The administration of Escherichia coli Nissle 1917 ameliorates irinotecan–induced intestinal barrier dysfunction and gut microbial dysbiosis in mice
Introduction
Irinotecan, a topoisomerase I inhibitor, is widely used in the treatment of advanced colorectal cancer. However, almost 50–80% of patients receiving irinotecan develop intestinal mucositis with diarrhea as the most common manifestation, which severely compromises the living quality of patients [1]. The intestinal damage induced by irinotecan is featured by increased apoptosis in crypts and loss of villi [2]. Irinotecan is metabolized mainly by hepatic carboxylesterases to form an active metabolite, SN-38, which is subsequently metabolized by uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1) into SN-38-glucuronide (SN-38G) [3]. After being secreted into the intestine, SN-38G is reactivated into SN-38 by β-glucuronidase-positive Enterobacteriaceae. It is believed that SN-38 is involved in the development of irinotecan-induced mucositis by damaging intestinal mucosal epithelial cells [4].
Tight junctions localized in the apical–lateral margin of adjacent enterocytes play a vital role in maintaining intact intestinal barrier function [5]. Tight junctions consist of transmembrane proteins (Occludin and Claudins) and peripheral membrane proteins (ZO). ZO proteins (ZO-1, ZO-2, and ZO-3) are cytosolic scaffolds that anchor peripherally located transmembrane proteins to the actin cytoskeleton and form the complete tight junction complex [6]. Alteration of tight junction contributed to intestinal barrier injury under pathological conditions, such as inflammatory bowel diseases [7] and intestinal infections [8]. In addition, a series of evidence suggested that the intestinal barrier injury caused by chemotherapeutic reagents is closely related to the disruption of tight junctions. Irinotecan significantly decreased Claudin-1 and Occludin expression in rats [9]. Besides, methotrexate treatment increased the intestinal permeability in rats, which is partially related to the reduction of Claudin-1 and Occludin expression [8].
Gut microbiota plays a vital role in maintaining the homeostasis of gastrointestinal tract. Natural intestinal microbiota interacts with the mucosal epithelium and performs diverse physiological functions such as regulation of immune and inflammatory response, modulation of integrity of the gut barrier and maintenance of substance metabolism [10]. Alteration of microbiota is associated with multiple diseases, such as gastrointestinal diseases [11] and metabolic diseases [12]. In addition, chemotherapy significantly induced the modification of gut microbiota. For instance, irinotecan increased the relative abundance of Fusobacteria and Proteobacteria in Sprague-Dawley rats [13]. Gut microbial dysbiosis is believed to be involved in the pathogenesis of intestinal damage induced by multiple factors [14]. Thus, balancing intestinal microbial homeostasis may be an alternative method to improve intestinal status.
Probiotics are defined as “Live microorganisms, which, when consumed in adequate amounts, confer a health benefit on the host” [15]. Recently, probiotics have been used to alleviate intestinal pathological state induced by chemotherapy. For example, Saccharomyces cerevisiae UFMG A-905 (Sc-905) prevented weight loss and intestinal lesions as well as maintained integrity of the mucosal barrier in irinotecan-treated mice [2]. Escherichia coli Nissle 1917 (EcN), a Gram-negative probiotic, has been widely studied for its therapeutic potential against a plethora of intestinal disorders,particularly in maintaining remission of ulcerative colitis [16]. The anti-inflammatory effect of EcN has been sufficiently illustrated [17]. In addition, EcN reinforces the intestinal epithelial barrier through upregulating tight junction proteins and increasing the expression of antimicrobial factors such as β-defensin-2 [5,[18], [19], [20]]. Furthermore, EcN was able to attenuate the DSS-induced gut microbial dysbiosis in mice, featured by an increase in bacterial diversity [21].
However, it still remains unclear whether EcN exerts protective effect against irinotecan-induced intestinal barrier dysfunction and gut microbial dysbiosis. Thus, we investigated the effect of EcN on the status of tight junction proteins and intestinal microbiota in mice receiving irinotecan. Besides, we further investigated the effect of EcN culture supernatant on epithelial barrier function in Caco-2 monolayers incubated with SN-38.
Section snippets
Reagents and probiotics
Escherichia coli Nissle 1917 was purchased from Ardeypharm (Herdecke, Germany), irinotecan and SN-38 were obtained from MedChem Express (Princeton, USA), FITC-Dextran (4000 Da) was provided by Sigma–Aldrich (USA). The primary antibodies were purchased from the following companies: ZO-1 (Invitrogen, USA); Occludin (Invitrogen, USA); Claudin-1 (Invitrogen, USA); GAPDH (CST, USA). Species-specific secondary antibodies were obtained as follows: anti-rabbit and anti-mouse antibodies (CST, USA);
EcN reduced weight loss and diarrhea in irinotecan-treated mice
As shown in Fig. 1A, compared to control and irinotecan groups, mice pre-treated with EcN in the first 9 days did not suffer weight loss. However, irinotecan significantly induced weight loss compared with control group, and EcN administration attenuated the weight loss at Day 14. In addition, diarrhea was first observed 4 days after the first dose of irinotecan injection in irinotecan-treated mice. While co-treatment with EcN remarkably attenuated the diarrhea severity compared to irinotecan
Discussion
Chemotherapy-induced gut toxicity is intensively related to the alteration in intestinal barrier function and gut microbiota. Recent studies indicated that EcN improved intestinal barrier function and regulated the disturbed gut microbiota in DSS mice [19,21]. So we hypothesized that EcN might protect against the gut damage resulted from irinotecan. In the present study, we showed that EcN ameliorated irinotecan-induced tight junction disruption and gut microbiota dysbiosis. In addition,
Conclusion
To our knowledge, this is the first study to analyze the protective effect of EcN against irinotecan-induced intestinal injury. Our data suggested that the prophylactic administration of EcN ameliorated irinotecan-induced intestinal barrier dysfunction and gut microbial dysbiosis. The protective effect of EcN against intestinal barrier dysfunction elicited by irinotecan might be mediated by the modulation of Claudin-1. Additionally, EcN restored the decreased diversity of gut microbiota and the
Acknowledgments
This work is supported by the grant for the protective effect of hydrogen sulfide on the intestinal barrier and multiple organ dysfunction in septic shock and the underlying mechanisms from The National Natural Science Foundation of China (8177030343).
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
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Contributed equally.