Fig. 9. Schematic representation of the proposed mechanism of GP on ameliorating intestinal inflammation.
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. Although the pathogenesis of IBD is ambiguous, studies have pointed out that autophagic dysfunction is a key factor in the persistence of intestinal inflammation (M. Zhou et al., 2018). Pioneer evidence from genome-wide association studies suggested that defective autophagy is associated with an increased risk of IBD (Retnakumar & Muller, 2019; Voinchet et al., 2010). Several roles of autophagy in gut inflammation have been summarized: elimination of pathogens, regulation of antigen presentation, governance of the secretion of cytokines, and maintenance of lymphocyte homeostasis. Notably, the accumulation of p62 in DSS-induced rats confirms the dysfunction of autophagy in the pathologicalprocess of intestinal inflammation. After treatment with GP, both groups of rats with intestinal inflammation presented diminished levels of phosphorylated mTOR, LC3B, and Beclin-1, which paralleled with a decrease of p62.
On the basis above, we further discussed the interaction between gut microbiome, TLR4 and autophagy, aiming to reveal the potential mechanism of the impact of GP on intestinal inflammation. In this study, 16S rRNA sequencing was used to evaluate the changes in intestinal flora before and after GP administration. The Spearman correlation coefficient was used to evaluate the correlation between high abundances of bacteria at the genus level and important characteristics of intestinal inflammation in order to explore the correlation between intestinal flora and autophagy, inflammation and oxidative stress. The results showed that the expression of TLR4 was positively correlated with four of the bacteria with high abundance in intestinal flora. p__ Verrucomicrobia−g__Akkermansia showed significant negative correlation with serum endotoxin, while displaying significant positive correlation with LC3BII/I. It can be seen that changes in intestinal flora, TLR4 pathway activation, and autophagy disorder are connected together in the occurrence and development of intestinal inflammation.
In recent years, the focus of research on intestinal flora has changed from association to modulation (Schmidt, Raes, & Bork, 2018). The most convincing experimental evidence of the role of intestinal microbiota in human diseases can be obtained from relevant experiments of intestinal flora transplantation (FMT) (Liu et al., 2021). However, there are few studies on the treatment of inflammatory bowel disease by FMT with the addition of GP. Therefore, indirect experimental animal models are needed to establish causal relationships between altered microbiomes and disease pathogenesis. In this study, FMT-related experiments showed that GP could improve the symptoms of DSS-induced intestinal inflammation by inhibiting the TLR4-NF-κB pathway and activating mTOR-dependent autophagy.
Recently, emerging research has focused on the connection between inflammation and autophagy (Deretic et al., 2013). Accumulating evidence indicates that inflammation and autophagy are linked by reciprocal regulation, and they are orchestrated by the upstream TLR4-mTOR pathway. It has been reported that inhibiting autophagy is a novel role of the TLR4-MyD88 in intestinal epithelial dysfunction. Subsequently, the release of NF-κB-induced cytokines was orchestrated by autophagy. In addition to previous research, our data also demonstrated that TLR4-MyD88 is involved in the regulation of mTOR phosphorylation, and that mTOR is a newfound intersection between inflammation and autophagy. Blocking the TLR4 pathway not only promotes the repair of autophagy, but also inhibits the disaggregation of IκBα from NF-κB. Consequently, cytokine release and oxidative stress were diminished. Importantly, autophagy also participates in reducing the degradation of the IκBα-NF-κB complex (R. Zhou, Yazdi, Menu, & Tschopp, 2011). Treatment with 3-MA deteriorated the LPS-induced inflammatory response in HT-29 cells. Meanwhile, rapamycin, an autophagy activator, ameliorated inflammation to a great extent, which further indicates that autophagy is involved in suppressing NF-κB activation.
Therefore, for the first time, this study elucidated that polysaccharide purified from P. ginseng ameliorates intestinal inflammation via the TLR4-autophagy axis through regulating gut microbiota by experiments with FMT. Our research revealed the mechanism concerning the intestinal anti-inflammatory effect of GP and provided a promising therapy for IBD.
Conclusion
The present results highlighted that the purified polysaccharides from P. ginseng showed intestinal anti-inflammatory effects in DSS-induced rats by regulating gut microbiota and mTOR-dependent autophagy. It was suggested that GP-treated intestinal flora transplantation elevated autophagy and suppressed TLR4-MAPK was associated with the downregulation of LPS-producing bacteria. Consequently, NF-κB-induced inflammation was attenuated via the activation of mTOR-dependent autophagy. The results suggested the view that GP may have potential in intestinal inflammation disease therapies, providing a foundation for the potential utilization of polysaccharide from P. ginseng for functional foods and complementary medicines.