Atractylodes macrocephala-derived extracellular vesicles-like particles enhance the recovery of ulcerative colitis by remodeling intestinal microecological balance

Abstract

Current treatment of ulcerative colitis (UC) remains challenging, with the mainstay of therapy being 5-aminosalicylic acid-based drugs, which have limited and inconsistent results. Atractylodes macrocephala (AM) is a traditional Chinese medicine commonly used in the clinical treatment of various inflammatory diseases. Herein, we demonstrate that AM-derived extracellular vesicle-like particles (AMEVLP) can effectively modulate the gut microbiota, thereby significantly improving the treatment efficiency of UC. This is achieved by enhancing the alpha diversity of the gut microbiota and re-establishing beneficial types, which in turn alter tryptophan metabolism, leading to an increase in indole derivatives within the gut. This process also protects the gut barrier and exerts anti-inflammatory effects. The mechanism behind these anti-inflammatory effects is closely associated with the Th17 cell differentiation signaling pathway. It is believed that the AMEVLP enable them to efficiently remodel gut microbiota, providing an avenue for the treatment of various inflammatory diseases. Significantly, preliminary clinical trials have shown that AMEVLP can substantially slow the progression of the disease in UC patients.

Keywords: Atractylodes macrocephala; Extracellular vesicle-like particles; Inflammation; Intestinal microecological; Ulcerative colitis.

Scheme 1

Diagram depicting the role of and the AMEVLP mechanism of action for the relief of UC

Fig. 1

Identification of AMEVLP. A. TEM images of AMEVLP. Scale bar indicates 200 nm and 50 nm. B. Nano FCM for detection of particle size distribution of AMEVLP. C. Triton X-100 detection of lipid membrane structure of AMEVLP. ***P < 0.001. D. Stability of AMEVLP in simulated gastric and small intestinal fluids. E. Agarose gel electrophoresis for identification of nucleic acids of AMEVLP. F. Lipid composition and metabolite composition mapping of AMEVLP

Fig. 2

In vitro anti-inflammatory and anti-oxidant effects of AMEVLP. A. Fluorescence signal detection of RAW264.7 cells after internalized uptake of Dil-labeled AMEVLP (0.25 µg/mL and 0.5 µg/mL) at different time points (2, 4, 8 h). B. Incubation of RAW264.7 cells with DiI-labeled AMEVLP (0.5 µg/mL). Fluorescence images for 2, 4, and 8 h. Scale bar indicates 20 μm. C-D. Fluorescence signal distribution and representative fluorescence images of DCF in RAW264.7 cells induced by H₂O₂ and treated with different protein concentrations of AMEVLP for 6 h. Scale bar indicates 100 μm

Fig. 3

AMEVLP alleviates DSS-UC and repairs colonic barrier function in mice. A. Experimental design of the mouse model of DSS-induced UC treated with AMEVLP. B. Body weight changes of each group were monitored daily and expressed as a percentage of initial body weight. C. Disease Activity Index (DAI) scores. n = 6. D. Morphological observation of the colons of representative mice in each group. E. Colonic lengths. n = 5. F. RT-qPCR showed that the administration of AMEVLP up-regulated the IL- 10 mRNA expression level. G. RT-qPCR showing inhibition of IL-1β, IL-6, IL-12. and TNF-α mRNA expression after AMEVLP administration. Data are shown as means ± standard deviation, n = 3. **P < 0.01, *** P < 0.001. H. Colonic endoscopic images of colonic inflammation in mice. I. H&E-stained colonic tissue sections. Scale bar indicates 200 μm. J. Images of immunofluorescence staining for the expression of colonic tight junction proteins (Occludin, ZO-1, and Claudin). Scale bar indicates 100 μm. K. Alcian blue stained images. Scale bars indicate 50 μm and 20 μm

Fig. 4

AMEVLP regulation of gut microbial and metabolite composition. A. Observed species index (Alpha diversity). B. Chao1 index (Alpha diversity). C. Shannon index (Alpha diversity). D. Principal component analysis (PCA). D. Intergroup comparison of relative abundance of gut microbiota at the gate level. E. Heat map of relative abundance of taxa at the genus level of each group abundance. F. Heat map of categorical relative abundance of groups at the genus level. G. Evolutionary dendrograms of species with significant differences. H. Relative abundance of beneficial flora with significant differences at the genus level. I. Relative abundance of harmful flora with significant differences at the genus level. Data are shown as means ± standard deviation, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. J. KEGG pathway enrichment bubble plots showing differential metabolite expression. K. Tryptophan metabolism pathway heat map. L. Steroid hormone biosynthesis pathway heat map

Fig. 5

AMEVLP alleviates UC in mice by regulating the Th17 cell differentiation signaling pathway. A. Heat map of AMEVLP and intestinal common differential metabolites. B. Correlation analysis of common differential metabolites and intestinal flora. C. KEGG pathway enrichment analysis of common differential metabolites and colon tissue transcriptome. D. KEGG pathway enrichment analysis of colon tissue transcriptome and intestinal flora. E. Immunohistochemical enrichment analysis of AMEVLP transcriptome and colon tissue transcriptome. F. Quantitative plot of immunohistochemical staining for cytokines associated with the Th17 cell differentiation signaling pathway, n = 3, **P < 0.01

Fig. 6

Clinical efficacy of AMEVLP. A. Comparison of colonoscopic findings after oral administration of AMEVLP in Patient A and Patient B. B. H&E-stained rectum tissue sections. Scale bars indicate 200 μm