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MiR-9394b from Lactobacillus paracasei extracellular vesicles targets iNOS to reprogram host NO-ornithine metabolism and ameliorate ulcerative colitis

ABSTRACT:

Background Probiotics have attracted considerable interest for ulcerative colitis (UC); however, their efficacy is highly strain-specific and the underlying molecular mechanisms remain incompletely understood.

Results Accumulating evidence indicates that bacterial extracellular vesicles (BEVs) function as important effector carriers by delivering regulatory cargos to host cells. In this study, we investigated lactobacilli isolated from infant intestinal contents and screened four strains in a DSS-induced murine colitis model. Oral administration of Lactobacillus paracasei (LPC) or its extracellular vesicles (LPC-EVs) markedly alleviated colitis severity, as evidenced by reduced histopathological damage, restoration of intestinal epithelial barrier integrity, and suppression of TLR4/NF-κB–associated inflammatory responses. Untargeted metabolomics further demonstrated that LPC-EVs reshaped host arginine metabolism, characterized by increased ornithine levels. In vitro assays showed efficient uptake of LPC-EVs by intestinal epithelial cells; their bioactivity was markedly diminished or abolished after RNase treatment, supporting an RNA cargo–dependent mechanism. miRNA sequencing identified miR-9394b as a highly enriched candidate key cargo in LPC-EVs. Mechanistic analyses suggested that miR-9394b can target inducible nitric oxide synthase (iNOS), thereby suppressing excessive nitric oxide (NO) production, enhancing ornithine availability, and promoting epithelial repair.

Conclusion In this study, we report for the first time that the Lactobacillus paracasei–EV–miR-9394b–iNOS/NO–ornithine metabolic regulatory axis contributes to UC amelioration, providing mechanistic and theoretical support for EV cargos targeting host metabolism and promoting mucosal repair.

INSTRUMENT(S): Liquid Chromatography MS - negative - reverse-phase, Liquid Chromatography MS - positive - reverse-phase

PROVIDER: MTBLS13753 | MetaboLights | 2026-01-23

REPOSITORIES: MetaboLights

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			Action	DRS
	a_MTBLS13753_LC-MS_negative_reverse-phase_metabolite_profiling.txt	Txt
	a_MTBLS13753_LC-MS_positive_reverse-phase_metabolite_profiling.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS13753_LC-MS_negative_reverse-phase_metabolite_profiling_v2_maf.tsv	Tabular
	m_MTBLS13753_LC-MS_positive_reverse-phase_metabolite_profiling_v2_maf.tsv	Tabular

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Project description:Human mesenchymal stem/stromal cells (hMSCs) hold significant potential for regenerative medicine due to their anti-inflammatory and pro-angiogenic secretome. In particular, 3D hMSC aggregates secrete EVs that have enhanced anti-inflammatory and immunomodulatory properties. However, safety and efficacy concerns associated with stem cell therapy have led to the discovery of cell-free approaches utilizing hMSC-derived extracellular vesicles (EVs). Despite their therapeutic promise, the clinical application of hMSC-EVs is limited by low production yield. This study investigates the scalable generation of hMSC-EVs from 3D aggregates in a novel Vertical-Wheel Bioreactor (VWBR) through shear stress-mediated biochemical cues, promoting cargo expression relevant to nerve regeneration and neuropathic treatment. Bone marrow-derived hMSCs were cultured as 3D aggregates in VWBRs and exposed to two different culture media—αMEM/FBS (serum-containing) and DMEM/F12/B27 (serum-free)—under three agitation speeds (25, 40, and 64 rpm). Metabolite and gene analyses were performed to assess EV biogenesis, focusing on ESCRT machinery markers. Results showed that hMSCs cultured in VWBR exhibited higher expression of EV biogenesis genes and glycolytic genes compared to static culture. αMEM/FBS (serum-containing) condition was more robust than DMEM/F12/B27 (serum-free) condition. EV yield (EV number per cell) increased by 3-10 fold (in serum-containing medium) in VWBR compared to static culture, with size of 120-180 nm and EV marker expression. microRNA-sequencing of the EVs shows the upregulation of miR-29a-3p, miR-451a, miR-224-5p, miR-16-5p, miR-133a-3p, and miR-143-3p, etc., reflecting a combination that enhances EV biogenesis, promotes metabolic reprogramming, and supports immunomodulatory behavior characteristics. Functional assays demonstrated that the generated EVs effectively modulated Schwann cell responses under neural inflammation, supporting their therapeutic potential. These findings highlight the role of VWBR-driven hydrodynamics in promoting EV production from 3D hMSC aggregates. This study advances the knowledge of dynamic aggregation and metabolic influence on 3D hMSC-EV production and fundamentally scale up the preconditioned techniques used to promote hMSC aggregation and the consequent EV secretion.

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MiR-9394b from Lactobacillus paracasei extracellular vesicles targets iNOS to reprogram host NO-ornithine metabolism and ameliorate ulcerative colitis

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