Extracellular Vesicles Derived from Kefir Grain Lactobacillus Ameliorate Intestinal Inflammation via Regulation of Proinflammatory Pathway and Tight Junction Integrity.
Department of Internal Medicine and Institute of Gastroenterology, Yonsei University College of Medicine, Seoul 03722, Korea.
Prostemics Research Institute, Seoul 04778, Korea.
Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul 03080, Korea.
Cellpark Clinic, Seoul 06029, Korea.
Abstract
The aim of this study was to demonstrate the anti-inflammatory effect of Lactobacillus kefirgranum PRCC-1301-derived extracellular vesicles (PRCC-1301 EVs) on intestinal inflammation and intestinal barrier function. Human intestinal epithelial cells (IECs) Caco-2 were treated with PRCC-1301 EVs and then stimulated with dextran sulfate sodium (DSS). Real-time RT-PCR revealed that PRCC-1301 EVs inhibited the expression of pro-inflammatory cytokines in Caco-2 cells. PRCC-1301 EVs enhanced intestinal barrier function by maintaining intestinal cell integrity and the tight junction. Loss of Zo-1, claudin-1, and occludin in Caco-2 cells and the colitis tissues was recovered after PRCC-1301 EVs treatment, as evidenced by immunofluorescence analysis. Acute murine colitis was induced using 4% DSS and chronic colitis was generated in piroxicam-treated IL-10-/- mice. PRCC-1301 EVs attenuated body weight loss, colon shortening, and histological damage in acute and chronic colitis models in mice. Immunohistochemistry revealed that phosphorylated NF-κB p65 and IκBα were reduced in the colon tissue sections treated with PRCC-1301 EVs. Our results suggest that PRCC-1301 EVs may have an anti-inflammatory effect on colitis by inhibiting the NF-κB pathway and improving intestinal barrier function.
Figure 1PRCC-1301 extracellular vesicles (EVs) inhibited pro-inflammatory cytokine gene expression in Caco-2 cells. (A) mRNA expression level of (A) IL-2, (B) IL-8, and (C) TNF-α in Caco-2 cells. The results are shown as mean ± SEM. ###p < 0.001 compared with control, * p < 0.05 and ** p < 0.01 and *** p < 0.001 compared with DSS group.Figure 2PRCC-1301 EVs recovered increase in intestinal permeability and disruption of tight junction complexes. In vitro permeability assay was performed in (A) Caco-2 and (B) HCT116 cells treated with TNF-α in the absence or presence of PRCC-1301 EVs. The results are shown as mean ± SEM. ###p < 0.001 compared with control, * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with TNF-α-treated group. (C) Expression of ZO-1 in Caco-2 cell monolayers incubated with 2.5% DSS in the absence or presence of PRCC-1301 EVs. (D) Quantification for ZO-1 fluorescence. The results are shown as mean ± SEM. ###p < 0.001 compared with control, ** p < 0.01 compared with DSS group. (E) Expression of ZO-1, claudin-1, and occludin was examined in control, DSS, and PRCC-1301 EV-treated mouse colon tissue. (F) Quantification for ZO-1, claudin-1, and occluding fluorescence, respectively. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). The results are shown as mean ± SEM. ###p < 0.001 compared with control, ** p < 0.01 and *** p < 0.001 compared with DSS group. Scale bar = 100 µm.Figure 3PRCC-1301 EVs prevented DSS-induced acute colitis and attenuated chronic colitis in IL10-/-. (A,D) Body weight, (B,E) colon length, and (C,F) histological evaluation in DSS‑induced and IL10-/- colitis, respectively. The results are shown as mean ± SEM. ###p < 0.001 compared with wild-type (WT), * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with vehicle group. Scale bar = 100 µm.Figure 4PRCC-1301 EVs suppressed NF-κB activation in DSS-induced acute colitis. Representative images of Immunohistochemistry (IHC) staining of phospho-NF-κB p65 and phospho-IκBα in 3 mg/kg of PRCC-1301 EV-treated mice. (A,B) The expression of phospho-NF-κB p65 in DSS-induced colitis mice. The arrow indicates phospho-NF-κB p65 positive cells. The expression of phospho-IκBα in (C,D) DSS-induced colitis. The results are shown as mean ± SEM. ###p < 0.001 compared with WT, *** p < 0.001 compared with vehicle group. Scale bar = 100 µm.
