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P067 The effect of a probiotic mix on mucosal healing and fibrotic responses in healthy colonic subepithelial myofibroblasts.

Tarapatzi, G.(1);Filidou, E.(1);Kandilogiannakis, L.(1);Arvanitidis, K.(1);Vradelis, S.(2);Kotzampassi, K.(3);Kolios, G.(1);

(1)Democritus University of Thrace, Laboratory of Pharmacology- Faculty of Medicine, Alexandroupolis, Greece;(2)Democritus University of Thrace, Second Department of Internal Medicine- University Hospital of Alexandroupolis, Alexandroupolis, Greece;(3)Aristotle University of Thessaloniki, Department of Surgery- AHEPA Hospital, Thessaloniki, Greece

Background

Various probiotic strains play a positive role in Inflammatory Bowel Disease and might support mucosal healing. Colonic Subepithelial Myofibroblasts (SEMFs) have a pivotal role in mucosal healing and fibrosis, by migrating to the trauma region and secreting collagen. Our study aimed to investigate whether different strains of probiotics can affect the migration capacity and the fibrotic phenotype of SEMFs isolated from healthy individuals.

Methods

The probiotic mix of Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium lactis and Saccharomyces boulardii was reconstituted in SEMFs culture medium, they were identified by Gram staining and their viability was assessed by Trypan Blue staining. Primary SEMFs were isolated from colonic biopsies from healthy individuals, and stimulated with 102, 104 or 108 cfu/ml of the mix for 6h or 48h; in 6h total RNA was collected and mRNA expression of collagen type I, III, fibronectin, tissue factor (TF) and α-SMA was measured by quantitative PCR, and in 48h their effect on migration rate through the Wound Healing Assay and collagen secretion via Sircol was assessed.

Results

Trypan Blue confirmed that the majority of the probiotic mix was alive, and all three bacterial strains and the yeast were stained Gram positive. Stimulation of SEMFs with the probiotic mix resulted in different effects depending on its concentration; 102 cfu/ml downregulated protein collagen secretion (3.4μg/ml, IQR: 2.7-5.0, p<0.01 vs control 6.3μg/ml, IQR: 5.2-6.7) and α-SMA mRNA production (0.7-fold, IQR: 0.7-0.8, p<0.01), 104 cfu/ml upregulated the Fibronectin mRNA expression (1.6-fold, IQR: 1.2-1.8, p<0.0001) and 108 cfu/ml downregulated collagen type I (0.2-fold, IQR: 0.1-0.8, p<0.01) and III mRNAs (0.3-fold, IQR: 0.1-0.8, p<0.01). In addition, all three concentrations resulted in the overall upregulation of TF mRNA expression. Regarding the effect of the probiotic mix on SEMFs migration capacity, 104 cfu/ml of the probiotic mix resulted in increased migration rate by 133.8% (IQR: 102.8-150.2, p<0.01) after 24h, while the use of 102 cfu/ml and 108 cfu/ml decreased it by 58.2% (IQR: 34.8-79.7, p<0.01) and by 46.8% (IQR: 34.3-72.1, p<0.01) respectively.

Conclusion

This study provides evidence that the probiotic strains L. plantarum, L. acidophillus, B. lactis and S. boulardii remain viable in SEMFs’ culture medium and have the ability to interact with and alter the behavior of SEMFs. Moreover, the effect of the probiotic mix on SEMFs fibrotic phenotype is dependent of the concentration of the probiotics used. Further preclinical research is needed in order to identify the effect of each strain, as well as the suitable dose and combinations in various pathological conditions.

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