45) in Caco-2 cells treated with L. plantarum MB452 (Table 3). Similarly, seven genes encoding for protein degrading proteasomes had decreased expression levels (fold change -1.21 to -1.28) in Caco-2 cells treated with L. plantarum MB452 (Table 3). Table 3 Caco-2 cell tubulin and proteasome genes that were differentially expressed (modified-P < 0.05) in the microarray analysis after co-culturing with L. plantarum MB452 (OD600 nm 0.9) for 10 hours. Gene Name Symbol Refseq ID Fold Change tubulin, alpha 1b TUBA1B NM_006082 -1.45 tubulin, alpha 1c

TUBA1C NM_032704 -1.35 tubulin, alpha 3d TUBA3D NM_080386 -1.22 tubulin, alpha 4a TUBA4A NM_006000 -1.27 tubulin, beta TUBB Anlotinib clinical trial NM_178014 -1.20 tubulin, beta 3 TUBB3 NM_006086 -1.20 tubulin, beta 6 TUBB6 NM_032525 -1.30 tubulin, beta 2c TUBB2C NM_006088 -1.35 proteasome, alpha subunit, 5 PSMA4 NM_002789 -1.24 proteasome, beta subunit, 1 PSMB1 NM_002793 -1.21 proteasome, beta subunit, 6 PSMB6 NM_002798 -1.22 proteasome, beta subunit, 7 PSMB7 NM_002799 -1.28 proteasome, 26 s subunit, 5 PSMC5 NM_002805 -1.24 proteasome, 26 s subunit non-ATPase, 12 PSMD12 NM_002816 -1.25 proteasome, activator subunit, 2 PSME2 NM_002818 -1.24 L. plantarum MB452 visually increased the abundance of tight junction proteins Using fluorescent microscopy the intensity of the immuno-stained ZO-1, ZO-2 occludin

and cingulin proteins appeared NCT-501 manufacturer higher in the learn more Caco-2 cells treated with L. plantarum MB452 than in the untreated controls (Figure 4). This indicated that the changes in gene expression observed were supported by changes in tight junction-associated protein intensity. Figure 4 Fluorescent microscopy images of immuno-stained tight junction proteins of confluent Caco-2 cells (6 days old) untreated or treated with L. plantarum MB452 (OD 600 nm 0.9) for 8 hours. Treatments were carried out in quadruplicate

and the images shown are typical. ZO-1: zonula occluden 1; ZO-2 zonula occluden 2; OCLN: occludin; check details CGN: cingulin. Discussion As hypothesised, this study showed that L. plantarum MB452 altered the expression levels of tight junction-related genes in healthy intestinal epithelial cells. Of the tight junction bridging proteins, occludin mRNA abundance was higher in the presence of L. plantarum MB452. The over-expression of the occludin protein has been linked to increased TEER [25], and based on the findings of this study, increased occludin gene expression may contribute to the ability of L. plantarum MB452 to enhance tight junction integrity. In support of this, genes encoding for the occludin-associated plaque proteins, ZO-1 and ZO-2 and cingulin, also had increased expression levels in the presence of L. plantarum MB452. The zonula occludens bind to the cytoplasmic end of occludin and form the scaffolding to link occludin to the actin cytoskeleton [26].