Inhibition of Cyclic Strain-Induced Endothelin-1 Secretion by Baicalein in Human Umbilical Vein Endothelial Cells

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Introduction

Baicalein (5,6,7-trihydroxyflavone) is a flavonoid extracted from the root of Scutellaria baicalensis Georgi, a medicinal plant traditionally used in Oriental medicine [1]. Baicalein is reported to act as a specific 12-lipoxygenase inhibitor and to possess many lipoxygenase-unrelated effects such as blocking calcium mobilization [2] and acting as an antioxidant [3]. Baicalein exhibits super- ior free radical scavenging activity among the flavonoid compo- nents of the herb [4] and has been shown to attenuate oxidative stress in cardiomyocytes [5]. Also, baicalein lowers blood pres-

sure in renin-dependent hypertension, and the in vivo hypoten- sive effect may be partly attributed to its inhibition of lipoxygenase, resulting in reduced biosynthesis and release of arachidonic acid-derived vasoconstrictor products [6]. However, the mecha- nism of action for baicalein allowing for its possible use in the prevention and treatment of cardiovascular diseases remains un- clear.

Endothelin-1 (ET-1) was originally isolated from a culture of por- cine endothelial cells [7]. Recently, numerous studies have shown that oxidative stress, represented by reactive oxygen spe-

Inhibition of Cyclic Strain-Induced Endothelin-1 Secretion by Baicalein in Human Umbilical Vein Endothelial Cells

Hsi-Hsien Chen¹ Hong-Jye Hong² Yu-Hsiang Chou³ Tzu-Hurng Cheng⁴ Jin-Jer Chen^{5, 6} Heng Lin^{6, 7}

Affiliation

1Department of Medicine, Taipei Medical University Hospital, Taipei, Taiwan, R.O.C.

2School of Chinese Medicine, China Medical University, Taichung, Taiwan, R.O.C.

3Department of Nuclear Medicine, Taipei Tzu Chi General Hospital, Taipei County, Taiwan, R.O.C.

4Department of Biological Science and Technology, China Medical University, Taichung, Taiwan, R.O.C.

5Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University Col- lege of Medicine, Taipei, Taiwan, R.O.C.

6Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan, R.O.C.

7Graduate Institute of Pharmacology and Toxicology, Tzu Chi University, Hualien, Taiwan, R.O.C.

Correspondence

Heng Lin, Ph.D. ´ Department of Medicine ´ Taipei Medical University Hospital ´ Taipei ´ Taiwan ´ Republic of China ´ Phone: +886-2-7899135 ´ Fax: +886-2-7858594 ´ E-mail: [email protected]

Received September 12, 2005 ´ Accepted August 21, 2006 Bibliography

Planta Med 2006; 72: 1318±1321 Georg Thieme Verlag KG Stuttgart ´ New York DOI 10.1055/s-2006-951702 ´ Published online October 20, 2006

ISSN 0032-0943 Abstract

Baicalein is a flavonoid extracted from the root of Scutellaria baicalensis Georgi, a medicinal plant traditionally used in Orien- tal medicine. Among its biological activities, baicalein has been reported to exhibit antioxidant effects. Endothelin-1 (ET-1) is a potent vasopressor synthesized by endothelial cells both in culture and in vivo. The aims of this study were to test the hypoth- esis that baicalein may alter strain-induced ET-1 secretion and to identify the putative underlying signaling pathways in endothelial cells. We show that baicalein inhibited strain-induced ET-1 secretion. Baicalein also inhibited strain-increased reactive oxygen species (ROS) formation and the extracellular signal-regula-

ted kinases (ERK) phosphorylation. Using a reporter gene assay, baicalein and the antioxidant Trolox also attenuated the strain- stimulated activator protein-1 (AP-1) reporter activity. We con- clude that baicalein inhibits strain-induced ET-1 gene expression, partially by interfering with the ERK pathway via attenuation of ROS formation. These results highlight the molecular pathways that may contribute to the beneficial effects of baicalein in the vascular system such as stroke prevention.

Key words

Endothelin-1 ´ baicalein ´ strain ´ endothelial cells ´ reactive oxygen species ´ extracellular signal-regulated kinases

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cies (ROS), is capable of significantly altering vascular function [8]. Both ET-1 and oxidative stress have been the subjects of in- tense investigation within the cardiovascular field over the past decade [9]. Endothelial cells are constantly under the influence of mechanical forces, including cyclic strain, as a consequence of vessel contraction and relaxation [10]. We have demonstrated that intracellular ROS mediate cyclic strain-induced ET-1 expression via the Ras/Raf/extracellular signal-regulated kinases (ERK) signaling pathway [11]. However, no studies exist that address the interference of baicalein on ET-1 expression in vascular endothelial cells. The present study aimed at investigating the effect of baicalein on the strain-induced ET-1 expression and to identify signaling protein kinase cascades that may be responsible for the putative effect of baicalein.

Material and Methods Materials

Imubind ET-1 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Amersham-Pharmacia (Amersham; Buck- inghamshire, U.K.). 2¢,7¢-Dichlorofluorescin diacetate (DCFH- DA) was obtained from Serva Co. (Heidelberg, Germany). Hydro- gen peroxide (H2O2) was purchased from Acros Organics (Pitts- burgh, PA, USA). Trolox was purchased from Calbiochem (San Diego, CA, USA). Baicalein and all other chemicals of reagent grade were obtained from Sigma (St. Louis, MO, USA).

Endothelial cell culture

Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords as described previously [11]. All procedures involving human samples were conducted according to the Guidelines for Animal and Human Experimentation of the Taipei Medical University. The transformed human cell line, ECV304 (ATCC CRL-1998), was purchased from the American Type Culture Collection (Bethesda, MD, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 U/mL), and streptomy- cin (100mg/mL) before subculturing.

In vitro cyclic strain on cultured endothelial cells

Endothelial cells cultured on the flexible membrane base were subjected to cyclic strain produced by a computer-controlled appli- cation of sinusoidal negative pressure as described previously [11].

Measurement of ET-1 concentration

ET-1 levels were measured in culture medium using a commer- cial enzyme-linked immunosorbent assay (ELISA) kit (Amer- sham-Pharmacia). Results were normalized to cellular protein content in all experiments and expressed as a percentage relative to the cells incubated with the vehicle.

Detection of intracellular ROS

Measurement of intracellular ROS formation in HUVECs was re- corded by monitoring changes in diclorofluorescein (DCF) fluorescence as described previously [12].

Western blot analysis

Rabbit polyclonal anti-phospho-specific extracellular signal- regulated kinases (ERK) antibody was purchased from New Eng-

land Biolabs (Beverly, MA, USA). Anti-ERK antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Western blot analysis was performed as described previously [11].

Luciferase assay

ECV304 cells plated on 3-cm diameter culture dishes were transfected with the luciferase reporter construct possessing consen- sus AP-1 (AP-1-Luc) binding sites (Stratgene; La Jolla, CA, USA) by the calcium phosphate method as described previously [12].

After incubation for 24 hours in 2% serum DMEM, ECV304 were cultured under different treatments as indicated for 48 hours.

ECV304 cells were assayed for luciferase activity with a luciferase reporter assay kit (Strategene). The firefly luciferase activities at AP-1 transcriptional activity were normalized for transfection efficiency to its respectiveb-galactosidase activity and expressed as relative activity to control.

Statistical analysis

Data are presented as mean SEM. Statistical analysis was performed using Student's t test and analysis of variance (ANOVA) followed by a Dunnett multiple comparison test using Prism ver- sion 3.00 for Windows (GraphPad Software; San Diego, CA, USA).

P values of less than 0.05 were considered to be statistically sig- nificant.

Results and Discussion

HUVECs cultured on flexible membrane bases were subjected to deformation to produce an average strain of 20 %. ET-1 released into the culture media was measured. HUVECs under cyclic strain for 24 hours increased their ET-1 secretion into the culture medium (Fig.1). Baicalein (1±100 mM) significantly inhibited strain-increased ET-1 secretion (Fig.1). These data indicate that baicalein inhibits strain-increased ET-1 secretion in endothelial cells.

We next examined whether baicalein prevents the strain-increased ROS formation. The addition of baicalein (1±100mM) to

Fig. 1 Baicalein inhibits strain-induced ET-1 secretion. HUVECs were pretreated with baicalein (1±100mM) 30minutes prior to strain treatment. Results are presented as mean SEM (n = 6). * P < 0.05 vs. un- strained control cells.^#P < 0.05 vs. strained cells (ANOVA).

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cultured HUVECs significantly inhibited strain-induced ROS formation as measured after strain treatment for 1 hour (Fig. 2A).

The pretreatment with baicalein (100mM) or the antioxidant Tro- lox (200 mM) in cultured HUVECs also significantly inhibited strain- or H2O2-induced ROS formation (Fig. 2B). These findings clearly demonstrate that baicalein inhibits strain-increased intracellular ROS levels in endothelial cells.

We further investigated whether baicalein inhibits the ERK pathway in strain-treated endothelial cells. HUVECs pretreated with baicalein (1±100mM) showed significantly decreased strain-induced ERK phosphorylation (Fig. 3A). Moreover, HUVECs pretreated with baicalein (100mM) or Trolox (200 mM) also showed significantly decreased strain- or H2O2-induced ERK phosphorylation (Fig. 3B). These findings imply that baicalein inhibits strain-activated ERK signaling pathway via attenuation of ROS formation in endothelial cells.

We next evaluated the effect of baicalein on AP-1 activation, which is involved in ET-1 gene induction [13]. The effects of baicalein on strain-induced AP-1 functional activity were as- sessed in a reporter gene assay. Both baicalein (100mM) and Trolox (200 mM) significantly attenuated strain- or H2O2-induced AP-1 reporter activation (Fig. 4). These results indicate that baicalein inhibits strain-increased AP-1 transcriptional activation.

The major new finding of this work is that baicalein inhibits strain-induced ET-1 secretion in endothelial cells. This is supported by the observations that baicalein inhibits strain-induced ET- 1 protein secretion in part via attenuation of ROS formation in endothelial cells. We have previously shown that cyclic-strain treatment of endothelial cells can induce intracellular ROS generation [12], [13]. Elevated ROS levels are involved in the release of ET-1 [12], [13], and this gene induction can be attenuated by antioxidant pretreatment of cells. The results of our present study demonstrate that baicalein reduced the strain-induced ROS generation in the endothelial cells. In particular, it has been demonstrated that activation of ERK is redox-sensitive and that suppression of ROS inhibits strain-induced ET-1 gene expression [11]. One possible explanation for the inhibitory effect of baicalein on strain-induced ET-1 gene expression may thus be its abil- ity to attenuate ROS formation. In our previous study, we found that the activation of AP-1 is redox-sensitive and might play a key role in ET-1 gene induction [12], [13]. Our present results indicate that baicalein inhibits strain-induced AP-1 reporter activity. The inhibitory effect of baicalein on strain-induced AP-1 transcriptional activation suggested that the attenuation of strain-induced ROS by baicalein leads to inhibition of AP-1.

Fig. 3 Inhibitory effects of baicalein on strain-increased ERK phosphorylation. A Effect of baicalein (1±100mM) on strain-activated ERK phosphorylation. B Effect of baicalein on strain- or H₂O2-induced phosphorylation of ERK. HUVECs were preincubated with either baicalein (100mM) or Trolox (200 mM) for 30minutes and stimulated with cyclic strain or H2O2 (25mM) for 30minutes. Data are represented as fold increase relative to control groups. The results show mean SEM (n = 6). * P < 0.05 vs. control; # P < 0.05 vs. strain (or H2O2) alone (ANOVA).

Fig. 2 Effects of baicalein on strain-increased ROS formation. A Effect of baicalein (1± 100mM) on strain-induced ROS generation. B Effect of baicalein (200mM) and Trolox (200 mM) on HUVECs from either control (C) cells or cells treated with cyclic strain or H2O2(25mM) for one hour.

Fluorescence intensities of cells are shown as relative intensity of ex- perimental groups compared with untreated control cells. The results show mean SEM (n = 6). * P < 0.05 vs. control; # P < 0.05 vs. strain (or H₂O₂) treated cells (ANOVA).

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From our results, the inhibitory effect of baicalein on strain-induced ET-1 expression is moderate. It had been reported that the H2O2 is produced during the auto-oxidation of baicalein [14]. These findings led us to speculate that the prooxidant effects of baicalein might be involved in its moderate inhibitory effect. However, similar pharmacological effects have also been reported for other flavonoids, such as resveratrol and tetramethylpyrazine in our previous studies [13], [15]. These compounds are believed to be beneficial for the prevention and treatment of cardiovascular diseases. In conclusion, the data obtained in the present study suggest that the baicalein-induced suppression of cyclic strain-induced ET-1 expression can be considered as one of the mechanisms responsible for the protective effect of baicalein in vascular vessels. These findings have highlighted the thera- peutic potentials of using plant-derived baicalein for the treatment of arteriosclerosis and hypertension.

Acknowledgements

This work was supported, in part, by a National Science Council Grant (NSC 92-2314-B-038-051-), Topnotch Stroke Research

Center Grant, Ministry of Education and Taipei Tzu Chi General Hospital Grant (TCRD-TPE-95-35), Taiwan, R.O.C.

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