Abstract
Background and Purpose: Polysaccharides from Panax ginseng C. A. Meyer (P. ginseng) are the main active component and exhibit significant intestinal anti-inflammatory activity. However, the unclear therapeutic mechanism of ginseng polysaccharide hinders the application for medicine or functional food.
Experimental Approach: In this study, a polysaccharide was isolated from P. ginseng (GP). The primary structure and morphology of GP were studied by HPLC, FT-IR spectra, and scanning electron microscopy (SEM). Further, its intestinal anti-inflammatory activity and its mechanism of function were evaluated in experimental systems using DSS-induced rats, fecal microbiota transplantation (FMT), and LPS-stimulated HT-29 cells.
Key Results: Results showed that GP restored mTOR-dependent autophagic dysfunction via modulating the structure of gut microbiota and blocking the TLR4-MyD88 pathway. Consequently, active autophagy suppressed inflammation through the inhibition of NF-κB, oxidative stress, and the release of cytokines. Conclusion and Implications: Therefore, our research provided a rationale for future investigations into the relationship between microbiota and autophagy via TLR4 and revealed the therapeutic potential of GP for inflammatory bowel disease.
Keywords: Panax ginseng C. A. Meyer; Polysaccharide; Intestinal inflammation; Gut microbiota; Autophagy.
Introduction
Inflammatory bowel disease (IBD) consists of a group of disorders including Crohn’s disease (CD) and ulcerative colitis (UC) and is a kind of recurrent, refractory gastrointestinal disease (Rooks & Garrett, 2016). At present, the incidence of IBD increases yearly. Inflammatory infiltration, redox imbalance, and gut microbiota dysbiosis are involved in the initiation, development, and exacerbation of intestinal inflammatory diseases. Intestinal epithelium plays an important role in maintaining gut homeostasis and is the main defense against pathogen invasion (Barker, 2014). It has been reported that the regulation of gut microbiota can alleviate the inflammatory response induced by dextran sodium sulphate (DSS) in mice. Lipopolysaccharide (LPS) is the main component of the cell walls of many Gram-negative bacteria. The inseparable relationship between microbiota dysbiosis, LPS content and abnormal immune response has been reported previously (Miller, Choi, Wiesner, & Bae, 2012; Rogers, Moore, & Cohen, 1985). Toll-like receptor 4 (TLR4), which is abundantly expressed in intestinal epithelial cells, is a gene coding receptor for bacterial LPS (Di Lorenzo et al., 2015; Medzhitov, Preston-Hurlburt, & Janeway, 1997). An abnormal microbiome, especially with microbes that produce LPS, triggers intestinal inflammation, and TLR4 might be the initial point of microbial interaction. Activated TLR4 recruits the downstream molecule MyD88 to trigger the phosphorylation of MAPKs (Q. Wang, Dziarski, Kirschning, Muzio, & Gupta, 2001), and it is indispensable in orchestrating the secretion of inflammatory cytokines and oxidative stress response throughout the initiation, development, and exacerbation of IBD (Russell et al., 2016; Tan, Gao, Guo, & Huang, 2014; Verkade et al., 2016).
Autophagy, a highly conserved process that evolved in eukaryotes, is involved in maintaining organism homeostasis via lysosome-mediated self-digestion and recycling of organelles and proteins (Dikic & Elazar, 2018; Mizushima, 2018). Cells trigger autophagy under various stress, such as exposure to toxic environments, starvation, and ischemiareperfusion (Netea-Maier, Plantinga, van de Veerdonk, Smit, & Netea, 2016). It has been reported that autophagy dysfunction increased susceptibility to inflammatory intestinal diseases (François et al., 2013; Hang, Lapaquette, Bringer, & Darfeuille-Michaud, 2013; Kuenzig et al., 2017; Strisciuglio et al., 2013). Repairing hampered autophagy normalized redox imbalance, increased the clearance of intracellular bacteria, and alleviated inflammation in intestinal mucosa (Schwerd et al., 2016); thus, it has become a new target of clinical drug development for IBD. Mounting evidence suggests the inseparable association between autophagy impairment and inflammation injury (Deretic, Saitoh, & Akira, 2013; Santeford et al., 2016; M. Zhou et al., 2018). mTOR, a highly conserved serine/threonine protein kinase, negatively regulates autophagy upstream, and it has demonstrated great autophagy activating and inflammation attenuating efficacy in silencing mTOR (Cosin-Roger et al., 2017). Interestingly, TLR4-MyD88-MAPK is one of the important pathways in mTOR regulation, and recent research clearly revealed the relationship between them (M. Zhou et al., 2018). Accumulating evidence indicates that autophagy and inflammation are linked by reciprocal regulation through the microflora-TLR4-mTOR axis. Mechanistically, microbiota dysbiosis activates the TLR4-MyD88-MAPK pathway, which is followed by the phosphorylation of mTOR to inhibit autophagy, thereby aggravating inflammatory injury and oxidative stress (M. Zhou et al., 2018). Although the roles of microflora in modulating inflammation and autophagy have had increased attention in regard to IBD, the development of related drugs is still in its infancy.
Panax ginseng C. A. Meyer (P. ginseng) has been widely used as an herb and functional food in the world (Li & Ji, 2018). Polysaccharide of P. ginseng has obvious beneficial effects, including gut microbiota regulation, intestinal mucosal barrier protection, autophagy promotion, and alleviation of inflammation and oxidative stress (Kim, Kim, & Park, 2020). However, the exact target and mechanism of the P. ginseng polysaccharide in gut microbiota and autophagy modulation is not well understood. Fecal microbiota transplantation (FMT) is one of the most effective ways to regulate the gut microbiota and can potentially reveal the function of microbiota and establish the causal relationship between flora and disease (Khoruts, 2018). At present, the use of FMT with P. ginseng polysaccharide to treat diseases through intestinal flora is still unknown. Therefore, the present study aims to purify crude polysaccharides from P. ginseng and to evaluate its gut microbiota and intestinal anti-inflammatory activity in dextran sulfate sodium (DSS)-induced rats with FMT. The mechanism of reciprocal regulation between inflammation and autophagy was estimated by LPS-induced inflammatory intestinal mucosal cells (HT-29 cells). Additionally, the changes in cytokines, reactive oxygen species (ROS), and autophagic proteins were detected in order to investigate the protective mechanism of the microbiota-autophagy relationship. Our study provides a reliable theoretical basis for the application of ginseng polysaccharide as a functional food material in the treatment of intestinal diseases.