Rosmarinic Acid Alleviates the Endothelial Dysfunction Induced by Hydrogen Peroxide in Rat Aortic Rings via Activation of AMPK

Abstract

Endothelial dysfunction is the key player in the development and progression of vascular events. Oxidative stress is involved in endothelial injury. Rosmarinic acid (RA) is a natural polyphenol with antioxidative, antiapoptotic, and anti-inflammatory properties. The present study investigates the protective effect of RA on endothelial dysfunction induced by hydrogen peroxide (H₂O₂). Compared with endothelium-denuded aortic rings, the endothelium significantly alleviated the decrease of vasoconstrictive reactivity to PE and KCl induced by H₂O₂. H₂O₂ pretreatment significantly injured the vasodilative reactivity to ACh in endothelium-intact aortic rings in a concentration-dependent manner. RA individual pretreatment had no obvious effect on the vasoconstrictive reaction to PE and KCl, while its cotreatment obviously mitigated the endothelium-dependent relaxation impairments and the oxidative stress induced by H₂O₂. The RA cotreatment reversed the downregulation of AMPK and eNOS phosphorylation induced by H₂O₂ in HAEC cells. The pretreatment with the inhibitors of AMPK (compound C) and eNOS (L-NAME) wiped off RA's beneficial effects. All these results demonstrated that RA attenuated the endothelial dysfunction induced by oxidative stress by activating the AMPK/eNOS pathway.

Figure 1

Effects of H₂O₂ exposure on PE- and KCl-induced contraction in rat aortic rings. (a) KCl induced contractile response in H₂O₂-treated aortic rings with (E+) or without (E−) endothelium. (b) PE induced contractile response in H₂O₂-treated aortic rings with (E+) or without (E−) endothelium. (c) ACh induced vasodilative response in H₂O₂-treated rat aorta with intact endothelium. (d) 5.0 mM H₂O₂ pulse treatment (10 min) induced the endothelium-dependent vasodilation impairments in rat aortic rings with intact endothelium. Data represents as means ± SD (n = 6). (a and b) ^∗P < 0.05, ^∗∗P < 0.01 versus the respective untreated group; ^##P < 0.01 versus the endothelium-intact aortic rings treated with the same concentration of H₂O₂; (c and d) ^∗∗P < 001 versus the untreated group.

Figure 2

Rosmarinic acid alleviates the endothelial dysfunction induced by H₂O₂ in endothelium-intact rat aortic rings. (a) Rosmarinic acid (RA) pulse exposure showed no effect on the contraction induced by KCl in rat aortic rings. (b) Rosmarinic acid (RA) pulse exposure showed no effect on the contraction induced by PE in rat aortic rings. (c) Rosmarinic acid (RA) preincubation alleviated the endothelium-dependent vasodilation impairments induced by H₂O₂. (d) Rosmarinic acid (RA) cotreatment inhibited the NBT reduction induced by H₂O₂ in the endothelium-intact aortic rings. The results were expressed as the means ± SD (n = 6). ^∗∗P < 0.01 versus the untreated control group; ^##P < 001 versus the H₂O₂-treated group.

Figure 3

eNOS activation mediated the protection of rosmarinic acid on the endothelial dysfunction induced by H₂O₂ in rat aortic rings. The rat aortic rings were cotreated with eNOS inhibitor L-NAME (2.0 μM) and RA (50 μM) for 10 min, then exposed to H₂O₂ (5.0 mM) for another 10 min. The endothelial function was assessed by the endothelium-dependent vasodilation induced by acetylcholine (ACh, 10 μM) (n = 6). (a) The relative endothelium-dependent vasodilation rate after exposure to RA, eNOS inhibitor L-NAME, and H₂O₂ in rat aortic rings. (b) The NBT reduction after exposure to RA, eNOS inhibitor L-NAME, and H₂O₂ in rat aortic rings. Data are presented as the means ± SD (n = 6). ^∗∗P < 0.01 versus the untreated control group; ^##P < 0.01 versus the H₂O₂-treated group; ^$P < 0.05 and ^$$P < 0.01 versus the H₂O₂- and RA-cotreated groups, respectively.

Figure 4

AMPK activation mediated the protection of rosmarinic acid against the endothelial dysfunction induced by H₂O₂ in rat aortic rings. The rat aortic rings were cotreated with AMPK activator AICAR (50 μM, 10 min) or inhibitor compound C (10 μM) and RA (50 μM) for 10 min, then exposed to H₂O₂ (5.0 mM) for another 10 min. The endothelial function was assessed by the endothelium-dependent vasodilation induced by acetylcholine (ACh, 10 μM). (a) The relative endothelium-dependent vasodilative rate after exposure to RA, AMPK modulator, and H₂O₂ in rat aortic rings. (b) The NBT reduction after exposure to RA, AMPK modulator, and H₂O₂ in rat aortic rings. Data are presented as the means ± SD (n = 6). ^∗P < 0.05 versus the untreated control group; ^∗∗P < 0.01 versus the untreated control group; ^##P < 0.01 versus the H₂O₂-treated group; ^$$P < 0.01 versus the H₂O₂- and RA-cotreated groups.

Figure 5

Rosmarinic acid induced the phosphorylation of AMPK and eNOS in HAEC cells. The HAEC cells were pretreated with the AMPK inhibitor compound C (10 μM) or activator AICAR (50 μM) combined with RA (50 μM) for 10 min prior to another 10 min exposure with H₂O₂ (5 mM). The total AMPK (t-AMPK), phosphorylated AMPK at Thr172 site (p-AMPK^Thr172), and the total eNOS (t-eNOS) and the phosphorylated eNOS at the Ser1177 site (p-eNOS^Ser1177) were determined by Western blot. The results is quantified as the relative ratio of the phosphorylated protein/total protein. The values are presented as the means ± SD; n = 3. ^∗∗P < 0.01 versus the untreated control group; ^##P < 0.01 versus the H₂O₂-treated group; ^$$P < 0.01 versus the H₂O₂- and RA-cotreated groups.