Upregulation of P-glycoprotein by probiotics in intestinal epithelial cells and in the dextran sulfate sodium model of colitis in mice

Abstract

P-glycoprotein (P-gp) mediates efflux of xenobiotics and bacterial toxins from the intestinal mucosa into the lumen. Dysregulation of P-gp has been implicated in inflammatory bowel disease. Certain probiotics have been shown to be effective in treating inflammatory bowel disease. However, direct effects of probiotics on P-gp are not known. Current studies examined the effects of Lactobacilli on P-gp function and expression in intestinal epithelial cells. Caco-2 monolayers and a mouse model of dextran sulfate sodium-induced colitis were utilized. P-gp activity was measured as verapamil-sensitive [(3)H]digoxin transepithelial flux. Multidrug resistant 1 (MDR1)/P-gp expression was measured by real-time quantitative PCR and immunoblotting. Culture supernatant (CS; 1:10 or 1:50, 24 h) of Lactobacillus acidophilus or Lactobacillus rhamnosus treatment of differentiated Caco-2 monolayers (21 days postplating) increased (∼3-fold) MDR1/P-gp mRNA and protein levels. L. acidophilus or L. rhamnosus CS stimulated P-gp activity (∼2-fold, P < 0.05) via phosphoinositide 3-kinase and ERK1/2 MAPK pathways. In mice, L. acidophilus or L. rhamnosus treatment (3 × 10(9) colony-forming units) increased mdr1a/P-gp mRNA and protein expression in the ileum and colon (2- to 3-fold). In the dextran sulfate sodium (DSS)-induced colitis model (3% DSS in drinking water for 7 days), the degree of colitis as judged by histological damage and myeloperoxidase activity was reduced by L. acidophilus. L. acidophilus treatment to DSS-treated mice blocked the reduced expression of mdr1a/P-gp mRNA and protein in the distal colon. These findings suggest that Lactobacilli or their soluble factors stimulate P-gp expression and function under normal and inflammatory conditions. These data provide insights into a novel mechanism involving P-gp upregulation in beneficial effects of probiotics in intestinal inflammatory disorders.

Fig. 1.

Effect of Lactobacillus acidophilus (LA) or Lactobacillus rhamnosus (LR) culture supernatant (CS) on P-glycoprotein (P-gp) activity and expression in Caco-2 cells. Overnight-serum-starved postconfluent Caco-2 cells were treated with a 1:50 dilution of L. acidophilus or L. rhamnosus CS in serum-free cell culture medium for 24 h. A: [³H]digoxin flux (1 μM). Results are expressed as percentage of control. Values are means ± SE of 5 separate experiments performed in triplicate. *P < 0.05 vs. untreated control. B: relative abundance of multidrug resistance 1 (MDR1) mRNA from control and treated Caco-2 cells normalized to histone mRNA (internal control). C: control and treated cell lysates were subjected to 7% SDS-PAGE and then transferred to a nitrocellulose membrane. Blot was probed with anti-MDR1 (P-gp) or anti-β-actin (β-actin) antibody. A representative blot of 3 different experiments is shown. Data were quantified by densitometric analysis and are expressed as percentage of control in arbitrary units. Values are means ± SE of 3 independent experiments performed in triplicate. *P < 0.05 vs. untreated control.

Fig. 2.

Effect of L. acidophilus or L. rhamnosus CS on MDR1 promoter activity in Caco-2 cells. Caco-2 cells were transiently transfected with MDR1 luciferase (Luc) promoter construct (−1073/+703) along with pCMVβ vector. Cells were treated with L. acidophilus or L. rhamnosus CS (1:10 dilution) for 24 h in medium containing 1% FBS after 24 h. At 48 h posttransfection, promoter activity was measured by luciferase assay. Values were normalized to β-galactosidase activity to correct for transfection efficiency. Results are expressed as percentage of control: transfected cells treated with L. acidophilus or L. rhamnosus CS vs. untreated cells (control). Values are means ± SE of 4 separate experiments with 3 independent observations for each experiment (n = 12). *P < 0.05 vs. control.

Fig. 3.

Effect of phosphoinositide 3-kinase (PI3K) or ERK1/2 MAPK inhibitor on L. acidophilus or L. rhamnosus CS-induced stimulation of P-gp activity in Caco-2 cells. Postconfluent Caco-2 cells were pretreated with the specific inhibitor of PI3K, LY-294002 (LY, 50 μM; A), or the specific inhibitor of ERK1/2 MAPK, PD-98059 (PD, 30 μM; B), for 60 min in the serum-free cell culture medium and then coincubated with 1:10 dilution of L. acidophilus or L. rhamnosus CS in serum-free cell culture medium for 24 h, and [³H]digoxin flux (1 μM) was measured. Results are expressed as percentage of control. Values are means ± SE of 4 separate experiments performed in triplicate. *P < 0.05 vs. untreated control.

Fig. 4.

P-gp expression in response to L. acidophilus or L. rhamnosus [3 × 10⁹ colony-forming units (CFUs)]. A: relative abundance of mdr1a mRNA from ileum and colon of control and treated mice normalized to histone mRNA (internal control). Total lysates extracted from mucosal tissues of ileum (B) and colon (C) of control and treated mice were subjected to 7% SDS-PAGE and then transferred to a nitrocellulose membrane. Blot was probed with anti-mdr1 (P-gp) or anti-β-actin (β-actin) antibody. A representative blot is shown. Data were quantified by densitometric analysis and are expressed as percentage of control in arbitrary units. Values are means ± SE of 3 mice. *P < 0.05 vs. untreated control.

Fig. 5.

L. acidophilus attenuates dextran sulfate sodium (DSS)-induced inhibition of P-gp expression in distal colon. A: change in body weight during DSS treatment. Mice were given 3% DSS in drinking water for 7 days. Control mice had only drinking water. L. acidophilus (3 × 10⁹ CFUs) was administered by gavage to control or DSS-treated mice for the first 2 days. Values are means ± SE (n = 4). B: mdr1a expression. Relative abundance of mdr1a mRNA from mucosal tissues of distal colon of control, DSS, or L. acidophilus + DSS mice was normalized to histone mRNA (internal control). C: P-gp expression. Total lysates were subjected to 7% SDS-PAGE and then transferred to a nitrocellulose membrane. Blot was probed with anti-mdr1 (P-gp) or anti-β-actin (β-actin) antibody. A representative blot is shown. Data were quantified by densitometric analysis and expressed as percentage of control in arbitrary units. Values are means ± SE of 4 mice. *P < 0.05 vs. untreated control. #P < 0.05 vs. DSS.