Marjoram Relaxes Rat Thoracic Aorta Via a PI3-K/eNOS/cGMP Pathway

Abstract

Despite pharmacotherapeutic advances, cardiovascular disease (CVD) remains the primary cause of global mortality. Alternative approaches, such as herbal medicine, continue to be sought to reduce this burden. Origanum majorana is recognized for many medicinal values, yet its vasculoprotective effects remain poorly investigated. Here, we subjected rat thoracic aortae to increasing doses of an ethanolic extract of Origanummajorana (OME). OME induced relaxation in a dose-dependent manner in endothelium-intact rings. This relaxation was significantly blunted in denuded rings. N(ω)-nitro-l-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) significantly reduced the OME-induced vasorelaxation. Cyclic guanosine monophosphate (cGMP) levels were also increased by OME. Moreover, wortmannin or LY294002 significantly reduced OME-induced vasorelaxation. Blockers of ATP-sensitive or Ca2+-activated potassium channels such as glibenclamide or tetraethylamonium (TEA), respectively, did not significantly affect OME-induced relaxation. Similarly, verapamil, a Ca²⁺ channel blocker, indomethacin, a non-selective cyclooxygenase inhibitor, and pyrilamine, a H1 histamine receptor blocker, did not significantly modulate the observed relaxation. Taken together, our results show that OME induces vasorelaxation via an endothelium-dependent mechanism involving the phosphoinositide 3-kinase (PI3-K)/ endothelial nitric oxide (NO) synthase (eNOS)/cGMP pathway. Our findings further support the medicinal value of marjoram and provide a basis for its beneficial intake. Although consuming marjoram may have an antihypertensive effect, further studies are needed to better determine its effects in different vascular beds.

Keywords: PI3-K; cGMP; hypertension; marjoram; nitric oxide; vasorelaxation.

Figure 1

Effect of Origanum majorana (OME) extract on the vasorelaxation of aortic rings. Cumulative dose response curve for OME-induced relaxation of rat aortic rings was determined. Data expressed are mean ± standard error of the mean (SEM, n = 7).

Figure 2

Role of endothelium in OME-induced relaxation. Cumulative dose-response curves for OME in isolated norepinephrine (NE)-pre-contracted rat aortic rings either with intact (+E; black) or denuded endothelium (-E; red). Data are expressed as mean ± SEM (n = 7; p < 0.01 for +E vs. -E).

Figure 3

Role of nitrous oxide (NO) or cGMP in OME-induced relaxation. (A) Endothelium-intact rings were treated with cumulative doses of OME in the presence (red) or the absence (black) of Nω-nitro-l-arginine methyl ester (L-NAME) (inhibitor of eNOS, 100 µM). Data represent mean ± SEM (p < 0.01 for OME alone vs. L-NAME plus OME; n = 7). (B) Endothelium-intact rings were treated with cumulative doses of OME in the presence (red) or absence (black) of ODQ (inhibitor of soluble guanylate cyclase, 1 µM). Data represent mean ± SEM (p < 0.01 for OME vs. ODQ plus OME; n = 6).

Figure 4

Effect of OME on cGMP levels. Rings were treated without (control) or with increasing concentrations of OME. cGMP levels were quantitated by an immunoassay. Data are shown as mean ± SEM, n = 5. (* p < 0.01; ** p < 0.001).

Figure 5

Effect of the PI3K pathway on OME-induced vasorelaxation. Rings with intact endothelium were treated with OME in the absence (black) or presence of phosphoinositide 3-kinase (PI3-K) inhibitors: Wortmannin (0.1 µM; red) or LY29400 (10 µM; blue). Data presented as mean ± SEM (n = 5; p < 0.01 for OME vs. Wortmannin + OME or LY29400 + OME).

Figure 6

Effect of potassium channel inhibitors on OME-induced vasorelaxation. Rings with intact endothelium were treated with OME in the absence (OME; black) or presence of (A) 10 µM of glibenclamide (Glib + OME; red), or (B) 100 µM of tetraethylammonium (TEA + OME; red). Data represent mean ± SEM (n = 6). p > 0.05 for OME alone vs. either Glib + OME or OME + TEA.

Figure 7

Role of calcium channels in OME-induced relaxation of aortic rings. Endothelium-intact aortic rings were pre-incubated with OME in the absence (OME; black) or presence of verapamil (verap) (1 µM; verap + OME; red). Data are presented as mean ± SEM (n = 5; p > 0.05).

Figure 8

Involvement of histaminic or muscarinic receptors in OME-induced relaxation. Rings with intact endothelium were incubated with cumulative doses of OME in the absence (OME; black) or presence of (A) 10 µM of atropine (natural alkaloid with antagonistic properties at muscarinic acetylcholine receptors; atropine + OME; red) or (B) 10 µM of pyrilamine (blocker of H₁ histamine receptors; pyrilamine + OME; red). Data showed represent mean ± SEM (n = 6 or 5 for atropine or pyrilamine-treated rings, respectively). p < 0.05 for OME alone vs. either atropine + OME; p > 0.05 for OME alone vs. either pyrilamine + OME.

Figure 9

Effect of cyclooxygenase inhibition by indomethacin on OME-induced aortic relaxation. Rings with intact endothelium were pre-treated without (OME; black) or with indomethacin (a cyclooxygenase (COX)1/2 inhibitor, 10 µM; INDO + OME; red) followed by the addition of cumulative doses of OME. Data showed represent mean ± SEM (n = 5; p > 0.05).