Tetramethylpyrazine Protects Endothelial Injury and Antithrombosis via Antioxidant and Antiapoptosis in HUVECs and Zebrafish

Chuanxiong Rhizoma, the dried rhizome of Ligusticum chuanxiong Hort., is a commonly used drug for promoting blood circulation and dissipating congestion. Tetramethylpyrazine (TMP), the main active ingredient of Ligusticum chuanxiong, has significant antioxidant, anti-inflammatory, and vascular protective effects. However, the protective properties and underlying mechanisms of TMP against endothelial injury-induced insufficient angiogenesis and thrombosis have not been elucidated. Therefore, we aimed to explore the protective effects of TMP on endothelial injury and its antithrombotic effects and study the mechanism. In vitro experiments showed that TMP could alleviate hydrogen peroxide– (H2O2–) induced endothelial injury of human umbilical vein endothelial cells (HUVECs) and the protective mechanism might be related to the regulation of MAPK signaling pathway, and its antioxidative and antiapoptotic effects. In vivo experiments showed that TMP restored PTK787-induced damage to intersegmental vessels (ISVs) in Tg(fli-1: EGFP)y1 transgenic (Flik) zebrafish larvae. Similarly, adrenalin hydrochloride– (AH–) induced reactive oxygen species (ROS) production and thrombosis in AB strain zebrafish were inhibited by TMP. RT-qPCR assay proved that TMP could inhibit the expression of fga, fgb, fgg, f7, and von Willebrand factor (vWF) mRNA to exert an antithrombotic effect. Our findings suggest that TMP can contribute to endothelial injury protection and antithrombosis by modulating MAPK signaling and attenuating oxidative stress and antiapoptosis.


Introduction
Thrombosis often causes cardiovascular diseases such as coronary heart disease, atherosclerosis, ischemic stroke, and myocardial infarction, which has a profound impact on people's quality of life [1]. At present, the most commonly used antithrombotic drugs are warfarin, aspirin, and heparin, but they are often accompanied by some side effects such as bleeding and drug resistance [2]. Therefore, it is necessary to study the molecular mechanism of thrombosis and explore alternative antithrombotic therapies.
It is generally accepted that oxidative stress and apoptosis are major factors associated with endothelial injury and thrombosis in cardiovascular diseases. Mitochondria are the main producers of reactive oxygen species (ROS), and the imbalance between the overproduction of mitochondrial ROS (mROS) and the antioxidant defense system leads to oxidative stress, which is a key initial step in the pathogenesis of many vascular diseases [3]. And oxidative damage is thought to lead to the initiation of endothelial injury and coagulation cascade [4]. On the other hand, excess ROS can lead to increased permeability of mitochondrial membrane, and various proteins and proteases related to apoptosis are released into the cytoplasm, leading to apoptosis and ultimately promoting thrombus formation. Therefore, studying oxidative stress and apoptosis in endothelial cells may provide a better understanding of the pathogenesis of thrombosis.
Chuanxiong Rhizoma, the dried rhizome of Ligusticum chuanxiong Hort., is one of the commonly used drugs for promoting blood circulation and removing blood stasis, and has protective effects on cardiovascular and cerebrovascular diseases [5]. Tetramethylpyrazine (TMP), as one of the important alkaloids extracted from Ligusticum chuanxiong, has pharmacological activities such as anti-inflammatory, antioxidation, antiplatelet aggregation, antiapoptosis, and improving microcirculation [6][7][8][9]. It is known that the MAPK pathway can be activated by hydrogen peroxide (H 2 O 2 ), thereby regulating various cellular activities, including proliferation, differentiation, survival, and death. However, whether TMP exerts its antithrombotic effect by protecting endothelial injury through the MAPK pathway has not been thoroughly studied. Therefore, in this study, human umbilical vein endothelial cells (HUVECs) were used to establish H 2 O 2 -induced a H 2 O 2 -induced oxidative damage model, and zebrafish larvae were used to establish thrombosis and angiogenesis deficiency models to evaluate the vascular endothelial protection and antithrombotic activities of TMP and lay a molecular basis and therapeutic potential for TMP to prevent cardiovascular diseases.
2.3. Determination of TMP Concentration. MTT assay was used to study the cytotoxicity of TMP on HUVECs. Cells were seeded at a density of 8 × 10 3 in sterile 96-well plates and incubated at 37°C for 24 h, using 10% FBS-1640 medium, 100 μL/well. Cells were treated with various concentrations of TMP (5-120 μg/mL) dissolved in DMSO, and medium containing 1% FBS was used. The control group only cultured 1640 medium containing 1% FBS in 5% CO 2 incubator at 37°C for 24 h. Then, add 20 μL MTT solution to each well, and incubate at 37°C for 4 h in the dark. After that, the MTT solution was discarded. Add 150 μL DMSO solution to each well at room temperature to lyse cells and mix for 5 min. Finally, the absorbance was determined at 570 nm with a BIO-RAD microplate reader (Benchmark Plus, USA).

Oxidative Stress Injury
Model. Cells were seeded in a sterile 96-well plates at a cell density of 8 × 10 3 for 24 h. HUVECs were exposed to H 2 O 2 (50-800 μM) for 24 h, and cell viability was detected by MTT as described in Section 2.3.

Cell Viability
Test. Cells were seeded in sterile 96-well plates at a cell density of 8 × 10 3 for 24 h. HUVECs were exposed to TMP (10, 20, and 40 μg/mL) and H 2 O 2 (100 μM) for 24 h, and cell viability was detected by MTT as described in Section 2.3.
2.6. Wound Healing Assay. HUVECs were seeded in 6-well plates and grown to about 80% fusion rate. Scratch with 200 μL sterile plastic pipette tip, and wash with PBS to remove non-adherent and damaged cells. The model group was treated with H 2 O 2 (100 μM), and the administration groups were treated with different concentrations of TMP (10, 20, and 40 μg/mL) and H 2 O 2 (100 μM) and then incubated with basal medium containing 1% FBS for 24 h. Image of the wounds were taken in five random fields (×100) by an inverted microscope (Leica DMI3000 B, Germany) at 0 h and 24 h. The wound healing areas were calculated by Image J, and the formula of wound healing ability is as follows: Wound closure% = ðwound areas on 0 h − wound areas on 12 hÞ/wound areas on 0 h × 100%.
2.13. ROS Analysis. Zebrafish larvae were grouped as described in Section 2.12.2. After treatment, zebrafish were washed 3 times and incubated with 25 ng/mL DCFH-DA solution for 30 min at 28.5°C in the dark. Afterwards, the embryos were rinsed with fresh medium and photographed under a fluorescence microscope. Finally, the fluorescence intensity of zebrafish larvae was analyzed by Image J software.
2.14. Total RNA Extraction, Reverse Transcription, and RT-qPCR Analysis. AB zebrafish larvae were grouped and administered as described in Section 2.12.2. Total RNA was extracted using Total Animal RNA Isolation kit (FORE-GENE), and the RNA concentration was measured at 260/ 280 nm by detecting OD. Then, the RNA was converted to single-strand cDNA with a cDNA Synthesis System for 3 Oxidative Medicine and Cellular Longevity RT-PCR (ABclonal). After that, RT-PCR was performed using an Applied Biosystems 7500HT Sequence Detection System. The RT-qPCR reaction conditions were set as follows: 95°C for 3 min, 40 cycles of 95°C for 5 s, and 60°C for 30 s. The expression of f7, fga, fgb, fgg, and von Willebrand factor (vWF) mRNA was analyzed according to the manufacturer protocol. GAPDH was used as an internal reference. All primer sequences (TsingKe) used are listed in Table 1.

Ethics Statement.
Studies on zebrafish were conducted following the Experimental Animal Ethical Committee of Chengdu University of Traditional Chinese Medicine.

Data Analysis.
All the values were presented as Mean ± S:D. The differences between groups were analyzed using Student's t-test when there were only two groups or assessed by one-way ANOVA when there were more than two groups. All statistical analyses were performed using Graph-Pad Prism 8.0 (GraphPad, San Diego, CA, USA). A twotailed value of P < 0:05 was considered statistically significant.

TMP Promoted Cell Proliferation and Inhibited H 2 O 2 -
Induced Injury in HUVECs. As shown in Figure 2(a), there was no significant cytotoxicity when TMP treatment in the concentration range of 2.5 μg/mL to 120 μg/mL for 24 h compared to the control group. Moreover, TMP showed significant proliferative ability in the concentration range of 10 μg/mL to 40 μg/mL. Therefore, 10 μg/mL, 20 μg/mL, and 40 μg/mL were selected as the optimal study concentrations.
The results of H 2 O 2 inducing HUVEC injury showed that the treatment with 50-800 μM H 2 O 2 for 24 h reduced cell viability. Among them, 100 μM H 2 O 2 reduced the survival rate of HUVECs to about 50% (P < 0:001), as shown in Figure 2(b). Based on this, 100 μM was selected as the modeling concentration to induce oxidative damage in HUVECs.

TMP Promoted HUVEC Migration.
Endothelial cell migration is critical for angiogenesis. The effect of TMP on the migration of HUVECs was assessed by wound healing assay. As shown in Figure 3, the migration rate of the control group was 60.03%. After H 2 O 2 (100 μM) treatment for 24 h, the mobility decreased to 17.43% (P < 0:001), which significantly inhibited the migration of HUVECs. However, compared with the model group, the cell migration rates in the TMP groups were 31.41%, 37.85%, and 48.27%, respectively, and the migration ability was significantly increased. The experimental results revealed that TMP could promote the migration of HUVECs.

TMP Suppressed mROS Production Induced by H 2 O 2 .
To investigate the antioxidant effect of TMP, the mROS production was assessed by MitoSOX Red. As shown in Figure 4, after H 2 O 2 treatment, the MitoSOX Red staining signal intensity was 285.14, which was significantly higher than that in the control group (P < 0:001), indicating that H 2 O 2 can induce oxidative damage in HUVECs. While the TMP treatment groups could significantly inhibit the overproduction of mROS induced by H 2 O 2 , the signal intensity decreased to 245.25, 124.97, and 125.29, respectively. These results showed that TMP could significantly reduce mROS overproduction in HUVECs after H 2 O 2 -induced oxidative damage.

TMP Regulated SOD and MDA in H 2 O 2 -Indced
HUVECs. MDA and SOD as the biomarkers of oxidative stress reflect the degree of damage to cell membrane function and integrity. As shown in Figure 5, compared with the control group, MDA was significantly increased (P < 0.05), and SOD activity was significantly decreased (P < 0:001) in the H 2 O 2 model group, indicating that the antioxidant capacity of HUVECs was impaired. After treatment with TMP (10, 20, and 40 μg/mL), the contents of MDA were significantly decreased, and the activity of SOD were significantly increased compared with the H 2 O 2 group (P < 0:01 and P < 0:001).

TMP Regulates the Expression Levels of MAPK Pathway-Related Proteins.
To investigate whether TMP can protect endothelial injury through the MAPK signaling pathway, we further performed western blot assay. As shown in Figure 6, compared with the control group, the model group significantly inhibited the expression ratios of p-p38/p38 Oxidative Medicine and Cellular Longevity (P < 0:05), p-MEK1/2/MEK1/2 (P < 0:05), and p-ERK1/2/ ERK1/2 (P < 0:001), while TMP treatment (10, 20, and 40 μg/mL) significantly increased the expression ratios of these proteins. Therefore, these results suggested that TMP could promote angiogenesis by activating the MAPK pathway in HUVECs.
3.6. TMP Inhibited Apoptosis in HUVECs. Inhibition of HUVEC apoptosis is an effective method to protect endothelial cell injury. Therefore, double staining with annexin V-FITC/PI was used to assess the apoptosis of HUVECs. As shown in Figure 7, H 2 O 2 stimulation significantly promoted the apoptosis of HUVECs compared with the control group. TMP could significantly inhibit apoptosis, including early and late apoptotic cells.

TMP Regulated the Expression Levels of Mitochondrial
Apoptosis Pathway-Related Proteins in HUVECs. Mitochondria play an important role in apoptosis. To further elucidate the potential molecular mechanism of TMP inhibiting the apoptosis of HUVECs, western blotting was used to detect the changes of TMP-related apoptosis proteins in the mitochondrial apoptosis signaling pathway in HUVECs. As shown in Figure 8, TMP significantly inhibited the expression of the proapoptotic protein Bax, cytochrome c, caspase-3, and cleaved caspase-3 and promoted the expression of the antiapoptotic protein Bcl-2, thereby exerting an antiapoptotic effect.
3.8. TMP Restored PTK787-Induced ISV Insufficiency in Zebrafish Embryos. As shown in Figure 9, 1 dpf zebrafish embryo treated with PTK787 for 24 h significantly inhibited the formation of ISVs. Therefore, we chose PTK787 to inhibit vascular structure to evaluate the proangiogenic activity of TMP in zebrafish embryos. The growth of ISVs was significantly inhibited to almost 60.87% after the treatment with 0.2 μg/mL PTK787 for 24 h in AB strain zebrafish. TMP (10, 20, and 40 μg/mL) treatment significantly promoted the recovery of ISVs and reversed the ISV deficiency induced by PTK787. Thus, TMP has a protective effect on PTK787-induced ISV insufficiency in zebrafish embryos. To study the antithrombotic effect of TMP in vivo, zebrafish were stained with o-anisidine. The antithrombotic activity of TMP was analyzed by calculating the erythrocyte staining intensity of the zebrafish caudal vein. As shown in Figure 10, compared with the control group, the staining intensity of caudal vein thrombus was significantly increased in the AH group (P < 0:001), indicating that AH can successfully induce the zebrafish thrombus model. Interestingly, the TMP (10, 20, and 40 μg/mL) treatment significantly improved caudal vein thrombosis, and the inhibition rates of thrombus were 58.09%, 61.70%, and 77.40%, respectively. These results suggest that TMP has a certain therapeutic effect on AH-induced thrombosis in zebrafish.

TMP Inhibited AH-Induced Oxidative Damage in
Zebrafish. Oxidative stress plays an important role in thrombosis. As shown in Figure 11, AH could significantly induce the production of ROS in zebrafish. Excessive production and accumulation of ROS can induce vascular endothelial cell damage in vivo, eventually leading to thrombosis. However, after treatment with different concentrations of TMP, the signal intensity of DCFH-DA was significantly attenuated, indicating that TMP can improve oxidative stress in zebrafish and inhibit thrombosis through antioxidation.

TMP Reduced the Expression of vWF, Coagulation
Factor (f7), and Fibrinogen (fga, fgb, fgg) in the Zebrafish Thrombosis Model. To further explore the possible antithrombotic mechanism of TMP, we quantitatively analyzed platelet activity and coagulation cascade-related mRNA by RT-qPCR. The results showed that the expression of fga, fgb, fgg, f7, and vWF mRNA was significantly upregulated after AH incubation and significantly decreased under TMP (10, 20, and 40 μg/mL) treatment, as presented in Figure 12. These results suggested that the mechanism of the antithrombotic effect of TMP may be related to inhibiting the related mRNA expression of platelet activity and coagulation cascade.

Discussion
Thrombosis is a common symptom of cardiovascular diseases with high morbidity and mortality. Vascular endothelial injury, slowed blood flow, and coagulation dysfunction   Oxidative Medicine and Cellular Longevity are important causes of thrombosis [11]. Among them, oxidative stress-induced endothelial injury, as one of the pathophysiological mechanisms that may cause thrombosis, can lead to venous wall damage. The functional integrity of endothelial cells in the vessel wall is an important protective mechanism against thrombosis [12]. It has been confirmed that endothelial injury can disrupt endothelial permeability and promote thrombosis in vivo [13]. Oxidative stress, apoptosis, and endothelial injury are inextricably linked to thrombosis. Once the pathological damage is induced by various stimuli such as oxidative stress in endothelial cells, the cell proliferation apoptosis and vascular damage will be affected through some signaling pathways [14]. Damaged vascular endothelial cells expose collagen, activate platelets, initiate coagulation, and gradually lead to thrombosis. Among them, oxidative stress is the initial event, and apoptosis is the core event of thrombosis. Interestingly, when ROS accumulate in vivo, these free radicals activate mitochondria to induce oxidative stress and then activate the MAPK signaling pathway, finally activating caspases to induce neuronal apoptosis [15]. Studies have demonstrated that the alleviation of endothelial cell oxidation and apoptosis inhibits thrombus development [16,17]  Oxidative stress is caused by an antioxidationprooxidation imbalance that can lead to elevated ROS and cell damage [18]. In vascular endothelial cells, ROS is a byproduct of mitochondrial metabolism and mainly originates from mitochondria [19]. In the cardiovascular system, ROS plays a role in regulating proliferation, endothelial function, and angiogenesis and is one of the important factors in thrombosis [20,21]. Previous studies confirmed the presence of high oxidative stress in thrombosis patients, suggesting that oxidative stress plays a key role in the pathological process of thrombosis [22]. Therefore, antioxidant effects can be used to prevent endothelial damage and ultimately improve thrombus formation. MDA is produced by the per-oxidation of polyunsaturated fatty acids on the cell membrane, which can further cause the tissue oxidative damage and is usually used as an indicator of oxidative stress in cell membranes [23]. Studies have shown that compared with healthy controls, patients with venous thrombosis have higher levels of MDA in the body [22]. SOD is the main antioxidant in vascular endothelial cells, which can remove lipid peroxides in the body and reduce peroxidative damage to cells. Studies have found that antioxidants can protect cells from oxidative stress by increasing SOD activity, thereby preventing thrombosis [24]. High concentrations of H 2 O 2 can be converted into oxygen free radicals and hydroxyl radicals in cells, promoting the accumulation of ROS in HUVECs to cause oxidative stress [25]. In the present study, we demonstrated that H 2 O 2 induced oxidative   9 Oxidative Medicine and Cellular Longevity signals originate from mitochondria and can be activated by a variety of stimuli including oxidative stress. Parts of the Bcl-2 family members, including proapoptotic (Bax) and antiapoptotic (Bcl-2, Bcl-xl) proteins, control mitochondrial membrane potential to regulate apoptosis signaling [27].
Among them, Bax stimulates the release of cytochrome c from mitochondria by enhancing the permeability of the mitochondrial outer membrane, thereby activating the downstream apoptosis executor caspase and initiating the apoptosis process [28]. Caspase-3, as an inactive dimer, is 10 Oxidative Medicine and Cellular Longevity activated to cleaved-caspase-3 by proteolytic cleavage of a subunit, which further cleaves different substrates, leading to the expansion of the protease cascade and ultimately cell death [29]. Once vascular endothelial cells are damaged or apoptotic, the expression of phosphatidylserine will be increased, the asymmetry of membrane phospholipids will 11 Oxidative Medicine and Cellular Longevity be deprived, and anticoagulant membrane components will be eliminated, thereby significantly promoting thrombosis [30]. The occurrence and regulation of apoptosis involve a variety of signaling pathways. Among them, PI3K/Akt, MAPK and Wnt signaling pathways have significant changes in thrombosis, and antiapoptotic signaling pathways can resist thrombosis by regulating endothelial cell status [14]. Therefore, modulation of apoptosis is a potential therapeutic approach for the treatment of thrombosis and cardiovascular diseases. Present studies showed that H 2 O 2 treatment increased the expression of Bax, cytochrome c, and caspase-3 proteins and decreased the level of Bcl-2 in HUVECs, which indicated that H 2 O 2 induced apoptosis. However, TMP reversed all these changes, suggesting that TMP has an antiapoptotic effect in endothelial cells.
Numerous studies have shown that MAPKs belong to the filaments/threonine protein kinases and play key roles in cell proliferation, migration, and angiogenesis [31]. MEK1/2 and ERK1/2 are members of the MAPK family, and once MEK1/2 is phosphorylated and activated, so will ERK1/2 [32]. Park et al. reported that rosin enhanced the migration and tube formation of HUVECs by regulating the MAPK pathway [33]. Furthermore, fucoidan has been shown to induce angiogenesis in HUVECs through the phosphorylation of MAPKs [31]. Recent studies have demonstrated that the activation of MAPKs can protect endothelial injury to resist thrombosis [34]. The results of this study demonstrated that the MAPK signaling was activated in HUVECs. Furthermore, TMP increased the phosphorylation of p38, MEK1/2, and ERK1/2, but did not affect the levels of p38, MEK1/2, and ERK1/2 proteins in H 2 O 2 -injured HUVECs. In vivo, a previous study showed that PTK787 can induce the deficiency in ISVs [35]. In order to study the protective effect of TMP on vascular endothelium in vivo, the proangiogenic effect of TMP was evaluated by PTK87-induced insufficiency of angiogenesis in Flik zebrafish, and the experimental data suggested that TMP has a proangiogenic activity. Therefore, the activation of the MAPK pathway may be the underlying cause of TMPmediated antioxidation, antiapoptosis, and protection from thrombosis.
At present, except for the mouse thrombosis model, there are limited ideal animal models for studying thrombosis. The zebrafish model we used is different from the existing thrombosis models in that its platelets are homologous to mammalian platelets, the hemostatic mechanism is similar to that of humans, and the embryo has high transparency. It was previously reported that AH promoted platelet aggregation and ultimately induced thrombosis [36]. This study established an AH-induced zebrafish tail vein thrombosis model, and the results were consistent with previous results. The results showed that thrombosis was significantly reduced after TMP treatment. Subsequently, the possible molecular mechanism of TMP in antithrombotic was further studied. Fibrinogen is a soluble substrate for fibrin-based coagulation, including fga, fgb, and fgg, with a central role in thrombosis [37]. In addition, vWF is one of the key players in thrombosis, mediating platelet adhesion to the exposed collagen of the damaged vascular endothelium [38]. Therefore, inhibition of vWF activity would be a new preventive measure to reduce thrombotic events. Our study found that TMP could significantly reduce AH-induced overexpression of fga, fgb, fgg, f7, and vWF mRNA, suggesting that TMP may be antithrombotic by inhibiting the coagulation cascade. However, this study also has limitations. First, although this study focused on endothelial cells and zebrafish, it is likely that the findings can be extended to other cell types and animal models, making the experiments more convincing. Secondly, future studies can verify the role of the MAPK pathway in protecting endothelial injury by using the pathway protein inhibitors. Furthermore, despite encouraging data from in vitro and in vivo studies of TMP, clinical studies of TMP are still lacking. Overall, our findings and hypotheses may at least partially reveal the potential role of TMP in the treatment of endothelial injury-related thrombosis.
To sum up, our study showed that TMP could effectively reduce the production of ROS, protect vascular endothelial injury through the MAPK pathway, and promote angiogenesis for antithrombosis. Therefore, TMP may be a potential candidate for preventing endothelial injury-induced cardiovascular diseases. These findings may provide an important scientific basis for further research on the blood-activating effect of Chuanxiong.

Conclusion
In conclusion, our study shows that TMP has a protective effect on H 2 O 2 -induced endothelial injury and protects zebrafish from angiogenesis deficiency and thrombosis. According to molecular biological analysis, the molecular mechanism of TMP involves activating the expression of the MAPK pathway proteins, antioxidation, and antiapoptosis and inhibiting the expression of thrombosis-related genes, as shown in Figure 13. This study may provide potential new ideas for TMP to treat thrombosis caused by endothelial injury.