Study On The Effect Of Hesperetin And Naringenin On Release Of Nitric Oxide From Endothelial Cells

Endothelium-derived nitric oxide (NO) plays an important role in the regulation of vasomotion and the balance between thrombosis and thrombolysis. The deficiency of endothelium-derived NO or inactivation of NO was the feature of endothelial dysfunction. It was also believed to involve in the pathogenesis of cardiovascular diseases. Fructus aurantii immaturus, a traditional Chinese herb has been used to improve circulation for centuries, but the corresponding pharmacological active components remains to be clarified. Hesperetin, naringenin and their glycosides are the most abundant flavonoid compounds in this herb (～10%). Some have reported that hesperetin or naringenin possess estrogenic activity. As we know, estrogen promotes the release of NO from endothelial cell, thus we supposed that effects of Fructus aurantii immaturus on circulation may relate to hesperetin and naringenin. The aim of this study is to prove the hypothesis mentioned above. The main findings are presented as below.The effect of hesperetin and naringenin on the proliferation of MCF-7, a estrogen receptor (ER) positive cell line was evaluated. When removing the endogenous estrogen from culture medium, both hesperetin and naringenin significantly promoted the proliferation of MCF-7 cells. At the concentration of 50uM, two flavonoids presented nearly equal results to that of estrodiol (10nM), which could be completely blocked by estrogenic receptor antagonists, namely ICI 182,780 and 4-hydroxyl-tamoxifen (TAM). However, hesperetin and naringenin appeared to compromise efficiency of estrodiol. These results indicated that hesperetin and naringenin possessed estrogenic activity. Further study using the reporter-gene assay discovered that both hesperetin and naringenin could induce the expression of reporter controlled by ERα, but only naringenin rather than hesperetin could active ERβ. This result indicated that hesperetin and naringenin was distinct to active subtypes of estrogenic receptor.The effect of hesperetin and naringenin on the release of NO in human umbilical vein endothelial cells (HUVEC) was investigated next. Fluorescence methods with 2,3-diamino- naphthalene (DAN) was employed to determinate the concentration of nitrite in culture medium, which indicated NO production in cells. As endogenous estrogen in culture medium was removed, hesperetin (12.5μM～100μM) promoted the release of NO in HUVEC in a dose-dependent manner. Whereas naringenin had no marked effect on NO production in the same range of this concentration. ICI 182,780 and TAM could completely or slightly inhibit the activity of hesperetin, respectively. When cells were cultured in the medium supplemented with normal FBS (high lever of endogenous estrogen), both hesperetin and naringenin were observed to decrease NO release. This result indicated that the activity of hesperetin, which promoting NO production was dependant on ER activation. The up-regulation or down-regulation of hesperetin on NO production in endothelial cells, which depended on the lever of endogenous estrogen, implied that this flavonoid possessed two-ways regulation activity.The effect of hesperetin and naringenin on the level of intracellular Ca²⁺ was measured using fluorescence dye Fluo-3AM. Meanwhile, the expression of endothelial nitric oxide synthase (eNOS) was evaluated by western-blotting method. Both of the two flavonoids raised intracellular Ca²⁺ lever in HUVEC rapidly. But only hesperetin could up-regulate the expression of eNOS. In addition, actinomycin D, a transcription inhibitor, inhibited the increase of NO induced by hesperetin. These results indicated that the effect of hesperetin on NO production in HUVEC involved in the regulation of transcription and expression of eNOS. Whereas naringenin had potency to active estrogenic receptor, it did not increase protein expression of eNOS.Taken together, these results demonstrate that both hesperetin and naringenin are selective estrogen receptor activators. Hesperetin, but not naringenin can promote the release of NO from HUVEC at relative low level of endogenous estrogen, and this effect is related with regulation of gene expression of eNOS. Under high level of endogenous estrogen, both of them inhibit the release of NO from HUVEC. We therefore suggested that the flavonoids from Fructus aurantii immaturus possesstwo-ways regulation activity on NO production in HUVEC.