2 Shandong University of Traditional Chinese Medicine, Jinan 250355, China;
3 Qingdao Hiser medical group, Qingdao 266034, China
Hypertension is an important risk factor for cardiovascular events, which are characterized by functional and structural vascular abnormalities. Vascular endothelium plays a fundamental role in modulating vascular tone and structure, and the endothelial dysfunction and resulting structural changes may be responsible for the adverse outcomes of hypertension . Angiotensin Ⅱ (Ang Ⅱ) can induce the expression of NADPH oxidase and increase the generation of intracellular reactive oxygen species (ROS). The increase in ROS results in the endothelial dysfunction, smooth muscle cell proliferation, lipid oxidation, and inflammatory reaction. The introduction of AngⅡ to vascular NADPH oxidase gene of p47phox or Nox2 knockout mouse model could not lead to hypertension or vascular hypertrophy, suggesting that ROS plays an important role in AngⅡ-induced hypertension . Although it is not recommended to supplying antioxidants in hypertension treatment, ROS is viewed as a new target for the treatment of hypertension.
Herbal medicines have stood the test of time for their safety, efficacy, and cultural acceptability. They are believed to have better compatibility with the human body . Tribulus terrestris (TT) is an herb (Zygophyllaceae family) with a wide distribution in all over the world . As a herbal remedy, it has been shown to have beneficial cardiotonic, diuretic, antioxidative and aphrodisiac effects in a number of animal experiments and human studies. TT is often used in the treatment of post-stroke syndrome, arteriosclerosis, edema, and coronary artery diseases in traditional Chinese medicine. However, little is known about the cellular and molecular mechanism underlying the action of TT. In the present study, we focused on the anti-hypertensive and vasoprotective effects of aqueous extracts of TT and the possible pharmacological mechanism for its effects against Ang Ⅱ-induced endothelial dysfunction.Materials and Methods Preparation of Tribulus terrestris
Tribulus terrestris L. (TT) were purchased from pharmacy of Affiliated Hospital of Shandong University of Traditional Chinese Medicine and identified by Prof. LI Feng in the Pharmacy College, Shandong University of Traditional Chinese Medicine, Jinan, China. The fruits of TT were air-dried and ultra-fine pulverized and the reflux extraction was performed twice using 10 volumes of water; the first extraction was decocted for 1.5 h, and the second was for 1 h. The extracts were combined, filtered, and concentrated under reduced pressure to a relative density of 1.20-1.25 (70-80 ℃). Excipient of powdered erythrose alcoholand stevioside (99 : 1) granules were then added and dried below 60 ℃ to obtain TT granules at 2 g raw herbs/g granules. For the experiments in vitro, the TT granules were dissolved in a phosphate buffered saline (PBS) and sterilized by passing through a 0.22-μm syringe filter. Valsartan was purchased from Novartis Pharma Ltd, Lot. # x1417 (Novartis, Beijing, China).Animals
The animal study was approved by the Faculty of Medicine & Health Sciences Ethics Committee for Animal Research, Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Jinan, China). Thirty-two male spontaneously hypertensive rats (SHRs), VAH/SPF level, 8-week-old, 164-190 g, and 8 age-matched normotensive Wistar Kyoto (WKY) rats were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. [certificate: SCXK (Beijing) 20120001]. The rats, housed under a 12 h/12 h light/dark cycle, were allowed food and water ad libitum with normal salt intake. SHRs were randomly divided into 4 groups: spontaneously hypertensive model group, high dose TT group (17.2 g raw herbs of TT·kg-1 body weight·d-1), low dose TT group (8.6 g raw herbs of TT·kg body weight·d-1), and valsartan group (13.35 mg valsartan·kg-1 body weight/d-1). The rats in each group were administered with corresponding drugs intragastricaly for 6 weeks, and the hypertensive model rats and WKY rats were used as controls and intragastricaly given the same volume of saline for the same duration.Detection of blood pressure and heart rate
Systolic pressure, diastolic pressure, mean arterial pressure and heart rate were detected daily by the non-invasive rat tail method. Rats were put in the ALC-HTP animal system, heated to dilate rat tail artery, and data were measured with ALC-NIBP noninvasive blood pressure analysis system . All rats were measured 5 times in parallel.Morphological observation of arterial endothelium
Rats were sacrificed at the end of the 6th week by overdose anesthesia with sodium pentobarbital (60 mg·kg-1, i.p.). After blood was collected by venipuncture, the thoracic aorta were taken on ice as soon as possible and cut into two parts, one part was fixed in neutral formaldehyde for HE staining and the other part was fixed in 2.5% glutaraldehyde solution, and then observed under scanning electron microscope (SEM) and transmission electron microscope (TEM) to observe the ultrastructural changes.Cell culture
Human umbilical vein endothelial cells (HUVECs, ScienCell, Santiago, California, USA) were cultured in a endothelial cell medium (ECM, ScienCell) with 5% FBS. The 3rd-8th passage cells were used in the experiments. HUVECs were pre-incubated for 60 min with TT (3 and 30 μg·mL-1 separately) or 10-5 mol·L-1 of valsartan (as positive controls). We then established the hypertensive endothelium model by applying 2 × 10-6mol·L-1 of AngⅡ (Sigma-Aldrich, St. Louis, MO, USA) for 24 h.Analysis of apoptosis in HUVECs
Annexin V/PI staining (Lifetechnologies, Eugene, OR, USA) was assessed to measure the level of early apoptosis. HUVECs were treated with drugs and then trypsinized and collected. The cells (2 × 105) were washed with ice-cold PBS, incubated with Annexin V and PI, and analyzed by flow cytometry (BD Accuri C6, Franklin L., NJ, USA).Enzyme-linked immunosorbent assay (ELISA) assay
The serum level of AngⅡ, endothelin-1 (ET-1), superoxide dismutase (SOD) and malonaldehyde (MDA), and the endothelial nitric oxide synthase (eNOS), ET-1, SOD and MDA in the cell culture supernatant were assayed using ELISA kits (Bioyun, Shanghai, China), according to the manufacturer's instructions. All samples were assayed in triplicate.Assessment of intracellular reactive oxygen species (ROS) generation
HUVECs in 6-well plates were loaded with fluorescent probe 2′, 7′-dichlorofluorescin diacetate (DCFH-DA, 10 μmol·L-1, Beyotime, Haimen, China) at 37 ℃ for 30 min and washed with PBS thrice to avoid high background fluorescence, and then observed in situ under a Zeiss Vert A1 fluorescence microscope (Zeiss, Jena, Germany).
The treated HUVECs were harvested and washed with calcium-and magnesium-free PBS (pH 7.4), and loaded with 10 μmol·L-1 DCFH-DA at 37 ℃ for 30 min and washed with PBS thrice . Fluorescence was measured using a flow cytometer (BD Accuri C6, USA); excitation was read at 488 nm and emission was detected at 525 nm. Relative ROS production was expressed as the percentage of fluorescence for the treated samples over fluorescence for the appropriate controls: (fluorescence treatment/fluo rescence control) × 100.Analysis of cell migration by Transwell assay
The cell migration assay was performed with a Transwell insert system (6.5 mm diameter inserts with 8.0 μm pores in a polycarbonate membrane situated in wells of 24 well polystrene, tissue culture-treated plates, Corning, Corelle, NY, USA). The Transwell inserts were precoated with matrigel (1 : 8) 4 ℃ overnight and hydrated with RPMI 1640 in 37 ℃, 5% CO2 incubator for 30 min. HUVECs suspension was added at 20 000 cells per insert. Drugs were added to the Transwell inserts, and ECM in the lower chamber. At the end of the observation, the cells on the upper surface of the insert were removed. The migrated cells on the bottom side were fixed in absolute ethyl alcohol and stained with hematoxylin-eosin (HE) staining. Cells were counted from four random fields under a microscope at 100 × magnification.Quantitative real-time PCR
The possible genes involved in hypertension (Akt1, Jak2, PI3Kα, Erk2, FAK, and NF-κB p65) were chosen for quantitative real-time PCR (qRT-PCR) to explore the possible mechanism of TT against Ang Ⅱ-induced endothelial dysfunction. The specific forward / reverse primer sequences (Sangon Biotech, Shanghai, China) were as follows: Akt1: 5′-GCCCAA CACCTTCATCATC-3′/5′-TCCTCCTCCTGCTT-CTTGAG-3′; JAK2: 5′-TTTGGGAGTGTGGAGATGTG-3′/5′-TGCTGTAG GGATTTCAGGATT-3′; PI3Kα: 5′-CGTT-TCTGCTTTGGG ACAAC-3′/5′-CCTGATGATGGTCGTGG-AG-3′; Erk2: 5′-G CGGTTAGTTCTCTCTTCT-3′/5′-GCTC-GACGCTTCGCGTTAC-3′; FAK: 5′-CTCCTACTGCCAAC-CTGGAC-3′/5′-GC CGACTTCCTTCACCATAG-3′; and NF-κB p65: 5′-GACCT GGAGCAAGCCATTAG-3′/5′-CACTGTCACCTGGAAGCAGA-3′. Total RNA was isolated using the TRIzol method (Invitrogen, Carlsbad, California, USA) and reverse-transcribed into cDNA with the PrimeScript RT reagent kit (TaKaRa, Dalian, Liaoning, China). Real-time PCR was performed with a LightCycler 480 SYBR Green Ⅰ Master kit (Roche, Mannheim, Germany). The reaction mixture contained SYBR Green Ⅰ Master, cDNA and the forward/reverse primers. Reaction mixture and amplification conditions were maintained according to the manufacturer's instructions. Each RNA sample was tested in triplicate, and the threshold cycle values were normalized to that of β-actin. The relative gene expressions (fold change) of each group was calculated with the 2-ΔΔCT method [6-7].Western blotting analysis
According to the results of qRT-PCR, the protein expression levels of Erk2, FAK and NF-κB p65 were determined by Western blotting analysis. In brief, the HUVECs were pre-treated with drugs for 1 h, induced by 2 × 10-6mol·L-1 Ang Ⅱ for 24 h, trypsinized, and collected. The cells were washed twice with 0.1 mol·L-1 PBS and then lysed in RIPA lysis buffer (Beyotime, Nantong, China). The protein concentration of the lysate was determined using a BCA protein assay kit (Beyotime, Nantong, China). Cell lysates containing 30 μg of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis using 12% polyacrylamide resolving gels. After electrophoresis, the proteins were transferred onto PVDF membranes, which were then blocked with 5% nonfat dry milk in PBS-0.05% Tween-20 (PBS-T) for 1 h at room temperature, and incubated at 4 ℃ with gentle shaking overnight with primary antibodies (rabbit anti-human Erk2, 1 : 3 000; mouse anti-human FAK, 1 : 2 000 and rabbit anti-human NF-κB p65, 1 : 2 000, Proteintech, Wuhan, China). After washed with PBS-T, the blots were incubated with horseradish peroxidase conjugated to goat anti-rabbit IgG (1 : 20 000) for 1 h. They were then incubated with 0.5 mL of immobilon Western chemiluminescent HRP substrate (Merck Millipore, Darmstadt, Germany) and exposed for 60 s under Fluor Chem Q. The optical density of the protein bands of interest relative to that of β-actin was analyzed by Image J (National Institutes of Health, Bethesda, Maryland, USA).Statistical analysis
Statistical analyses were performed by using SPSS 19.0. The values are presented as means ± standard deviation. Unless otherwise stated, statistical comparisons were made using a single-factor analysis of variance (ANOVA). P-values less than 0.05 were considered statistically significant.Results Blood pressure and heart rate
Blood pressure began to decrease after rats had been treated with drugs for about ten days (P < 0.05). The anti-hypertensive effect reached the highest point at the end of the 5th week, compared with that of untreated SHRs (P < 0.05). Systolic blood pressure decreased about 30 mmHg in valsartan group, 24 mmHg (3 199.74 Pa) in high dose TT group, while low dose TT group was less significantly (17 mmHg) (Fig. 1A). Diastolic pressure decreased about 18 mmHg in high dose TT group, 16 mmHg in valsartan group, and 11 mmHg in low dose TT group (Fig. 1B). Mean arterial pressure decreased by 15-22 mmHg in treatment groups at the end of 6-week-treatment (Fig. 1C). Only high dose TT demonstrated effects on heart rate. Heart rate in high dose TT group decreased in the 2nd, 3rd and 6th week. The heart rate showed no significant difference in other groups during the 6-week study (Fig. 1D).
The vascular cord of WKY rats arranged in neat rows, structural integrity, intercellular connection integrity, and mucosa smooth, with no obvious fibrous or plaque attached. The large elastic arteries were damaged seriously in SHRs. The vascular endothelia of SHRs sheded significantly, aggregated, the cable partly disordered, and cell-connection lost, with some holes or honeycombs shaped endometrium and with attachments adhesive to membrane. The vascular smooth muscle layer hyperplasia of SHRs was notable, and the blood vessels became hypertrophy. Cell organelles were abundant, mitochondria and endoplasmic reticulum were expanded, which demonstrated "secretory type", and the stroma increased in the vascular endothelium of SHRs. Endometrial integrity and shedding state of endothelial cells improved obviously after drug treatment. The high dose TT demonstrated a similar protective effect on endothelium as valsartan. The efficacy of low dose TT was a little lower than that of the other two drugs. (Fig. 2).
The serum levels of AngⅡ, ET-1, and MDA were higher, meanwhile SOD was lower (P < 0.05) in SHRs than that of WKY rats. The level of all the biomarkers of endothelial damage and oxidative stress normalized in different degrees after all the drug treatments (Fig. 3). Valsartan demonstrated excellent efficacy on down-regulation of AngⅡand ET-1, high dose TT showed good efficacy on regulation of serum level of MDA and SOD.
The apoptosis rate increased (21.2 fold) after treating the HUVECs with AngⅡ for 24 h (P < 0.05). All of the drugs suppressed apoptosis rate to different degree. TT (30 μg·mL-1 TT group) demonstrated the better efficacy than valsartan for suppressing apoptosis (Figs. 4A and B).
AngⅡ decreased the concentration of eNOS and SOD and increased ET-1, and MDA in the culture supernatant significantly (P < 0.05). The level of all the biomarkers of endothelial damage and oxidative stress normalized in different degrees with the treatments (Fig. 5). High dose TT demonstrated even better efficacy than valsartan on regulation of level of oxidative stress in vitro.
AngⅡ increased ROS generation significantly (P < 0.05, from 3.19% ± 0.37% to 9.41% ± 0.89%). TT and valsartan effectively improve the ROS generation of HUVECs (P < 0.05; Fig. 5).
HUVECs migration capability was decreased by 41.6% (P < 0.05) after induction with AngⅡ for 24 h. In contrast, the number of the migrated cells was obviously increased in the presence of TT or valsartan (P < 0.05; Fig. 6).
To explore the pharmacological mechanism of TT as vascular protective agent, the possibly involved genes were assayed. AngⅡ increased the gene expression of Akt1, Jak2, PI3Kα, Erk2, FAK, and NF-κB p65, with the most notable changes being with Erk2, FAK and NF-κB p65 (P < 0.05). Then, we verified the gene expression results by Western blotting. The changes in protein expression were consistent with gene expression. These AngⅡ-induced changes were prevented by TT and valsartan to different degrees (P < 0.05; Fig. 7).
Tribulus terrestris (family Zygophyllaceae) has been used for a long time in Chinese medication systems for treatment of various kinds of diseases. Its various parts contain a variety of chemical constituents which are medicinally important, such as flavonoids, flavonol glycosides, steroidal saponins, and alkaloids. It has diuretic, aphrodisiac, antiurolithic, immunomodulatory, antidiabetic, absorption enhancing, hypolipidemic, cardiotonic, central nervous system, hepatoprotective, anti-inflammatory, analgesic, antispasmodic, anticancer, and antibacterial activities . For the last few decades, extensive research work has been done to prove its biological pharmacological activities of its extracts. Though TT is touted as a testosterone booster and remedy for impaired erectile function, the dried fruit of the herb is usually used in the treatment of hypertension, post-stroke and genitourinary tract disorders in the clinical application of traditional Chinese medicine. In the present study, we aimed to confirm the anti-hypertensive effects of TT on SHR and explore the possible cellular mechanism. Our results indicated that high dose TT (17.2 g·kg-1·d-1) notably attenuated the diastolic pressure and heart rate of SHR. The pharmacological mechanism needs to be validated in the following study.
Many of Ang Ⅱ's pathologic effects in the vasculature occur via activation of NADPH oxidases and generation of ROS . Increasing evidences indicate that oxidative stress has a pivotal role in atherogenesis, hypertension and age-related vascular dysfunctions. The oxidative stress leads to an exaggerated production of O2-, decreases the NO bioavailability and consequently endothelial dysfunction [10-11]. ROS have been identified as major contributors to biological damage in organisms . Experimental genetic models of arterial hypertension (SHR) are characterized by endothelial dysfunction, which is a primary contributor to the increased generation of ROS, leading to NO breakdown [13-14]. In the present study, TT reduced the endothelial injury, prevented aorta remodeling under HE staining, SEM and TEM observation, decreased ROS generation in vitro and demonstrated good efficacy on regulation of level of MDA and SOD both in vivo and in vitro, suggesting the vasoprotective effects against oxidative stress of TT.
One of the significant results from the present study was that high dose TT (30 mg·mL-1) suppressed AngⅡ-induced HUVEC proliferation and apoptosis rate. And TT prolonged the HUVEC survival time and postponed the cell decaying stage notably. TT also improved the migration of HUVECs. This suggested that the stabilization of endothelial cells and improvement of endothelial function might be one of the cellular mechanisms by which TT alleviates hypertensive endothelial injury.
Akt1, Jak2, PI3Kα, Erk2, FAK and NF-κB p65 are representative regulatory molecules associated with the hypertensive endothelial injury . Therefore, they were chosen to explore the possible pharmacological mechanism of TT. According to the RT-PCR results, TT decreased the mRNA expression of Akt1, Jak2, PI3Kα, Erk2, FAK, and NF-κB p65, with the most notable regulation being on Erk2, FAK and NF-κB. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are core factors that regulate cell hypertrophy and proliferation [16-17]. Initiation of FAK signaling facilitates cell adhesion, migration, proliferation, and differentiation [18-19]. NF-κB is an oxidant-sensitive transcriptional factor, which plays a crucial role in the expression of many genes involved in vascular inflammation and remodeling . They cross talk with each other in pathology of hypertension. The Western blot results from the present study showed TT decreased the protein expression of Erk2, FAK and NF-κB p65. We think that Erk2, FAK and NF-κB may be the possible targets of the pharmacological mechanism of TT as the vascular protective agent.
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