Apoptosis, Dysbiosis and Expression of Inflammatory Cytokines are Sequential Events in the Development of 5‐Fluorouracil‐Induced Intestinal Mucositis in Mice

Abstract

The chemotherapeutic agent 5-fluorouracil (5-FU) causes intestinal mucositis with severe diarrhoea, but the pathogenesis is not fully understood. In this study, we investigated the pathogenic effects of 5-FU in mice, focusing on apoptosis, enterobacteria and inflammatory cytokines. Repeated administration of 5-FU caused severe intestinal mucositis on day 6, accompanied by diarrhoea and body-weight loss. TNF-α expression increased 1 day after exposure to the drug, and spiked a second time on day 4, at which point myeloperoxidase activity and IL-1β expression also increased. Apoptotic cells were observed in intestinal crypts only on day 1. 5-FU also induced dysbiosis, notably decreasing the abundance of intestinal Firmicutes while increasing the abundance of Bacteroidetes and Verrucomicrobia. Twice-daily co-administration of oral antibiotics significantly reduced the severity of intestinal mucositis and dysbiosis, and blocked the increase in myeloperoxidase activity and cytokine expression on day 6, without affecting apoptosis and TNF-α up-regulation on day 1. In cultured colonic epithelial cells, exposure to 5-FU also up-regulated TNF-α expression. Collectively, the data suggest that crypt apoptosis, dysbiosis and expression of inflammatory cytokines are sequential events in the development of intestinal mucositis after exposure to 5-FU. In particular, 5-FU appears to directly induce apoptosis via TNF-α and to suppress intestinal cell proliferation, thereby resulting in degradation of the epithelial barrier, as well as in secondary inflammation mediated by inflammatory cytokines.

Probiotic Bifidobacterium bifidum G9‐1 attenuates 5‐fluorouracil‐induced intestinal mucositis in mice via suppression of dysbiosis‐related secondary inflammatory responses

“…Repeated administration of 5‐FU significantly decreased the abundances of Firmicutes and Actinobacteria , which are mostly comprised of gram‐positive bacteria, while significantly increasing the abundances of Bacteroidetes and Proteobacteria , which are mostly gram‐negative bacteria. Similar alterations to the intestinal microbiota were recently reported in human subjects during cancer chemotherapy and in experimental animals receiving 5‐FU treatment . Toll‐like receptor 4 signalling is reportedly involved in the pathogenesis of anti‐cancer agent‐induced gastrointestinal toxicities .…”

“…We previously showed that apoptosis can be detected in the intestinal crypt within 24 hours of the first injection of 5‐FU, suggesting that apoptosis is a critical event for the initiation of 5‐FU‐induced intestinal mucositis . In addition, secondary inflammatory responses are believed to occur resulting in the disruption of the epithelial barrier, which is induced by apoptosis and hypoproliferation . Indeed, several studies have demonstrated that inflammatory responses such as increases in MPO activity and inflammatory cytokine expression are involved in the pathogenesis of 5‐FU‐induced intestinal mucositis .…”

“…These findings suggest that BBG9‐1 cannot prevent the induction of apoptosis but can suppress the secondary inflammatory responses during the progression of 5‐FU‐induced intestinal mucositis. We recently showed that antibiotics significantly reduced the severity of intestinal mucositis, mainly by suppressing secondary inflammatory responses, without any effect on the induction of apoptosis or early TNF‐α upregulation on the day following the onset of 5‐FU treatment . Thus, it can be assumed that the preventive effect of the BBG9‐1 occurs through the prevention of microbiota‐related secondary inflammatory responses.…”

“…The entire small intestine (from pylorus to ileocecal junction) was collected 24 hours after the final injection of 5‐FU (on day 6 following the onset of 5‐FU treatment), and the length of the small intestine was measured. Our recent study showed that severe intestinal mucositis can be observed on day 6 following the onset of 5‐FU treatment . Since further treatment with 5‐FU often causes death, we evaluated the severity of intestinal mucositis on day 6.…”

“…Our previous study also indicated that apoptosis plays a central role in the initial event that results in the development of 5‐FU‐induced intestinal mucositis via the production of ROS and TNF‐α . Further, inflammatory cytokines including TNF‐α are involved in the secondary inflammation that occurs after the disruption of the intestinal barrier, which is caused by apoptosis …”