Effects of Saccharides from Arctium lappa L. Root on FeCl3-Induced Arterial Thrombosis via the ERK/NF-κB Signaling Pathway

Saccharides from Arctium lappa. L. root (ALR-S) is a high-purity fructosaccharide separated from the medicinal plant Arctium lappa. L. root. These compounds showed many pharmacological effects in previous studies. In the present study, the antithrombotic effects of ALR-S in arterial thrombosis via inhibiting platelet adhesion and rebalancing thrombotic and antithrombotic factor expression and secretion were found in rats and human aortic endothelial cells (HAECs). This study also showed that inhibition of oxidative stress (OS), which is closely involved in the expression of coagulation- and thrombosis-related proteins, was involved in the antithrombotic effects of ALR-S. Furthermore, studies using FeCl3-treated HAECs showed that ALR-S induced the abovementioned effects at least partly by blocking the ERK/NF-κB pathway. Moreover, U0126, a specific inhibitor of ERK, exhibited the same effects with ALR-S on a thrombotic process in FeCl3-injured HAECs, suggesting the thrombotic role of the ERK/NF-κB pathway and the antithrombotic role of blocking the ERK/NF-κB pathway by ALR-S. In conclusion, our study revealed that the ERK/NF-κB pathway is a potential therapeutic target in arterial thrombosis and that ALR-S has good characteristics for the cure of arterial thrombosis via regulating the ERK/NF-κB signaling pathway.


Introduction
Arterial thrombosis is one of the main underlying causes of cardiovascular disease. The endothelium is responsible for many functions, such as the control of vascular permeability and tone, maintenance of vessel integrity, regulation of vascular inflammation, and prevention of thrombosis [1,2]. Endothelial dysfunction is the hallmark and indeed a predictor of cardiovascular disease. In atherosclerosis, diabetes, and other vascular pathology, oxidative stress signals activate vascular endothelial cells (VECs), inducing the expression of reactive oxygen species (ROS) and decreasing vessel endothelial integrity. VECs can synthesize and release antithrombotic factors, including prostacyclin (PGI 2 ), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM), and platelet endothelial cell adhesion molecule-1 (PECAM-1), which inhibit blood coagulation and platelet adhesion and aggregation and finally prevent thrombosis [3,4]. PGI 2 can relax blood vessels and inhibit platelet aggregation. The FXa-TFPI-FVIIa-TF tetramer was combined by TFPI with tissue factor (TF), then coagulated factors VII(FVII) and X(FX), thus inactivating tissue factor pathway and inhibiting coagulation reaction. TM can reversibly bind to the endothelial cell protein C receptor and reduce the level of free thrombin, thus preventing thrombin from cleaving fibrinogen or activating platelets. After endothelial cell injury, TM on the endothelial cell surface is released into the blood, which reduces the thrombin binding rate and the antithrombotic effect of endothelial cells [5]. Therefore, plasma TM content is a gold-standard indicator of endothelial cell injury. PECAM-1, synthesized by endothelial cells, is a key factor involved in platelet adhesion and aggregation. PECAM-1 can inhibit platelet aggregation and thrombosis by regulating the phosphorylation of intracytoplasmic signaling molecules to restrict communication of the activation response between cells. When endothelial cells are damaged, PECAM-1 is highly expressed, which initiates a negative feedback mechanism and inhibits thrombosis [6].
When VECs are damaged, they slough off, leading to exposure of subendothelial tissue and potentially the release of a large amount of TF. The TF complex with FVII in the blood activates the exogenous coagulation pathway to produce thrombin. Thrombin activates XIII and causes fibrin to form blood clots. In addition, when VECs are injured, a large amount of vWF is released to stimulate platelet activation and produce platelet aggregation. At the same time, activated platelets release TXA 2 , ADP, and other substances to further promote platelet adhesion, aggregation, and thrombosis.
After vascular injury, VECs, vascular smooth muscle cells, leukocytes, fibroblasts, and platelets are important sources of ROS, which can regulate platelet activation [7,8], triggering a vicious circle that leads to thrombosis. NADPH oxidase is the main regulator in the antioxidant system in organisms [9][10][11][12][13]. NOX4 is the primary subtype of NADPH oxidase expressed in VECs and participates in the production of ROS in VECs [14]. If a large amount of ROS cannot be eliminated quickly from the body, the high ROS levels can activate NF-κB, AP-1, and/or other signaling pathways through extracellular regulated protein kinase 1/2 (ERK1/2). These activation events cause lipid peroxidation, which injures VECs and promotes the overexpression of thrombosis factors, inflammatory factors, and adhesion factors. These factors can further injure VECs, cause thrombosis, and induce cardiovascular disease [15,16].
In addition to treating thrombosis with antithrombotic agents, there is increasing interest in the use of natural food products and biologically active ingredients for the prevention and treatment of thrombosis. ALR-S contain fructosaccharides that can be separated at high purity. These polysaccharides have many pharmacological effects, such as anti-inflammatory and immune regulatory activity [17,18]; antifatigue [19], anticancer [20], and hypolipidemic effects [21]; and protection against alcohol damage [22]. They also have obvious antioxidant effects in vivo and in vitro [23].
In this study, we used a FeCl 3 -induced rat arterial thrombosis model and a human aortic endothelial cell (HAEC) injury model to explore whether ALR-S can inhibit excessive OS and regulate the expression of thrombosis-associated factors through the ERK/NF-κB signaling pathway, leading to antithrombotic effects.

Materials and Methods
2.1. Preparation of ALR-S. ALR-S was extracted and purified from Arctium lappa L. root in our laboratory. The dry ALR powder was macerated in 80% alcohol at 60°C for 6 h and refluxed in aether petrolei at 45°C for 1.5 h to remove soluble contaminants. The residues were vacuum-dried and were macerated in distilled water at 95°C for 3 h; then, the filtrate was concentrated. The Sevag method was used to remove proteins [24]. The deproteinized solution was precipitated by 75% alcohol for more than 12 h, then centrifuged at 5000 rpm, 15 min, and the precipitation was extracted in water. Then, the polysaccharide precipitation was collected, washed with anhydrous alcohol and acetone, and freezedried to produce the crude ALR-S. The crude ALR-S was filtered twice by Sephadex G-50 column chromatography to obtain the purified ALR-S. Purity and molecular weight of ALR-S were detected by a high-performance gel permeation chromatography (HP-GPC) method. Infrared spectroscopy and liquid chromatography-mass spectrometry (LC-MS) were used for detecting fructose configuration and content. The results showed that the average molecular weight was 2135 Da and the purity was 97.7%. The content of fructose was about 86%, mainly in the form of fructofuranose.

Animals and Treatment.
Male Sprague-Dawley (SD) rats weighing 180 g to 220 g were purchased from the Animal Center of Dalian Medical University (Dalian, China). The rats were housed under 12 h light/12 h dark cycles at 21 ± 3°C and 55 ± 5% relative humidity.
All the groups of male SD rats were anesthetized with 20% urethane (50 mg/kg, i.p.). Different concentrations of ALR-S, heparin, or normal saline were injected, respectively, into the right femoral vein at 2 ml/kg for each group. Twenty minutes later, the common carotid artery thrombosis model was prepared according to Kurz et al.'s method [25,26]. In all groups, a 1 cm portion of the right common carotid artery was exposed by careful blunt dissection after a midline cervical incision. A small piece of plastic film was placed underneath the artery to separate it from surrounding tissues. A small piece of filter paper (5 mm × 5 mm) was soaked in 10 μl solution of 25% FeCl 3 and then applied for 5 min on the right common carotid artery to induce thrombosis. Then, the filter paper was removed, and the surgical site was rinsed with sterile saline to remove excess FeCl 3 . The control group underwent the same surgery, but FeCl 3 was replaced with normal saline.
After 10 minutes, blood samples were collected from the rats via the abdominal aorta. The blood was anticoagulated by 3.8% sodium citrate and then centrifuged (4°C, 3000 rpm, 10 min) to obtain plasma. The supernatant (plasma) was stored at -80°C.
The same length of the right common carotid artery segment containing the thrombus of all rats was dissected and weighed. Then, the arterial thrombus were carefully removed and the blood vessels were stored at -80°C. HAECs were divided into five groups: (1) control group (serum-free DMEM), (2) model group (0.4 mmol/l FeCl 3 ), (3) low-dose ALR-S group (0.1 mg/ml ALR-S), (4) middledose ALR-S group (0.2 mg/ml ALR-S), and (5) high-dose ALR-S group (0.4 mg/ml ALR-S). HAECs (1 × 10 5 ) were seeded on six-well plates and pretreated at 90% confluence with different concentrations of ALR-S or vehicle for 4 h and then exposed to 0.4 mmol/l FeCl 3 for 2 h.
Platelets ROS were detected using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe according to the instructions in the detection kit (Beyotime Institute of Biotechnology, China). Briefly, the platelets were incubated with the probe (5 μM) for 30 min at 37°C in the dark. The relative fluorescence units of the platelets were measured by a fluorescence microplate reader (Thermo Scientific, USA) using excitation at 488 nm and emission at 525 nm.

Measurement of Intracellular ROS. Intracellular ROS in
HAECs were detected with the same detection kit as Section 2.5. Each group of cells was collected and incubated with the DCFH-DA probe (10 μM) for 30 min at 37°C in the dark. Fluorescence in each group was measured by flow cytometry (Becton Dickinson, USA) using excitation at 488 nm and emission at 525 nm. Cell fluorescence was also observed by inverted fluorescence microscopy.
On the second day, the appropriate secondary antibodies were incubated at room temperature for 4 h. The target proteins were exposed to chemiluminescence plus reagents (BioTools, USA). Emitted light was measured by a multispectral imaging system (Upland, CA), and a Gel-Pro Analyzer (Version 4.0, Media Cybernetics, Rockville, USA) was used for analyzing bands of each group.

Enzyme-Linked Immunosorbent Assay (ELISA) Analysis.
The supernatant was obtained from centrifuged rat plasma (4°C, 3000 rpm, 5 min) or from cell culture medium (4°C, 3000 rpm, 20 min). The contents of TXB 2 , 6-keto-PGF1α, vWF, and PECAM-1 in the supernatant were detected according to the instructions of the ELISA kits (Dongge Company, Beijing, China). The OD values were measured at 450 nm using a microplate reader (Thermo Scientific, USA). The contents of TXB 2 , 6-keto-PGF1α, vWF, and PECAM-1 were calculated according to the standard curve.

Transmission Electron Microscopy (TEM) Analysis.
HAECs were washed with PBS, collected by centrifugation (room temperature, 2500 rpm, 10 min), and placed in 2.5% glutaraldehyde overnight at 4°C. The cells were rinsed three times with 0.1 M phosphate buffer for 15 min each, fixed for 1 hour with 1% osmium acid, and rinsed again as before. The cells were blocked with 1% uranium acetate for 2 hours and then dehydrated with an acetone gradient (50%, 70%, 80%, 90%, and 100%) for 15 minutes at each concentration and twice with 100% acetone for 10 minutes each. The cells were immersed consecutively in the following solutions: acetone : embedding solution (1 : 1) for 2 hours at 37°C; acetone : embedding solution (1 : 4) overnight at 37°C; and pure embedding solution for 2 hours at 45°C. Finally, the blocks of cells were sectioned and observed.
2.10. Platelet Adhesion Test. PRP, PPP, and washed platelets were prepared as described in Section 2.5. Washed platelets were suspended in PPP, and the counts were adjusted to Oxidative Medicine and Cellular Longevity 2.11. Statistical Analysis. Statistical analysis was performed by IBM SPSS 21.0 (Chicago, USA). All values are presented as the mean ± SD. T-test, one-way ANOVA, and the Student-Newman-Keuls (SNK) method were used for data analysis. p < 0:05 referred that the difference was statistically significant.

ALR-S Inhibited FeCl 3 -Induced Common Carotid Artery
Thrombosis in Rats. Compared with the control group, the model group showed a significant increase in thrombus weight (Figure 1(a)). The ALR-S and heparin groups showed a significant decrease in thrombus weight compared with the model group.

The Antioxidative Effects of ALR-S on FeCl 3 -Induced
Common Carotid Artery Thrombosis in Rats. One of the most important mechanisms of thrombosis was oxidative stress [30]. Antioxidants have shown potential antithrombotic effects in previous studies [31,32]. Plasma LDH, MDA, and H 2 O 2 levels in the model group were obviously increased compared with those in the control group, and the levels were obviously decreased in rats treated with ALR-S compared with model rats (Figures 2(a)-2(c)). The protein expression of NOX4, the primary subtype of NADPH oxidase, showed similar trends with those of plasma LDH, MDA, and H 2 O 2 (Figure 2(d)). However, the plasma SOD levels in the model group were significantly decreased compared with those in the control group and increased in rats treated with ALR-S compared with

The Effects of ALR-S on Platelet Adhesion to FeCl 3 -Injured HAECs.
After incubation with FeCl 3 for 2 h, the cells were observed by TEM (Figure 3(a)). The FeCl 3 treatment group showed nucleus expansion or even fragmentation, mitochondrial swelling, the decrease or disappearance of mitochondrial cristae, and vacuolization, whereas ALS-OS pretreatment decreased nuclear fragmentation, mitochondrial swelling, and vacuolization and induced an obvious improvement in cell morphology. The platelet adhesion to HAECs was determined by platelet fluorescence intensity. The FeCl 3 -treated group increased the platelet fluorescence intensity significantly compared to the control group, while the ALR-S pretreatment obviously reversed the FeCl 3 -induced platelet adhesion (Figure 3(b)).
The factors relative to platelet adhesion were determined. The medium levels of vWF were significantly increased in the FeCl 3 group compared to the control group and decreased in the ALR-S groups (Figure 3(c)). The medium levels of 6-keto-PGF 1α were significantly decreased in the FeCl 3 group and increased in the ALR-S groups (Figure 3(d)). The intracellular protein expression levels of PECAM-1 and TF were significantly increased in the FeCl 3 group and decreased in the ALR-S groups. The intracellular protein expression levels of TFPI and TM were decreased in the FeCl 3 group and increased in the ALR-S groups (Figure 3(e)).

The Effects of ALR-S on Oxidative Stress in FeCl 3 -Injured
HAECs. As shown in Figure 4, compared with the control group, the FeCl 3 treatment group showed significantly decreased GSH and SOD activities and NO release, but increased NOX4 protein expression, LDH release, and ROS generation, while ALR-S treatment reversed these changes induced by FeCl 3 .

The Effects of ALR-S on the ERK/NF-κB Signaling
Pathway in FeCl 3 -Injured HAECs. The ERK/NF-κB pathway mediates dysfunction of VECs and the release of thrombosis or coagulation-related factors [29,33,34]. p-ERK and nuclear P65 levels were significantly increased, and cytoplasmic P65 levels were significantly decreased in the FeCl 3treated group compared with the control group. ALR-S treatment significantly reversed these changes induced by FeCl 3 (Figures 4(g) and 4(h)).
3.6. The Effects of ALR-S and U0126 on Platelet Adhesion to FeCl 3 -Injured HAECs. U0126 is a specific inhibitor of ERK. U0126 decreased the platelet adhesion to HAECs compared to FeCl 3 treatment alone ( Figure 5(a)). U0126 also reversed the abnormal expression of TF, TFPI, TM, and PECAM-1 induced by FeCl 3 (Figure 5(b)). Further experiments showed that U0126 significantly inhibited ERK phosphorylation and nuclear P65 levels compared to the FeCl 3 group (Figure 5(c), 5(d)). At same time, ALR-S exhibited the same effects on the abovementioned indices with U0126 ( Figure 5(a)-5(d)).

Discussion
Thrombotic disorders are one of the most common causes of morbidity, disability, and mortality in the world. Arterial thrombosis may cause ischemic stroke (IS) and ischemic heart disease (IHD), even acute myocardial infarction, which are often life-threatening. Polysaccharides, one of the most important active ingredients of traditional Chinese medicine decoction, have attracted more and more attention. ALR-S, extracted from a traditional medicinal plant, showed multiple pharmacological activities. However, the effects and mechanism of ALR-S on thrombosis have not been reported.
The present study demonstrated that ALR-S effectively alleviated arterial thrombosis by rebalancing thrombotic/antithrombotic factors and inhibiting platelet adhesion. The underling mechanism is related to the inhibition of the OS and ERK/NF-κB pathway, leading to VEC protection. VECs are very important to maintain blood homeostasis. VEC dysfunction caused by various stimuli promotes thrombosis in the body. Oxidative stress is a common and important cause of endothelial dysfunction. FeCl 3 is widely used to establish a thrombus model for scientific researches in vivo, and previous reports indicated that FeCl 3 -mediated thrombosis involves inducing VEC oxidative injury and  7 Oxidative Medicine and Cellular Longevity platelet adhesion [35,36]. Therefore, FeCl 3 -induced arterial thrombosis model and FeCl 3 -induced HAEC injury model were used, respectively, in in vivo and in vitro experiments to investigate the effects and mechanism of ALR-S on arterial thrombosis.
VECs are extensively involved in blood homeostasis by synthesis and secretion of many active substances to regulate platelet activation, coagulation, and fibrinolysis. VEC damage is an initial stage of FeCl 3 -induced thrombus formation [36]. TF is released on VEC destruction and initiates the extrinsic coagulation process, leading to fibrin clot and thrombus formation [37,38]. TFPI, a specific inhibitor of extrinsic coagulation, inactivates TF by forming a FXa-TFPI-FVIIa-TF tetramer [39]. vWF, stored in Weibel-Palade bodies, is secreted at high levels on VEC damage to directly recruit platelets to adhere to VECs [40]. TM inhibits the fibrin clot formation and platelet activation. Plasma TM and vWF are also thought as key indicators of VEC damage. PGI 2 and PECAM-1 are secreted on stimuli to inhibit platelet aggregation. This study showed that ALR-S significantly inhibited FeCl 3 -induced arterial thrombosis in rats by rebalancing the abovementioned thrombotic/antithrombotic factors, including vWF, TF, TXB 2 (the metabolite of TXA 2 ), 6-keto-PGF1α (the metabolite of PGI 2 ), TM, TFPI, and PECAM-1. In vitro studies also showed that ALR-S inhibited platelet adhesion to HAECs and improved thrombosisrelative imbalance (vWF, TF, PECAM-1, 6-keto-PGF1α, TFPI, and TM) induced by FeCl 3 injury in HAECs. These results suggest that ALR-S exert the antithrombotic effects mainly by protecting VECs and improving VEC dysfunction.
Endothelial OS state was examined further to investigate the mechanism of ALR-S on arterial thrombosis. OS plays an important role in the early regulation of thrombosis by activating VECs. Activated VECs then produce a large amount of ROS, leading to a vicious circle of EC dysfunction [41]. The present study showed that ALR-S significantly downregulated plasma levels of H 2 O 2 , LDH, and MDA and the expression of endothelial NOX4 and upregulated SOD activities in FeCl 3 -induced artery thrombosis in rats. These results indicated that ALR-S inhibited OS via regulating the relative enzymes, as evidenced by decreased ROS production and lipid peroxidation. In vitro studies showed that ALR-S inhibited OS in HAECs by reversing the FeCl 3 -induced changes in the production of ROS, the activities of GSH and SOD, and the expression of endothelial NOX4. These results in in vivo and in vitro  Oxidative Medicine and Cellular Longevity investigations suggest that ALR-S exert the role of protecting VECs and improving VEC dysfunction mainly by inhibiting endothelial OS. As previously reported, iron can induce cell oxidative damage and produce higher ROS levels to activate ERK1/2 phosphorylation. NF-κB is then activated by p-ERK and translocates into the nucleus to bind to specific DNA fragments and regulate gene and protein expression [42][43][44], leading to EC damage, thrombosis, and other cardiovascular disease. The present study showed that ALR-S significantly downregulated the expression of p-ERK1/2 and nuclear P65, suggesting the blocking effects of ALR-S on the ERK/NF-κB pathway. U0126, the accepted specific inhibitor of ERK, is frequently used to block the ERK/NF-κB pathway. Here, U0126 was used for further clarifying the role of the ERK/NF-κB pathway in mediating ALR-S action. As expected, U0126 demonstrated the same effects with ALR-S on FeCl 3 -injured HAECs, including inhibiting platelet adhesion and rebalancing the factors of TF, TFPI, PECAM-1, and TM. Moreover, both U0126 and ALR-S inhibited the nuclear translocation of NF-κB, as evidenced by the decreased expression of nuclear P65. These results suggest that ALR-S may inhibit the ERK/NF-κB pathway and then the NF-κB translocation into the nucleus, leading to the downregulated expression of thrombotic factors and upregulated expression of antithrombotic factors.

Conclusion
In conclusion, our research revealed the effects and mechanism of ALR-S against arterial thrombosis in vitro and in vivo. ALR-S inhibited platelet adhesion to VECs and rebalanced thrombotic/antithrombotic factor expression and secretion by inhibiting the oxidative stress and ERK/NF-κB pathway in VECs, which may lead to the VEC protection and the alleviation of arterial thrombosis. The study also indicated that the ERK/NF-κB pathway may be a potential therapeutic target in arterial thrombosis and ALR-S could