Abstract
Worldwide the incidence of cancer has been continuing increasing. Mucositis of the gastrointestinal tract is a common side effect in patients under chemotherapy. Anticancer drug busulfan, used for treating chronic myeloid leukemia especially in pediatric patients, causes mucositis of the gastrointestinal tract. Alginate oligosaccharides (AOS) are natural products with attractive pharmaceutical potentials. We aimed to investigate, at the single-cell level, AOS preventing small intestine mucositis induced by busulfan. We found that busulfan disturbed the endoplasmic reticulum and mitochondria of cells in the small intestine, damaged cell membranes especially cell junctions, and disrupted microvilli; all of which were rescued by AOS. Single-cell RNA sequencing analysis and functional enrichment analysis showed that AOS could recover small intestinal function. Deep analysis found that AOS improved the expression of transcriptional factors which explained AOS regulating gene expression to improve small intestine function. Further investigation in IPEC-J2 cells found that AOS acts its function through mannose receptor signaling pathway. Moreover, the improved blood metabolome confirmed small intestinal function was recovered by AOS. As a natural product with many advantages, AOS could be developed to assist in the recovery of intestinal functions in patients undergoing anticancer chemotherapy or other treatments.
Introduction
The incidence of cancer has been continuing increasing worldwide.1,2,3,4Many investigations have reported that mucositis of the gastrointestinal (GI) tract is a common side effect and occurs in ~40% of cancer patients under chemotherapy.1,2,3,4 Intestinal mucositis is characterized by decreased villi length, and disruption of crypt cell homeostasis and tight junction proteins in the small intestinal mucosa.2,4 The epithelium of the mammalian small intestine is a highly ordered and structured tissue with repeated crypt-villus units along the axis. Intestinal stem cells are located at or near the base of crypts and divide to produce transit-amplifying cells (TAs). TAs then develop, following proliferation and differentiation, into five main cell types (enterocytes, goblet cells, Paneth cells, enteroendocrine (EED) cells, and tuft cells).5,6,7,8Enterocytes, the most numerous villus cell type, produce the digestive enzymes and transporters for the digestion and absorption of nutrients, respectively, and also protect the body from the harsh bacterial-rich environment.5,9,10 Goblet cells and Paneth cells play very important roles in mucosal defense because they are mucus-secreting cells and defensin-secreting cells, respectively. EED cells regulate hormone secretion to control GI processes. Tuft cells are chemosensory cells expressing taste receptors like α-gustducin and TRPM5.11 All these five types of cells are tightly structured in the crypt-villus.5,6,11 Mucositis may lead to morbidity and even mortality, because the GI tract is a barrier that protects the body from pathogenic microbes,6,9,10,12,13,14 and it plays vital roles in the digestion and absorption of nutrients, the secretion of mucus and hormones, and interaction with commensal microbiota.6,10
Alginate oligosaccharides (AOS) are excellent natural products derived from the degradation of alginate. They are attracting great attention from a pharmaceutical perspective15,16,17 because of their following benefits: anti-inflammatory,16 anti-apoptosis,18 anti-proliferation,19antioxidant activities,15,18,20 and even anti-cancer properties.21 AOS benefits intestinal morphology and barrier function by increasing the length of intestinal villi, the content of secretory immunoglobulin A, and the number of Goblet cells.22 However, the underlying mechanisms of how AOS improve small intestine morphology and function from the single intestinal cell level is unknown.
Busulfan, an alkylating agent and an effective chemotherapeutic drug, has been used for patients with chronic myeloid leukemia especially for children (under 3 years of age). Moreover, it has been used for myeloablative-conditioning regimens before stem cell transplantation.12,13,23 Busulfan was used to produce the small intestine mucositis animal model in current investigation because it causes mucositis in patients.12,13,14 Many investigations have attempted to reduce chemotherapy-induced intestinal disruption by using prebiotics, probiotics, selenium, volatile oils, and others,1,2,24,25 however, these efforts have not been successful.26,27 Therefore, new approaches or new medicines are urgently needed to assist in the recovery following mucositis in cancer patients (especially pediatrics) under chemotherapy. The purpose of this investigative was to explore the improvement of small intestine by AOS after busulfan treatment and the underlying mechanisms at the single-cell level.
Results
AOS rescued the cellular damage caused by busulfan
There were four treatment groups (AOS 0, AOS 10, B + A 0, B + A 10 mg/kg body weight) in this investigation as stated in the “Materials and methods” section. AOS 10 mg/kg had some effects on the small intestine at the histopathological and ultrastructural levels, and gene expression levels. However, the beneficial effects on murine intestine was not so obvious as in the mice treated by busulfan (B + A 10 mg/kg). In order to show the rescue effects of AOS, AOS 10 mg/kg was removed from the following data analysis. From the ultrastructure of the small intestine, it was clear that busulfan treatment damaged the small intestinal cells, causing swelling of the ER and mitochondria, a decrease in the number of desmosomes on the cell membrane (cell–cell junctions), and a reduction in the density of microvilli (Fig. 1a). AOS (B + A 10) rescued the busulfan induced damage by reducing swelling in the ER and mitochondria, assisting recovery in the number of desmosomes on cell–cell junctions, and elevating the density of microvilli (Fig. 1a). Histopathology of small intestinal tissues are presented in Fig. S1a. The cellular damage caused by busulfan was also shown by an increase in the protein levels of caspase 8 and p-PTNE, with a concurrent decrease in the protein levels of Bcl-2 (Fig. 1b). However, the protein levels of caspase 8 and p-PTEN were decreased while the protein level of Bcl-2 was increased by AOS in B + A 10 (Fig. 1b). The microvilli also were recovered under AOS treatment in B + A 10 with an increase in the protein level of villi (Fig. 1c). The desmosome protein DSG2 (desmoglein 2) was significantly increased by AOS in B + A 10 compared to busulfan treatment alone (B + A 0 group; Fig. 1d) which indicated that desmosome recovery was assisted by AOS. The data here suggested that busulfan caused the small intestine mucositis which was prevented by AOS. Next, we set out to explore the underlying mechanisms by which AOS rescues cellular damage caused by busulfan.