St. John's Wort Extract Increases Pgp Expression in the Brain but Not in the Small Intestine or the Liver of Wistar Rats

Abstract

St John's Wort (SJW), commonly used to treat mild depression, is known to pose a risk of drug-herb interactions through hyperforin-mediated activation of the pregnane X receptor (PXR). This induces transcription and expression of PXR target genes, including the efflux transporter P-glycoprotein (Pgp). While the activation of human PXR by the SJW constituent hyperforin is well established, there are contradictory findings on rodent PXR target genes. This study aimed to further investigate SJW effects on Pgp expression in rats. Male Wistar rats were treated for 10 days with the two commercial SJW formulations, Hyperiplant and Rebalance, which differ in their hyperforin content. Quantitative real-time PCR, western blot analysis, and immunohistochemical staining were applied to test for Pgp mRNA expression and protein abundance in the small intestine (jejunum), liver, and brain (cerebrum). Treatment with the hyperforin-rich Hyperiplant increased protein levels in the brain. However, it did not affect mRNA levels. Besides, there was no impact on Pgp protein abundance in the small intestine or the liver. The hyperforin-poor formulation Rebalance did not affect Pgp expression in any of the investigated tissues. Taken together, our results show that there is a modulation of brain Pgp protein abundance in Hyperiplant-treated animals. As such, we conclude that the inducing effect is governed by a so far unknown regulatory mechanism that most likely does not affect transcription of the transporter.

FIGURE 1

Localization of Pgp in small intestine (A), liver (B), and brain (C) sections of differently treated rats. Immunohistochemical staining was applied to organ sections of animals that were orally treated with 400 mg/kg with the formulations Hyperiplant or Rebalance or the suspension mixture as control on 10 consecutive days. In negative control samples, the primary antibody was omitted. Nuclei were counterstained using Mayer's hemalum solution (white bar scale = 100 μm for small intestine and liver, 50 μm for brain).

FIGURE 2

mRNA expression of Abcb1a and Abcb1b in the small intestine, liver, and brain of differently treated animals. Quantitative real‐time PCR was applied to determine Abcb1a (A, C, E) and Abcb1b (B, D, F) mRNA expression in the mentioned organs of rats treated with the suspension mixture (control), Hyperiplant, or Rebalance. Each rat received 400 mg/kg of the formulations or the control on 10 consecutive days. β‐Actin was used as housekeeping gene. Data are reported as mean ± SD and are shown as fold of study mean, each value was measured in duplicates (n = 4–6 animals). *p ≤ 0.05, one‐way ANOVA followed by Tukey's multiple comparisons test.

FIGURE 3

Protein expression of Pgp in the small intestine, liver, and brain of rats treated with the St. John's wort formulations Hyperiplant or Rebalance. Rats were orally treated with 400 mg/kg of the respective formulation or the suspension mixture (control) on 10 consecutive days. (A) Western blots of three randomly selected control‐, Hyperiplant‐, or Rebalance‐treated animals. Actin served as loading control. (B) Densitometric analysis of Pgp protein expression in the different organs obtained by western blot analysis. Data are shown as normalized arbitrary units ± SD. *p ≤ 0.05, one‐way ANOVA followed by Tukey's multiple comparisons test was performed for n = 6 animals.

FIGURE 4

Pgp protein expression in fractions of the small intestine of Hyperiplant‐treated rats. Animals were treated on 10 consecutive days with 400 mg/kg before organ harvest. Membranes of the small intestine were obtained before adding MgCl₂ to separate between apical and basolateral membranes. Three randomly picked samples were used of Hyperiplant‐treated animals. BCRP served as apical marker and Na⁺/K⁺ATPase was used as loading control.