Cytochrome P450 3A4 suppression by epimedium and active compound kaempferol leads to synergistic anti-inflammatory effect with corticosteroid

Abstract

Introduction: Cytochrome P450 (CYP) 3A4 is a major drug metabolizing enzyme for corticosteroids (CS). Epimedium has been used for asthma and variety of inflammatory conditions with or without CS. It is unknown whether epimedium has an effect on CYP 3A4 and how it interacts with CS. We sought to determine the effects of epimedium on CYP3A4 and whether it affects the anti-inflammatory function of CS and identify the active compound responsible for this effect. Methods: The effect of epimedium on CYP3A4 activity was evaluated using the Vivid CYP high-throughput screening kit. CYP3A4 mRNA expression was determined in human hepatocyte carcinoma (HepG2) cells with or without epimedium, dexamethasone, rifampin, and ketoconazole. TNF-α levels were determined following co-culture of epimedium with dexamethasone in a murine macrophage cell line (Raw 264.7). Active compound (s) derived from epimedium were tested on IL-8 and TNF-α production with or without corticosteroid, on CYP3A4 function and binding affinity. Results: Epimedium inhibited CYP3A4 activity in a dose-dependent manner. Dexamethasone enhanced the expression of CYP3A4 mRNA, while epimedium inhibited the expression of CYP3A4 mRNA and further suppressed dexamethasone enhancement of CYP3A4 mRNA expression in HepG2 cells (p < 0.05). Epimedium and dexamethasone synergistically suppressed TNF-α production by RAW cells (p < 0.001). Eleven epimedium compounds were screened by TCMSP. Among the compounds identified and tested only kaempferol significantly inhibited IL-8 production in a dose dependent manner without any cell cytotoxicity (p < 0.01). Kaempferol in combination with dexamethasone showed complete elimination of TNF-α production (p < 0.001). Furthermore, kaempferol showed a dose dependent inhibition of CYP3A4 activity. Computer docking analysis showed that kaempferol significantly inhibited the catalytic activity of CYP3A4 with a binding affinity of -44.73kJ/mol. Discussion: Inhibition of CYP3A4 function by epimedium and its active compound kaempferol leads to enhancement of CS anti-inflammatory effect.

Keywords: CYP3A4; anti-inflammation; drug-drug interaction (DDI); epimedium; kaempferol.

FIGURE 1

Epimedium inhibition of CYP3A4 activity: The inhibition of CYP3A4 was measured by commercially available Vivid assay kit as per manufacturers instruction. Epimedium showed dose-dependent suppression of CYP3A4 enzyme activity (A). The IC₅₀ and IC₉₀ were calculated to be 95.73 pg/mL and 216.1 pg/mL respectively (B,C). Data represents triplicate experiments and expressed as mean ± SD. np < 0.01; ***p < 0.001 vs. control.

FIGURE 2

Epimedium suppressed CYP3A4 mRNA expression in human liver cell lines: HepG2 cells were cultured with Epimedium (EPI) and Dexamethasone (DEX) either alone or in combination. Rifampin (RI F)was used as a CYP3A4 enhancer whereas Ketoconazole (KETO) as a CYP3A4 inhibitor. The expression levels were determined by qRT-PCR. EPI alone inhibited CYP3A4 mRNA expression comparable to KETO whereas DEX enhanced CYP3A4 mRNA levels comparable to RIF. Coculture EPI and DEX remarkably reduced DEX enhancement of CYP3A4 mRNA levels (A). Cell proliferation assay measured using MTT assay showed no different in proliferation between groups (B). Data represents triplicate experiments and expressed as mean ± SD. *p < .05 vs. control (0.1%DMS0), #p < 0.05 vs. DEX.

FIGURE 3

Epimedium inhibits CYP3A4 protein expression. HepG2 cells were cultured with Epimedium for 48 h and protein expression was determined using western blotting. (A). Western Bands (B). CYP3A4 protein expression. Data represents triplicate experiments and expressed as mean ± SD. *p < .05 vs. control (0.1%DMS0).

FIGURE 4

Epimedium and dexamethasonesynergistically suppressed TNF-α production: RAW cells were cultured with Epimedium either alone or in combination with Dexamethasone for 24 h. The cells were stimulated with LPS (0.5 pg/mL). The supernatant were harvested and TNF-a levels were measured by ELISA. (A) Dexamethasone (10⁻⁶ M, 10⁻⁷ M) and Epimedium (125 pg/m1) significantly reduced TNF-α levels. When Epimedium was added to dexamethasone, the TNF-α levels were suppressed by 88%. Data represents triplicate experiments and expressed as mean ± SD. ***p < 0.001 vs. control (0.1%DMS0),"#p < 0.001 vs. Dex (10–7 M).

FIGURE 5

Effect of four epimedium candidate compounds on IL-8 production: HepG2 cells were cultured in presence of four main Epimedium candidate compound for 48 h. After which cells were stimulated with IL-1 β (5 ng/m1). The supernatant were harvest and IL-8 levels were measured by ELISA. (A) Out of four main candidates, Kaempferol significantly reduced IL-8 levels. (B) Kaempferol showed a dose dependent inhibition of IL-8 production by stimulated HepG2 cells. (C) No cell cytotoxicity was observed with Kaempferol at different doses. Data represents triplicate experiments and expressed as mean ± SD. "p < 0.01; p < 0.001; vs. control (0.1%DMSO). 0.D; optical density.

FIGURE 6

Effect of Kaempferol and dexamethasone co-culture on TNF- α production by RAW cells: Epimedium and dexamethasone synergistically suppressed TNF-α production RAW cells were cultured with Epimedium either alone or in combination with Dexamethasone for 24 h. The cells were stimulated with LPS (0.5 pg/m1). The supernatant were harvested and TNF-α levels were measured by ELISA. (A) Dexamethasone (10^–7,10^–6 M) and Kaempferol (40 pg/m1) significantly reduced TNF-α levels. When Kaempferol and dexamethasone was added together, the TNF-α levels were suppressed by 97%. (B) Cell viability with CCK8 assay showed no cell cytotoxicity. Data represents triplicate experiments and expressed as mean ± SD. "'p < 0.001 vs. control (without LPS), "p < 0.05; "p < 0.01 vs. Dex (10⁻⁷ M).

FIGURE 7

Kaempferol dose dependent inhibition of CYP3A4 activity: The inhibition of CYP3A4 was measured by commercially available Vivid assay kit as per manufacturers instruction. (A) Kaempferol showed dose-dependent suppression of CYP3A4 enzyme activity. The IC₅₀ and IC₉₀ were calculated to be 9.8 pg/mL and 29.54 pg/mL respectively (B,C). Data represents triplicate experiments and expressed as mean ± SD. "p < 0.01; ***p < 0.001 vs. control.

FIGURE 8

The Binding conformations of compound Kaempferol with enzyme CYP3A4. (A) 3D Binding pose of Kaempferol in inhibited binding domain of CYP450 3A4. For Kaempferol, carbon and oxygen are highlighted in blue and red, respectively. For amino acids of protein, carbon, oxygen and nitrogen are displayed by yellow, red and blue, respectively. For HEM molecule, the orange, blue and yellow color stand for carbon, nitrogen and iron ion. (B) Binding interactions between Kaempferol with residues of CYP450 3A4. The green and pink lines stand for hydrogen bonds and hydrophobic interactions. The docking score was −44.7332 kJ/md, showing good binding affinity between kaempferol and CYP3A4.