apoptosis, Gram‐negative Enterobacteria, and Inflammatory Cytokines in the Pathogenesis of 5‐Fluorouracil‐induced Intestinal Mucositis in Mice

Abstract

Background & AimCancer chemotherapy frequently causes intestinal mucositis accompanied by severe diarrhea. While several pathogenic factors such as apoptosis, enterobacteria, various inflammatory responses, and hypoproliferation are considered to be involved in the pathogenesis of intestinal mucositis, the detailed mechanisms have not been investigated. 5‐fluorouracil (5‐FU), a widely used chemotherapeutic agent, induces clinical manifestations of intestinal mucositis in 50–80% of treated patients. The present study investigated the pathogenic mechanisms of 5‐FU–induced intestinal mucositis in mice, especially in relation to apoptosis, enterobacteria, and inflammatory responses.MethodsIntestinal mucositis was induced in male C57BL/6 mice by repeated administration of 5‐FU (50 mg/kg, i.p.) for 6 days. Ampicillin (250 mg/kg), a broad‐spectrum antibiotic, and aztreonam (50 mg/kg), a gram‐negative bacteria‐selective antibiotic, were administered p.o. twice daily for 6 days. The severity of mucositis was evaluated histologically while apoptosis, myeloperoxidase (MPO) activity, and cytokine expression were determined during 5‐FU treatment. The intestinal microbiota was analyzed for 16S ribosomal RNA with a next‐generation sequencer. The effect of 5‐FU on TNF‐α expression was examined in cultured colonic epithelial cells (YAMC: Young adult mouse colon).ResultsRepeated administration of 5‐FU caused severe intestinal mucositis, characterized by shortening of villi and destruction of crypts by day 6. TUNEL‐positive apoptotic cells were detected in crypts on day 1 but this response was diminished from day 2 and thereafter. The evident increase in MPO activity and interleukin (IL)‐1β expression was observed from day 4 while upregulation of TNF‐α expression was detected on day 1 and further augmented from day 4. During 5‐FU treatment, daily administration of ampicillin and aztreonam significantly reduced the severity of intestinal mucositis, the increase in MPO activity, and cytokine upregulation on day 6 without any effect on apoptosis induction and upregulation of TNF‐α on day 1. The analysis of intestinal microbiota showed that 5‐FU treatment caused disruption of the microbiota, notably decreasing Firmicutes and increasing Bacteroides, but these changes were improved by daily administration of ampicillin. The exposure of 5‐FU to YAMC cells upregulated the expression of TNF‐α mRNA.ConclusionThese findings suggest that crypt apoptosis, gram‐negative bacteria, and inflammatory cytokines are sequentially involved in the occurrence of 5‐FU‐induced intestinal mucositis. The apoptosis induces microbiota abnormality via disruption of epithelial barrier, resulting in upregulation of inflammatory cytokines. The early upregulation of TNF‐α, which is directly induced by 5‐FU, may lead to apoptosis via an intrinsic cytokine‐mediated pathway.Support or Funding InformationWe thank Dr. Robert Whitehead for permission to use and providing YAMC cells. We also thank Biofermin Co., Ltd. for technical assistance in analyzing intestinal microbiota.