Ginseng exosomes modulate M1/M2 polarisation by activating autophagy and target IKK/IкB/NF-кB to alleviate inflammatory bowel disease

Abstract

Background: Exosomes are involved in intercellular communication and regulation of the inflammatory microenvironment. In a previous study, we demonstrated that fresh ginseng exosomes (GEs) alleviated inflammatory bowel disease. However, the precise mechanism by which GEs activate the immune system and subsequently inhibit the formation of intestinal inflammatory microenvironment remains unknown.

Methods: Herein, we investigated the effects of GEs on autophagy, macrophage polarisation, intestinal inflammation, and the epithelial barrier by means of transcriptome sequencing, network pharmacology, transmission electron microscopy, immunoblotting, flow cytometry and small molecule inhibitors.

Results: GEs significantly activated autophagy and M2-like macrophage polarisation, which could be blocked by the autophagy inhibitor 3-methyladenine. In the co-culture system of macrophages and intestinal epithelial cells, macrophages treated with GEs secreted more interleukin-10 (IL-10) and significantly reduced Nitric oxide (NO) levels in intestinal epithelial cells in vitro. Furthermore, GEs acted directly on intestinal epithelial cells through the IKK/IкB/NF-кB signalling pathway to reduce inflammation and restore the intestinal barrier. Orally administered GEs could restore disrupted colonic barriers, alleviate inflammatory bowel responses, and regulate the polarisation of intestinal macrophages in vivo.

Conclusion: In summary, GEs may be a potential treatment for inflammatory bowel disease, and targeting autophagy and macrophage polarisation may help alleviate intestinal inflammation.

Keywords: Autophagy; Ginseng exosomes; Intestinal barrier; M1/M2 polarisation.

Fig. 1

Isolation and characterisation of GEs. A Schematic diagram of the process of isolating GEs from ginseng. B Specific morphology of GEs observed using transmission electron microscopy (TEM). Scale bar: 200 nm. C Potential of GEs. D Particle size of GEs

Fig. 2

Small RNA transcriptome sequencing of GEs. A Length distribution statistics of the total sRNAs fragments. B, C Secondary structures of known and unknown miRNAs, respectively. D First nucleotide bias of known miRNAs (18–30 nt) in GEs. The horizontal coordinate represents the length of the miRNA, and the vertical coordinate is the percentage of the first base in the miRNA of that length in which A/U/C/G occurs (the value at the top of the bar represents the total number of miRNAs of that length). E miRNA nucleotide bias at each position. The horizontal coordinate represents the base position of the miRNA, and the vertical coordinate is the percentage of bases A/U/C/G occurring in the miRNA at that position

Fig. 3

Detection of macrophage polarisation modulation by GEs. A Protein expression of β-actin, CD86, and CD206 was measured using western blotting (WB). B Changes in intracellular CD86, INOS,CD206 and TGF-β mRNA levels in LPS-stimulated RAW264.7 cells treated with and without different doses of GEs. Values are shown as the mean ± SD. n = 3. ^#p < 0.05, ^###p < 0.05 vs. Control; **p < 0.01, ***p < 0.001 vs. Model

Fig. 4

Determining the regulatory role of GEs in macrophage autophagy. A Confocal microscopy to detect the formation of GFP-LC3 spots. Scale bar: 10 µm. B Protein expression of β-actin, Beclin1, Atg7, LC3I/II, m-TOR, p-m-TOR, AKT, and p-AKT was measured using WB. C Protein expression of autophagy factors LC3 at different time points. D GEs increased the intracellular autophagic lysosomal activity. Scale bar: 5 µm or 2 µm. Values are shown as the mean ± SD. n = 3. ^##p < 0.01 vs. Control; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Model

Fig. 5

Activation of autophagy leads to macrophage polarisation. A Expression of CD80 and CD206 in the presence of 3-MA. B Quantile of M1/M2 with 3-MA participation. C, D TNF-α and IL-10 concentrations in macrophage supernatants. E NO levels in the supernatant of Caco-2 cells. F Protein levels of NF-κB in Caco-2 cells. G Schematic diagram of the autophagy-regulated polarisation process. Values are shown as mean ± SD. n = 3. ^#p < 0.05, ^##p < 0.01 vs. Control; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Model

Fig. 6

GEs attenuate cellular inflammation by targeting the IKK/IкB/NF-кB signalling pathway. A Cells were untreated, treated with 5 μg/mL LPS alone for 24 h, or co-treated with 5 μg/mL LPS and GEs (5, 10, or 20 μg/mL) for 24 h. WB analysis was conducted to determine protein expression. Relative expression levels of P-IKKα/β, P-IĸBα, NF-κB, Cox-2 and TNF-α . B Expression levels of the NF-кB nuclear translocator protein in intestinal epithelial cells. C After treatment with asperuloside, the expression levels of NF-кB and β-actin were measured using WB and respective antibodies. β-actin was used as a protein loading control. D FITC-dextran flux assay of Caco-2 cells after GEs treatment. Values are shown as the mean ± SD. n = 3. ^#p < 0.05, ^##p < 0.01 vs. Control; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Model

Fig. 7

A Histological staining. Scale bar: 100 µm. B Mouse intestinal length. C DAI index and Survival rate in mice. D, E Levels of IL-1β and IL-10 in intestinal tissue. Values are shown as the mean ± SD. n = 3. ^##p < 0.01, ^###p < 0.001 vs. Control; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Model

Fig. 8

GEs protect the intestinal mucosa to repair the intestinal barrier. A FITC-dextran flux. B Plasma levels of D-lactate. C Colonic tissue and serum DAO levels. D 5-HT and (E) SP. (F) Immunofluorescence detection of mouse intestinal tight junction protein (ZO-1). Scale bar: 100 µm. G Protein expression levels of the tight junction protein occludin. Values are shown as the mean ± SD. n = 3. ^#p < 0.05, ^##p < 0.01 vs. Control; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Model

Fig. 9

Effects of GEs on intestinal cell polarisation and intestinal autophagy. A Expression of CD86 and CD206 in intestinal tissue. Scale bar: 100 µm. B Expression of CD80 and CD206 in the intestinal macrophages. C Quantitative immunohistochemistry of the intestinal tissue. D Quantification of macrophage polarisation detected using flow cytometry. E WB analysis of protein expression and relative expression levels of Atg7 and LC3. Values are shown as the mean ± SD. n = 3. ^##p < 0.01, ^##p < 0.001 vs. Control; **p < 0.01, ***p < 0.001 vs. Model

Fig. 10

Ginseng exosomes alleviate IBD through the IKK/IкB/NF-кB signalling pathway. A WB analysis of protein expression and relative expression levels of P-IKKα/β, P-IĸBα, and NF-κB. B Expression levels of NF-кB nuclear transporter protein in intestinal tissues. Values are shown as the mean ± SD. n = 3. ^##p < 0.01, ^###p < 0.001 vs. Control; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Model