Tetramethylpyrazine Protects against Hydrogen Peroxide-Provoked Endothelial Dysfunction in Isolated Rat Aortic Rings: Implications for Antioxidant Therapy of Vascular Diseases

Background and Objectives. Oxidative stress can initiate endothelial dysfunction and atherosclerosis. This study evaluated whether tetramethylpyrazine (TMP), the predominant active ingredient in Rhizoma Ligustici Wallichii (chuanxiong), prevents endothelial dysfunction in a rat model of oxidative stress. Methods. Isolated rat aortic rings were pretreated with various drugs before the induction of endothelial dysfunction by hydrogen peroxide (H2O2). Changes in isometric tension were then measured in acetylcholine- (ACh-) relaxed rings. Endothelial nitric oxide synthase (eNOS) expression was evaluated in the rings by Western blotting, and superoxide anion (O2 ∙−) content was assessed in primary rat aortic endothelial cells by dihydroethidium- (DHE-) mediated fluorescence microscopy. Results. ACh-induced endothelium-dependent relaxation (EDR) was disrupted by H2O2 in endothelium-intact aortic rings. H2O2-impaired relaxation was ameliorated by acute pretreatment with low concentrations of TMP, as well as by pretreatment with catalase and the NADPH oxidase inhibitors, apocynin and diphenyleneiodonium (DPI). TMP, apocynin, and DPI also reduced O2 ∙− accumulation in endothelial cells,but TMP failed to alter eNOS expression in aortic rings incubated with H2O2. Conclusions. TMP safeguards against oxidative stress-induced endothelial dysfunction, suggesting that the agent might find therapeutic utility in the management of vascular diseases. However, TMP's role in inhibiting NADPH oxidase and its vascular-protective mechanism of action requires further investigation.


Introduction
Excessive production of reactive oxygen species (ROS) by vascular endothelial cells contributes to endothelial dysfunction [1] which then initiates atherosclerosis [2]. Atherosclerosis is a progressive process that gradually leads to artery blockage. The clinical progression of atherosclerosis culminates in a number of devastating vascular events, including myocardial infarction and stroke [3]. Accordingly, patients with an ischemic complication of atherosclerosis experience a particularly vulnerable disease phase [4]. Currently, prevention and management of cardio-cerebrovascular diseases encompass the control of risk factors, as well as antithrombotic interventions [5,6]. Additional efforts are directed toward interrupting ongoing oxidative processes in the vessel wall so as to prevent ROS-provoked endothelial impairment [7]. In this regard, antioxidants, which directly remove ROS threats, are emerging as one of the more promising treatments for vascular diseases in the recent years [8].
Rhizoma Ligustici Wallichii (Chinese name, chuanxiong), or lovage tuber, is commonly used in traditional Chinese medicine to promote blood circulation. Tetramethylpyrazine (TMP) is the predominant active component of Rhizoma Ligustici Wallichii [9], and the compound is noted 2 Evidence-Based Complementary and Alternative Medicine for its potent vasodilating properties [10]. Like the parental herb, TMP (also called ligustrazine) is widely used to treat vascular diseases in China, with few adverse reactions in humans [11]. TMP has a long history of use, and the latest studies report its antioxidant proficiency in animal models of ischemic reperfusion [12], atherosclerosis [13], and cerebral vasospasm [14]. However, the underlying mechanism of TMP to combat detrimental ROS production and to rescue endothelial dysfunction in artery walls remains unclear.
Hydrogen peroxide (H 2 O 2 ) acts as a relatively benign cell signaling molecule at low concentrations [15], but exposure of blood vessels to excessive amounts of H 2 O 2 results in severe endothelial impairment [16]. In addition, H 2 O 2 released during the progression of atherosclerosis is likely to worsen vascular damage [17]. In light of the protective effect of TMP in clinical and animal studies, we hypothesized that the administration of this compound to H 2 O 2 -compromised blood vessels could prevent endothelial dysfunction. The actions of acute TMP pretreatment on endothelium-dependent relaxation (EDR) and the protein expression levels of endothelial nitric oxide synthase (eNOS) were, therefore, evaluated in isolated rat aortic rings under various experimental conditions. We also explored the preventative effect of TMP against H 2 O 2 -provoked generation of the superoxide anion (O 2 •− ), a particularly harmful ROS, in primary cultures of endothelial cells established from the rat aorta.

Blood Vessel Preparation.
All animal experiments were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong (Approval no. 4362/04 M). Male Sprague Dawley rats aged 6-7 months (260-280 g) were supplied by the Animal Service Center for the Chinese University of Hong Kong and euthanized by carbon dioxide inhalation. Aortas were isolated and dissected as previously described [18].
Briefly, the thoracic segment of each aorta was excised, removed of the surrounding adipose tissue, and cut into rings approximately 3 mm long in ice-cold oxygenated Krebs solution that consisted of 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1 mM MgCl 2 , 25 mM NaHCO 3 , 1.2 mM KH 2 PO 4 , and 11 mM D-glucose. The rings were then suspended between stainless stain wires in 10-mL organ baths containing Krebs solution bubbling with 95% O 2 plus 5% CO 2 at 37 ∘ C. A basal tension of 20 mN was applied to all rings. The rings were exposed to 0.3 M Phe to test smooth muscle contractility and then were relaxed by the addition of 3 M ACh to assess the integrity of the endothelium. In some cases, the endothelial layer was mechanically disrupted by gently rubbing the lumen against the tips of a pair of forceps. Removal of the endothelium was confirmed by the lack of relaxation in the presence of 3 M ACh. The rings were rinsed several times in prewarmed Krebs solution until baseline tension returned and then were used in isometric tension measurement experiments, as described below.

Isometric Tension Measurement.
Contraction was once again induced in the aortic rings by exposure to 0.3 M Phe. A gradient of ACh (3 nM-10 M) was cumulatively added to induce relaxation of the rings when the contraction response reached a plateau, so as to obtain a concentration-response curve for ACh-mediated relaxation. The rings were again rinsed in prewarmed Krebs solution until baseline tension returned, followed by pretreatment with various compounds, as described below, and exposure to H 2 O 2 (200 M, 60 min). Thereafter, a second Ach concentration-response curve was obtained to examine any changes in the EDR imposed by H 2 O 2 or the drugs.
The first series of experiments was designed to investigate whether TMP pretreatment could rescue endothelial dysfunction provoked by H 2 O 2 . The aortic rings were incubated with various concentrations (100 M, 1 M, or 10 nM) of TMP for 60 min and subsequently treated with H 2 O 2 (200 M) for 60 min. For comparison, the effects of losartan (1 M, 30 min), a newly identified antioxidant agent [19], and vitamin E (100 M, 30 min), a classical antioxidant [20], were also tested.
The next series of experiments was designed to explore the underlying mechanism of H 2 O 2 -induced EDR impairment. The rings were separately pretreated for 30 min with catalase (20000 U/mL), an enzyme that catalyzes the decomposition of H 2 O 2 to water and molecular oxygen [21], and two inhibitors of NADPH oxidase, apocynin (10 M) and DPI (10 nM) [22,23]. After catalase, apocynin, or DPI pretreatment, the rings were exposed to H 2 O 2 (200 M) for 60 min.

Western
Blotting. Western blotting analysis was performed as previously described [24]. Briefly, aortic rings were snap frozen in liquid nitrogen and homogenized in ice-cold RIPA lysis buffer. The lysates were centrifuged, and the supernatants were collected. Protein concentrations in each sample were determined by the Lowry method. Equal amounts of protein (50 g per sample) were resolved in 10% sodium dodecyl sulfate-(SDS-) polyacrylamide gels and then transferred to nitrocellulose immobilon-P polyvinylidenedifluoride membranes. The membranes were blocked for 1 h at room temperature with 1% bovine serum albumin and incubated overnight at 4 ∘ C with a primary Evidence-Based Complementary and Alternative Medicine 3 antibody against eNOS (1 : 3000) (BD Transduction Laboratories, Lexington, KY, USA) and a primary antibody against the loading control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1 : 3000) (Ambion, Austin, TX, USA). The membranes were then incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (DakoCytomation, Carpinteria, CA, USA) for 1 h at room temperature. Immunoreactive polypeptide bands were visualized by using an enhanced chemiluminescence detection system, followed by exposure to X-ray film. Densitometry was performed by using a FluorChem documentation program (Alpha Innotech Corp., San Leandro, CA, USA) to measure the eNOS and GAPDH integrated density values (IDVs) of the aortic rings for each experimental condition.

Rat Aortic Endothelial Cell
Culture. Primary rat aortic endothelial cells were cultured as previously described [25]. Briefly, aortas were isolated, cut open longitudinally, and digested with 0.2% collagenase directed against type 1A collagen (Sigma-Aldrich) for 15 min in a 37 ∘ C shaking water bath. RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin was added to the cell suspension, and the cell suspension was then centrifuged at 1500 ×g for 10 min. The cell pellet was resuspended in the supplemented RPMI medium, and the cells were plated into culture flask (25 cm 2 ) and allowed to settle for 1 h. At this time, the medium was changed to remove nonadherent cells. After the adherent cells reached 80% confluence, they were seeded onto coverslips in 6-well trays, pretreated with various drugs as described below, exposed to After incubation with DHE, the cells were rinsed with phosphate buffered saline, and the fluorescence of DNAbound ethidium was measured under a FluoView Confocal Microscope (Olympus, Center Valley, PA, USA). The microscope was equipped with a 585-nm long pass filter and operated at an excitation wavelength of 515 nm. The fluorescence intensity of each sample was analyzed by using FluoView FV10-ASW software, version 1.5 (Olympus). The summarized data represent fold-changes in fluorescence intensity relative to that of the control, which was set to a value of 100%.

Statistical Analysis.
All quantifiable data are expressed as the mean ± the standard error of the mean (SEM). GraphPad Prism software, version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA), was used for data analysis. The vasorelaxing effect of TMP and other drugs was expressed as the percentage reduction of the Phe-evoked contraction. Nonlinear regression curve fitting was performed on individual cumulative Ach concentration-response curves to estimate the max (maximal response) and the pD 2 (negative logarithm of the EC 50 , where the EC 50 represents the drug concentration that induces 50% of max ) for each experimental agent (H 2 O 2 or drug) or combination of agents. Thus, max is a measure of effectiveness and pD 2 is a measure of potency.
The concentration-response curves were analyzed by two-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. The statistical significance of the Western blotting data and the DHE-mediated fluorescence microscopy data was determined by one-way ANOVA followed by the Bonferroni post hoc test. In each case, a value of less than 0.05 was considered significant.  Figure 1). However, TMP pretreatment for 60 min at concentrations of 10 nM, 1 M, and 100 M significantly reversed the endothelial impairment provoked by H 2 O 2 . TMP and losartan, the newly identified antioxidants, showed similar effects at 1 M (Table 1), while pretreatment with 100 M vitamin E, a classical antioxidant, did not greatly reverse the endothelial damage (Table 1, Figure 1).    Figure 2).

TMP Does Not Rescue EDR by Altering eNOS Expression.
Nitric oxide acts as a vasodilator and is produced through the actions of eNOS in the endothelium. Therefore, we reasoned that agents with the capacity to influence the EDR in rat aortic rings might have an effect on eNOS expression.  Figure 3) and by comparing the eNOS IDV/GAPDH IDV ratio for each experimental condition (Table 3, Figure 3). On the other hand, the endothelium-denuded group with no drug treatment showed significantly decreased eNOS expression relative to any group with an intact endothelium (Table 3, Figure 3).   Figure 4).

Discussion
Vascular events (death from all vascular causes, nonfatal stroke, or nonfatal myocardial infarction) are complex occurrences caused by multiple factors, and ROS-induced endothelial injury is suggested to be the common denominator in most vascular conditions [26]. Consequently, antioxidant therapy, which directly targets the common mediator of vascular disorders, shows great promise for the prevention and management of cardio-cerebrovascular diseases.
The current study explored the capacity of acute pretreatment with TMP, an antioxidant constituent of Rhizoma Ligustici Wallichii, to overturn the H 2 O 2 -mediated impairment of ACh-induced relaxation in the rat aorta. We found that the exposure of rat aortic rings to TMP prior to H 2 O 2 treatment did indeed thwart the actions of H 2 O 2 to promote endothelial dysfunction (Table 1, Figure 1). TMP was effective at surprisingly low doses (i.e., nM levels), yet vitamin E, a classical antioxidant, did not provide the same protective benefits at a considerably higher concentration (100 M) (Table 1, Figure 1). This may be due to the use of high concentration of H 2 O 2 (200 M) to induce impaired EDR and the potent antioxidant effect of TMP. The protective effect of TMP by reduction of oxidative stress in the present study is consistent with other studies working in different systems [12,13,[27][28][29].
Evidence-Based Complementary and Alternative Medicine H 2 O 2 is a relatively stable ROS with dual functions in the vasculature, depending on its effective concentration. For example, low levels of H 2 O 2 maintain the physiology of vascular endothelial cells [30], and H 2 O 2 seldom exerts vascular damage in the absence of unpaired electrons [31]. Therefore, drugs which are capable of stimulating superoxide dismutase to transform O 2 •− into oxygen and H 2 O 2 are regarded as protective agents [32]. However, increasing evidence demonstrates that substantial exposure to H 2 O 2 at high concentrations (i.e., >100 M) contributes to vascular injury and associated inflammatory responses, vascular endothelial cell cytotoxicity and apoptosis, and even vascular events [33]. The present study therefore exposed the isolated rat aortic rings to high levels of H 2 O 2 (200 M) for 60 min, with the goal of establishing an in vitro model of oxidative stressinduced endothelial dysfunction. Given that ACh-induced relaxation is endothelium-dependent and reflects normal endothelium functionality [34], H 2 O 2 -induced disruption of ACh-stimulated EDR mimics the pathophysiology of ROStriggered vascular diseases. Interestingly, TMP or losartan pretreatment of H 2 O 2exposed aortic rings did not rescue EDR by altering eNOS expression (Table 3, Figure 3), although H 2 O 2 reportedly affects endothelial function by modulating Enos content in the rabbit aorta [35]. In particular, low levels of the ROS exert vasorelaxing effects via eNOS upregulation of nitric oxide [35], suggesting that high concentrations of H 2 O 2 , such as that used herein, might impair EDR by downregulating eNOS content. Nonetheless, this was not the case (Table 3, Figure 3). The protective effect of TMP to H 2 O 2 -induced endothelial dysfunction in the present study may be due to the reported TMP-mediated activation of eNOS activity through PI3K/Akt and phosphorylation of eNOS at Ser1177 for increased generation of NO in myocardial ischaemia reperfusion [12]. At the same time, TMP can alleviate oxidative stress by increased total antioxidant activity and SOD1 activity [13]. Furthermore, catalase, a strong H 2 O 2 scavenger [21], only partially reversed the impairment of EDR by H 2 O 2 (Table 2, Figure 2). Hence, H 2 O 2 may indirectly as well as directly harm the vascular endothelium.
An earlier investigation indicated that H 2 O 2 can activate NADPH oxidase to produce O 2 •− in vitro [36]. We therefore pretreated rat aortic rings with two different NADPH inhibitors, apocynin and DPI, prior to H 2 O 2 exposure and found that the drugs significantly safeguarded the aortas from ROS insult, and rescued EDR (Table 2, Figure 2 Figure 4). This exciting finding implies that O 2 •− is produced in response to exposure of the vascular endothelium to oxidative stress, which is potentially mediated via the stimulation of NADPH oxidase. On the other hand, it is still possible that H 2 O 2 exposure to rat aortic rings may increase ROS generation from mitochondria due to increased oxidative stress. TMP could improve the impaired EDR by reduction of mitochondrial ROS production [27].  We cannot exclude this possible mechanism although its contributory role may not be significant. At the same time, it has been shown that the antioxidant effect of TMP likely is due to its ability of the mitochondrial biogenesis [28].
The possibility that TMP averts H 2 O 2 -provoked endothelial dysfunction by inhibiting NADPH oxidase is supported by our findings that its actions were similar to those of apocynin and DPI in both isolated aortic rings (compare Table 1 with Figure 1 and Table 2 with Figure 2) and primary aortic endothelial cells (Table 4, Figure 4). NADPH oxidase is a key generator of ROS in blood vessel walls during the progression of vascular disease [37]. An unequivocal demonstration of the TMP-mediated suppression of NADPH oxidase expression/activity under pathological conditions, with a concomitant reduction in the overproduction of O 2 •− and other ROS, would undeniably strengthen our proposal that TMP might serve as a new antioxidant agent for the management of vascular diseases. However, this hypothesis requires further investigation, especially in regard with determining the NADPH oxidase and O 2 •− levels in rat aortic rings post-TMP treatment.
Other possible alternative mechanisms may be offered by TMP to protect endothelial dysfunction. The protective effect of TMP may be due to its action to induce antiinflammatory effect in endothelium. TMP has been shown to reverse the decrease of NO production induced by TNFand inhibit the downregulated expression of intracellular adhesion molecular-1 and heat shock protein 60 mediated by TNF-, suggesting that TMP may protect endothelium through inhibition of immunological reactions [29]. In addition, TMP treatment has been shown to prevent the increment of both inducible NO synthase and TNF-expression [38], improving survival of rodent model of endotoxic shock induced by lipopolysaccharide [39]. TMP has also been demonstrated to protect streptozotocin-induced diabetic rats by downregulated expression of vascular endothelial growth factor [40].

Conclusions
In conclusion, the present study showed that TMP prevented oxidative stress-induced endothelial dysfunction in isolated rat aortic rings without affecting eNOS expression. In addition, TMP reduced O 2 •− accumulation in H 2 O 2 -exposed primary rat aortic endothelial cells. The NADPH oxidase inhibitors, apocynin and DPI, displayed the same actions as TMP in both in vitro models. These results suggest that TMP might find therapeutic utility as an efficacious antioxidant for the treatment of human cardio-cerebrovascular diseases and warrant further exploration of its actions in whole-animal models and preclinical studies. However, the role of TMP in inhibiting NADPH oxidase remains to be elucidated.