Vasodilatory Effect of Phellinus linteus Extract in Rat Mesenteric Arteries

Abstract

Phellinus linteus is a well-known medicinal mushroom that is widely used in Asian countries. In several experimental models, Phellinus linteus extracts were reported to have various biological effects, including anti-inflammatory, anti-cancer, hepatoprotective, anti-diabetic, neuroprotective, and anti-angiogenic activity. In the present study, several bioactive compounds, including palmitic acid ethyl ester and linoleic acid, were identified in Phellinus linteus. The intermediate-conductance calcium-activated potassium channel (IK_Ca) plays an important role in the regulation of the vascular smooth muscle cells' (VSMCs) contraction and relaxation. The activation of the IK_Ca channel causes the hyperpolarization and relaxation of VSMCs. To examine whether Phellinus linteus extract causes vasodilation in the mesenteric arteries of rats, we measured the isometric tension using a wire myograph. After the arteries were pre-contracted with U46619 (a thromboxane analogue, 1 µM), Phellinus linteus extract was administered. The Phellinus linteus extract induced vasodilation in a dose-dependent manner, which was independent of the endothelium. To further investigate the mechanism, we used the non-selective K⁺ channel blocker tetraethylammonium (TEA). TEA significantly abolished Phellinus linteus extract-induced vasodilation. Thus, we tested three different types of K⁺ channel blockers: iberiotoxin (BK_ca channel blocker), apamin (SK_ca channel blocker), and charybdotoxin (IK_ca channel blocker). Charybdotoxin significantly inhibited Phellinus linteus extract-induced relaxation, while there was no effect from apamin and iberiotoxin. Membrane potential was measured using the voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC₄(3)) in the primary isolated vascular smooth muscle cells (VSMCs). We found that the Phellinus linteus extract induced hyperpolarization of VSMCs, which is associated with a reduced phosphorylation level of 20 KDa myosin light chain (MLC₂₀).

Keywords: Kca channels; Phellinus linteus; mesenteric artery; relaxation; vasodilation.

Figure 1

Gas chromatogram of the compounds in Phellinus linteus extract.

Figure 2

Phellinus linteus extract induces vasodilation in mesenteric arteries of rats. (A₁–A₃), data showing responses to cumulative administration of Phellinus linteus (50 ng/mL–800 ng/mL) on U46619 (A₁) and phenylephrine (A₂)-induced contraction. Statistical analysis of the relaxation response to Phellinus linteus (A₃). (B₁–B₃), data showing responses to cumulative administration of acetylcholine (10⁻⁹ M–10⁻⁵ M) on U46619-induced contraction in endothelium intact (B₁) and endothelium denuded (B₂) mesenteric arteries. Statistical analysis of the relaxation response to acetylcholine (B₃). Inset, representative trace showing responses to vehicle DMSO (0.01–0.4%). Mean ± SD (n = 5). * p < 0.05 for endothelium intact vs. endothelium denuded. (PLE: Phellinus linteus extract, W/O: wash out).

Figure 3

Involvement of endothelium in Phellinus linteus extract-induced relaxation. (A) Relaxation by Phellinus linteus extract in endothelium intact mesenteric artery pre-contracted with U46619 (1 μΜ). (B) Relaxation by Phellinus linteus extract in endothelium denuded mesenteric artery pre-contracted with U46619 (1 μΜ). (C) Relaxation by Phellinus linteus extract in mesenteric artery in the presence of l–NNA (300 μM). (D) Statistical analysis of the relaxation response of Phellinus linteus extract. Relaxation of arteries is expressed as the percentage of the contraction induced by U46619 (1 μΜ). Mean ± SD. (n = 5). (l–NNA: nomega–nitro–l–arginine).

Figure 4

Involvement of K⁺ channel in Phellinus linteus extract-induced relaxation. (A) Effect of Phellinus linteus extract in the mesenteric artery pre-contracted with U46619 (1 μΜ) in the presence of TEA (2 mM). (B) Effect of Phellinus linteus extract in the mesenteric artery pre-contracted with U46619 (1 μΜ) in the presence of apamin (50 nM). (C) Effect of Phellinus linteus extract in the mesenteric artery pre-contracted with U46619 (1 μΜ) in the presence of iberiotoxin (100 nM). (D) Effect of Phellinus linteus extract in the mesenteric artery pre-contracted with U46619 (1 μΜ) in the presence of charybdotoxin (20 nM). (E) Statistical analysis of the relaxation response of Phellinus linteus extract in the presence of various blockers. Relaxation of arteries is expressed as the percentage of the contraction induced by U46619 (1 μΜ). Mean ± SD (n = 5). * p < 0.05 for control versus TEA or charybdotoxin. (TEA: tetraethylammonium, IBTX: iberiotoxin, CBTX: charybdotoxin).

Figure 5

Effect of Phellinus linteus extract on the membrane potential and phosphorylation of 20 KDa myosin light chain (MLC₂₀). (A) Representative images of fluorescence intensity of DiBAC₄(3) in control VSMCs, VSMCs treated with U46619 (1 μΜ), and VSMCs co-treated with U46619 (1 μΜ) and Phellinus linteus extract (200 ng/mL) (scale bar: 50 μm). (B) Representative western blot analysis and quantitative data for phosphorylated MLC₂₀ (P–MLC₂₀) and total MLC₂₀ (T–MLC₂₀) in control VSMCs, VSMCs treated with U46619 (1 μΜ), and VSMCs co-treated with U46619 (1 μΜ) and Phellinus linteus extract (200 ng/mL). * p < 0.05 for VSMCs treated with U46619 vs. VSMCs co-treated with U46619 and Phellinus linteus extract. (n = 4).