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. 2019 Aug 15;9:11929. doi: 10.1038/s41598-019-47457-1

Figure 8.

Figure 8

Pre-treatment with metformin or resveratrol partially rescues impairment in mitochondrial biogenesis and restores epithelial barrier integrity following 3-oxo-C12-HSL treatment and prevents bacterial transmigration. (A,B) BEAS-2B cells were pre-treated with either metformin 1 mM (A) or resveratrol 20 μM (B) for 24 hours prior to 12-hour treatment with 100 μM 3-oxo-C12-HSL and relative mRNA expression of TFAM was measured by QPCR. The attenuation in TFAM expression caused by 3-oxo-C12-HSL was blocked by prior treatment with metformin or resveratrol. (C) Calu-3 cells grown on transwell inserts were pretreated with either vehicle control, metformin 1 mM, or resveratrol 20 μM overnight and then inoculated with PAO1 (MOI 1) in the apical media for 6 hours. The basolateral media was collected, serially diluted, and cultured to quantify cfu. Resveratrol or metformin pretreatment significantly decreased the degree of bacterial transmigration. (D) BEAS-2B cells pretreated with either metformin1 mM or resveratrol for 24 hours prior to overnight treatment with 100 μM 3-oxo-C12-HSL were examined by immunofluorescence microscopy staining for the tight junction protein, ZO-1. Treatment with 3-oxo-C12-HSL distorted intercellular distribution of ZO-1. These changes were attenuated in cells pretreated with resveratrol or metformin. 60x magnification, Scale bar = 50 μm. (E–J) Calu-3 were cells grown on transwell inserts and pre-treated overnight with either vehicle control, metformin 1 mM (D–F) or resveratrol 20 μM (G–I). Transepithelial electrical resistance (TEER) was then measured at baseline and at regular intervals following treatment with 100 μM 3-oxo-C12-HSL (E,H). 3-oxo-C12-HSL significantly reduced TEER within 2 hours and this effect persisted for at least 48 hours. Pre-treatment with metformin lead to restoration of barrier integrity at 24 (E) and 48 (F) hours despite 3-oxo-C12-HSL treatment. A similar effect was seen with resveratrol at 24 (H) and 48 (I) hours. (E–J) Primary NhBEs cultured on transwell inserts were pretreated with either vehicle control, metformin 1 mM (K-M), or resveratrol 20 μM (N–P). TEER was then measured before and at 3, 6, 12, and 24 hours following treatment with 100 μM 3-oxo-C12-HSL. Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test, n = 5 independent experiments (A–C) or two-way ANOVA with Tukey’s multiple comparisons test, n = 5 independent experiments (E–J) n = 6 (K–P).