Quercetin and its principal metabolites, but not myricetin, oppose lipopolysaccharide-induced hyporesponsiveness of the porcine isolated coronary artery

Abstract

Background and purpose: Quercetin is anti-inflammatory in macrophages by inhibiting lipopolysaccharide (LPS)-mediated increases in cytokine and nitric oxide production but there is little information regarding the corresponding effect on the vasculature. We have examined the effect of quercetin, and its principal human metabolites, on inflammatory changes in the porcine isolated coronary artery.

Experimental approach: Porcine coronary artery segments were incubated overnight at 37°C in modified Krebs-Henseleit solution with or without 1µg·mL(-1) LPS. Some segments were also co-incubated with quercetin-related flavonoids or Bay 11-7082, an inhibitor of NFκB. Changes in isometric tension of segments to vasoconstrictor and vasodilator agents were recorded. Nitrite content of the incubation solution was estimated using the Griess reaction, while inducible nitric oxide synthase was identified immunohistochemically.

Key results: Lipopolysaccharide reduced, by 35-50%, maximal contractions to KCl and U46619, thromboxane A(2) receptor agonist, and impaired endothelium-dependent relaxations to substance P. Nitrite content of the incubation medium increased 3- to 10-fold following exposure to LPS and inducible nitric oxide synthase was detected in the adventitia. Quercetin (0.1-10µM) opposed LPS-induced changes in vascular responses, nitrite production and expression of inducible nitric oxide synthase. Similarly, 10µM Bay 11-7082, 10µM quercetin 3'-sulphate and 10µM quercetin 3-glucuronide prevented LPS-induced changes, while myricetin (10µM) was inactive. Myricetin (10µM) prevented quercetin-induced modulation of LPS-mediated nitrite production.

Conclusion and implications: Quercetin, quercetin 3'-suphate and quercetin 3-glucuronide, exerted anti-inflammatory effects on the vasculature, possibly through a mechanism involving inhibition of NFκB. Myricetin-induced antagonism of the effect of anti-inflammatory action of quercetin merits further investigation.

Figure 1

The effect of overnight exposure of the porcine coronary artery to 1 µg·mL⁻¹ LPS with or without 10 µM Bay 11-7082 (with subsequent removal) on (A) KCl- and (B) U46619-induced contraction. In some experiments responses to (C) KCl and (D) U46619 were also examined in the absence and presence (‘post-incubation’) of 10 µM 1400 W. Bay 11-7082 was included in the incubation medium 1 hour before overnight exposure to LPS, while 1400 W was added to the organ bath 20 min prior to construction of the concentration response curves. The responses shown are as mean ± SEM of 11–12 observations. *P < 0.05, significant difference between the responses for the paired LPS-treated preparations. LPS, lipopolysaccharide.

Figure 2

The effect of overnight exposure of the porcine coronary artery to 1 µg·mL⁻¹ LPS, in the presence or absence of either (A, B) 10 µM quercetin or (C, D) 10 µM myricetin, on responses elicited by KCl and U46619. The responses shown are as mean ± SEM of 7–13 observations. *P < 0.05, significant difference between the responses for the paired LPS-treated preparations. LPS, lipopolysaccharide.

Figure 3

The effect of overnight exposure of the porcine coronary artery to 1 µg·mL⁻¹ LPS, in the presence or absence of either (A, B) 0.1 µM quercetin or (C, D) 1 µM quercetin, on responses elicited by KCl and U46619. The responses shown are as mean ± SEM of 13–26 observations. *P < 0.05, significant difference between responses for the paired LPS-treated preparations. LPS, lipopolysaccharide.

Figure 4

The effect of overnight exposure of the porcine coronary artery to 1 µg·mL⁻¹ LPS in the presence or absence of either 1 µM quercetin, or 1 µM quercetin and 10 µM Bay K 11-7082 on responses to either (A) KCl or (B) U46619. The responses are shown as the mean ± SEM of 10 observations. *P < 0.05, **P < 0.01 significant differences between the maximum responses in preparations treated with LPS: anova followed by Dunnett's post hoc test. LPS, lipopolysaccharide.

Figure 5

Immunohistochemical localization of (upper panels) PECAM-1 (CD31) and (lower panels) inducible nitric oxide synthase (iNOS) in the porcine isolated coronary artery following incubation in modified Krebs-Henseleit solution for 16–18 h at 37°C without (Control) or with either 1 µg·mL⁻¹ LPS or 1 µg·mL⁻¹ LPS and 10 µM quercetin (quercetin added 60 min before LPS). Evidence for the presence of these proteins in either the endothelium or tunica adventitia is shown by the presence of red staining and the bar on each panel represents either 100 µm or 50 µm. LPS, lipopolysaccharide.

Figure 6

The effect of 24 h exposure to 1 µg·mL⁻¹ LPS (A) 1 µg·mL⁻¹ LPS plus 10 µM quercetin and (B) 1 µg·mL⁻¹ LPS plus 10 µM myricetin on nitrite ion production in porcine coronary artery segments incubated in DMEM. The flavonoids were added to DMEM 60 min before LPS. The values shown are the mean ± SEM of 8–12 observations. **P < 0.01 significant difference from control, anova. DMEM, Dulbecco's modified Eagle's medium; LPS, lipopolysaccharide.

Figure 7

The effect of 24 h exposure to 1 µg·mL⁻¹ LPS, 1 µg·mL⁻¹ LPS plus 1 µM quercetin (Quer) and 1 µg·mL⁻¹ LPS with a combination of 10 µM myricetin (Myr) and 1 µM quercetin on nitrite ion production in porcine coronary artery segments incubated in DMEM. Myricetin was added to DMEM 60 min before quercetin, which in turn was added 60 min prior to the addition of LPS. The values shown are the mean ± SEM of 12 observations. **P < 0.01 significant difference from control by anova. DMEM, Dulbecco's modified Eagle's medium; LPS, lipopolysaccharide.