Leonurine Ameliorates Oxidative Stress and Insufficient Angiogenesis by Regulating the PI3K/Akt-eNOS Signaling Pathway in H2O2-Induced HUVECs

Thrombus is considered to be the pathological source of morbidity and mortality of cardiovascular disease and thrombotic complications, while oxidative stress is regarded as an important factor in vascular endothelial injury and thrombus formation. Therefore, antioxidative stress and maintaining the normal function of vascular endothelial cells are greatly significant in regulating vascular tension and maintaining a nonthrombotic environment. Leonurine (LEO) is a unique alkaloid isolated from Leonurus japonicus Houtt (a traditional Chinese medicine (TCM)), which has shown a good effect on promoting blood circulation and removing blood stasis. In this study, we explored the protective effect and action mechanism of LEO on human umbilical vein endothelial cells (HUVECs) after damage by hydrogen peroxide (H2O2). The protective effects of LEO on H2O2-induced HUVECs were determined by measuring the cell viability, cell migration, tube formation, and oxidative biomarkers. The underlying mechanism of antioxidation of LEO was investigated by RT-qPCR and western blotting. Our results showed that LEO treatment promoted cell viability; remarkably downregulated the intracellular generation of reactive oxygen species (ROS), malondialdehyde (MDA) production, and lactate dehydrogenase (LDH); and upregulated the nitric oxide (NO) and superoxide dismutase (SOD) activity in H2O2-induced HUVECs. At the same time, LEO treatment significantly promoted the phosphorylation level of angiogenic protein PI3K, Akt, and eNOS and the expression level of survival factor Bcl2 and decreased the expression level of death factor Bax and caspase3. In conclusion, our findings suggested that LEO can ameliorate the oxidative stress damage and insufficient angiogenesis of HUVECs induced by H2O2 through activating the PI3K/Akt-eNOS signaling pathway.


Introduction
As a chronic multifactorial disease, thrombosis refers to blood clots forming in arteries or veins. It is considered the pathological phenomenon of cardiovascular disease and thrombotic complications as it often causes myocardial infarction, ischemic stroke, coronary heart disease, acute atherosclerotic syndrome, and pulmonary embolism. Additionally, the death and prognosis of the current pandemic caused by SARS-CoV-2 (the aetiological agent of COVID-19) were proved to be related to thrombosis [1]. Therefore, the thrombus is seriously threatening people's life and health [2,3].
Vascular endothelial cells, the critical regulator to maintain vascular health and normal function, together with platelets and circulating coagulation proteins, are crucial mediators of thrombosis. Vascular endothelial cells are considered to be the center of vascular diseases as they have anticoagulant, antithrombotic, and plasminogen properties and play an indispensable role in regulating vascular tension and maintaining homeostasis [4]. Vascular endothelial cell injury may expose fibrinogen, induce monocyte/macrophage aggregation and adhesion, promote coagulation and platelet aggregation [5], also increase the release of ET-1 and platelet IV factor, and reduce the release of PGI 2 and NO [6], thus inducing or accelerating the formation of thrombosis. At present, many antithrombotic drugs can significantly reduce cardiovascular adverse events. However, the curative effect and prognosis are still limited due to varying degrees of adverse reactions, such as bleeding, liver and kidney dysfunction, or stomachache [7]. Therefore, the research and development of effective drugs to protect endothelial cells have an extensive prospect in preventing and treating thrombotic diseases.
Oxidative stress refers to excessive production of high activity enzymes such as living nitrogen free radicals or reactive oxygen species (ROS), which leads to the imbalance of cellular antioxidant capacity. Substantial evidence suggested that oxidative damage was crucial in both vascular endothelial cell injury and insufficient angiogenesis in the process of tissue repair, which may lead to aggravating thrombosis [4]. As a kind of reactive oxygen species (ROS) produced by the body, H 2 O 2 produces a large amount of ROS at high concentrations, which may cause oxidative damage to endothelial cells [8,9]. Therefore, H 2 O 2 is widely used to induce oxidative damage and replicate the apoptosis model [10].
Leonurus japonicus Houtt is a traditional herb, which has a significant effect on promoting blood circulation and removing blood stasis syndrome. It has been widely used in the treatment of blood stasis syndrome for thousands of years. LEO is a unique alkaloid isolated from Leonurus japonicus. Leonurus japonicus Houtt is a common medicine of TCM for promoting blood circulation, resolving stasis, and regulating menstruation, diuresis, and detumescence. It has been widely used for centuries to treat dysmenorrhea, menstrual disorders, and other gynecological diseases [11]. LEO is a specific alkaloid only found in Leonurus japonicus Houtt. Modern pharmacological studies have shown that it has a variety of biological activities such as vasodilation [12], antiplatelet aggregation and inhibition of vasoconstriction [13][14][15][16], anticoagulant [17], anti-inflammatory [18], antioxidative [19], anti-ischemia, antiapoptosis [20,21], and heart protection [22]. However, whether it can repair vascular endothelial cell injury and promote angiogenesis through antioxidant stress has not been clarified.
In this study, we established an H 2 O 2 -induced oxidative injury model of HUVECs and explored the effects of LEO on the repairing and angiogenesis after oxidative stress injury. Our results showed that LEO protected HUVECs from H 2 O 2 -induced endothelial dysfunction by improving the oxidative stress index (ROS, LDH, MDA, and SOD) and cell apoptosis. Besides, LEO potently stimulated eNOS activation and endothelial NO production by activating the PI3K/Akt-eNOS signaling pathway, which may benefit antithrombosis.

Determination of LEO Concentration.
The MTT cell assay was taken to study the cytotoxic effect of LEO on HUVECs. Cells were seeded in 96-well plates at a density of 8 × 10 3 cells per well, 100 μl per well with 10% FBS culture medium. After 24 h incubation, cells were treated with different concentrations of LEO (0-1000 μM) that dissolve in DMSO and dilute with 1640 medium containing 1% FBS. The control group was treated only with the 1640 medium containing 1% FBS at 37°C in a 5% CO 2 incubator for 24 h. Then, MTT solution (20 μl) was added to each group, and cells were incubated at 37°C for another 4 h. After that, the MTT solution was discarded. Cells were then dissolved by adding DMSO (150 μl per well), and the solutions were  HUVECs were plated and treated as described in Section 2.6. Then, cells were washed with PBS and incubated with 1% 1640 medium with 5 μM DCFH-DA in the dark at 37°C for another 0.5 h. Subsequently, cells were washed three times with precooled PBS to remove DCFH-DA that failed to enter cells. Images were taken under a fluorescence microscope (Leica DMI3000 B, Germany). The green fluorescence intensity of each group was analyzed to quantify ROS production by Image-Pro Plus 6.0.
2.10. LDH, MDA, SOD, and NO Analysis. HUVECs were plated and treated as described in Section 2.6. Then, cells in each group were digested and collected with PBS (0.01 M, pH 7.4), and the cell homogenate was obtained by ultrasonic crushing. The activities of LDH, SOD, and NO in cell homogenate were detected according to the instructions on the corresponding kits. The absorbance of LDH, MDA, SOD, and NO was measured at 450 nm, 532 nm, 550 nm, and 550 nm following the manufacturer's instructions. Each experiment was performed in triplicate.
2.11. Total RNA Extraction, Reverse Transcription, and RT-qPCR Analysis. Total RNA was extracted by the Cell Total RNA Isolation Kit according to the manufacturer's instructions. Cells were treated as described in Section 2.6. The optical density (OD) at 260/280 nm was measured for RNA purity detection with the Nucleic Acid/Protein Analyzer and then converted to single-strand cDNA with a cDNA Synthesis System for RT-qPCR (abm). The RT-qPCR reaction conditions were set as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 30 s. For RT-qPCR reactions, three independent biological samples were used for each experiment. The relative mRNA expression levels were calculated by the 2-ΔΔ CT method. All primers used in RT-qPCR were designed using the Prime-BLAST (NCBI) and synthesized in TSINGKE Biological Technology (Chengdu, China). The gene primer sequences are listed in Table 1.
2.12. Western Blot Assay. Cells were treated as described in Section 2.6. The HUVECs were washed twice with ice-cold PBS and then lysed with RIPA lysis buffer (RIPA lysis buffer : PMSF : protein phosphatase inhibitor : protein mixing enzyme inhibitor = 100 : 1 : 1 : 1) at 4°C. The protein concentration of each sample was quantified using the BCA protein assay kit according to the manufacturer's instructions. Then, adjust the protein concentration to the same by the lysis buffer. Protein loading buffer was added (total protein : loading buffer = 4 : 1) and heated for 5 min at 100°C. Then, equal amounts of protein from each group were loaded onto 10% SDS-PAGE and transferred to PVDF membranes. Then, the membranes were blocked with 5% skimmed milk for 2 h at room temperature followed by incubation overnight at 4°C with a primary antibody (GAPDH, β-actin, PI3K, Akt, eNOS, phospho-PI3K, phospho-Akt and phospho-eNOS, Bax, Bcl2, and caspase3) 3 Oxidative Medicine and Cellular Longevity at a dilution of 1 : 1000. Subsequently, the membranes were washed three times with TBST and incubated with the appropriate HRP-conjugated goat anti-rabbit IgG (1 : 10000) for 2 h at room temperature. The protein band was detected by the ECL kit and quantified by ImageJ. GAPDH and β-actin were used as a standard reference. The relative density of each protein band was normalized to GAPDH or β-actin.

Data Analysis
All statistical analyses were performed using GraphPad Prism Version 8.00 (GraphPad Software, Inc.). Values were presented as the mean ± S:D: The differences were analyzed by a t-test when there were only two groups or assessed by one-way ANOVA when there were more than two groups. The P value (P < 0:05) was considered statistically significant among all the analyses.

LEO Promoted Cell Proliferation and Inhibited H 2 O 2 -
Induced Injury in HUVECs. The results showed that there was no significant change in cell morphology and no apparent cytotoxicity when treated with LEO at the concentration range of 0.78 μΜ-100 μΜ for 24 h compared with the control group. LEO at the concentration range of 3.125 μΜ to 12.5 μΜ showed a substantial effect on promoting cell proliferation in a dose-dependent manner, and the ability to promote cell proliferation was strongest at the concentration of 12.5 μM. In contrast, when the concentration of LEO reached 200 μM, it could significantly inhibit the proliferation of cells (P < 0:01), as shown in Figure 2(a). Therefore, 2.5 μM, 5 μM, and 10 μM were selected as the optimal concentration for the study.
The results of the study on the role of H 2 O 2 in inducing HUVEC injury showed that treatment with 50-1200 μM H 2 O 2 for 24 h decreased the survival rate of HUVECs in a concentration-dependent manner. 200 μM H 2 O 2 reduced the survival rate of HUVECs to about 50% (P < 0:001), as shown in Figure 2(b). The reduction degree is moderate, and the reproducibility was stable. Based on these results, 200 μM H 2 O 2 was selected as the moulding concentration for subsequent experiments to induce HUVEC oxidative injury.
To assess the protective effect of LEO on H 2 O 2 -induced injury, HUVECs were exposed to H 2 O 2 (200 μM) and LEO (2.5, 5, and 10 μM) for 24 h. As shown in Figure 2

LEO Suppressed ROS Production Induced by H 2 O 2 .
To investigate the effect of LEO on antioxidation, the intracellular ROS generation was evaluated by the DCFH-DA assay. As shown in Figure 5, results indicated that the signal intensity of DCFH-DA staining increased significantly compared with the control group (P < 0:001) after treatment with H 2 O 2 , NO is an essential part of maintaining and improving the local blood flow and inhibiting thrombus formation. The decrease in its bioavailability is one of the important features of vascular endothelial cell injury. Malondialdehyde (MDA) production, lactate dehydrogenase (LDH), and superoxide dismutase (SOD) are biomarkers of oxidative stress. They reflect the damage degree of the cell's membrane function and integrity [25]. As shown in Figure 6, compared with the control group, the contents of LDH and MDA in cell homogenate were significantly increased (P < 0:001, P < 0:01), and the activity of NO and SOD was dramatically decreased in the H 2 O 2 group (P < 0:001), indicating that the antioxidant capacity of HUVECs was unbalanced and significantly impaired. After treatment with LEO (2.5, 5, and 10 μM), the contents of LDH and MDA were significantly decreased, and the activity of NO and SOD was significantly increased compared with the H 2 O 2 group (P < 0:001, P < 0:001).

LEO Inhibited HUVEC Apoptosis Induced by H 2 O 2 via
Bax/Bcl2/Caspase3 Signaling Pathway. To further explore the antiapoptosis effect of LEO, the expression of apoptosis-related genes and proteins was detected by RT-

LEO Regulated Oxidative Stress and Angiogenesis of HUVECs Induced by H 2 O 2 via PI3K/Akt-eNOS Signaling
Pathway. Previous research suggested that the PI3K/Akt pathway was associated with cell proliferation, survival, metabolism, and finally regulating endothelial function. To investigate whether LEO can inhibit oxidative stress and promote angiogenesis through the PI3K/Akt signaling pathway, we further performed western blot assays. As shown in Figure 8, H 2 O 2 downregulated phospho-PI3K, phospho-Akt, and its downstream target phospho-eNOS compared   Figure 4: Evaluation for the tube formation in HUVECs. Images for the in vitro formed tubes in HUVECs. Values are presented as means ± S:D: (n = 3). # P < 0:05, ## P < 0:01, and ### P < 0:001 vs. control group; * P < 0:05, * * P < 0:01, and * * * P < 0:001 vs. H 2 O 2 group. The bar chart shows quantitative data for HUVEC tube formation with the treatment of different concentrations of LEO. 6 Oxidative Medicine and Cellular Longevity with the control group (P < 0:001), while those protein levels were all upregulated in a dose-dependent manner following the treatment of different concentrations of LEO (2.5, 5, and 10 μM). Collectively, these results showed that LEO could regulate the apoptosis and hypoangiogenesis of HUVECs induced by H 2 O 2 through activating the PI3K/Akt-eNOS pathway.

Discussion
Due to the characteristics of multitarget therapy, TCM has become a potential treatment for various diseases. At present, the antithrombotic treatment of TCM has been widely used in the clinic, especially in cardiovascular diseases. The significant efficacy of TCM in promoting blood circulation and removing blood clots has been recognized by the majority of patients, such as safflower injection, Danhong injection, and safflower yellow pigment injection. Vascular endothelial cells can secrete vasoactive substances and regulate vascular function, and its injury will affect the normal proliferation, migration, and apoptosis of endothelial cells. Meanwhile, the injured endothelial cells can also cause plasma extravasation and angiogenesis disturbance and then lead to local circulation disturbance and thrombus [26]. Therefore, the repair of vascular endothelial injury can restore the homeostasis of vascular endothelial cells, thus improving the progress of thrombus-related diseases and restoring blood flow [27,28] by promoting the formation of new blood vessels around the thrombus. Therefore, the repair of endothelial injury and promotion of angiogenesis can be used as indicators to evaluate the effi-cacy of drugs for promoting blood circulation and removing blood clots.
Oxidative stress is caused by the presence of ROS. A high level of ROS is one of the main factors causing oxidative stress and inducing endothelial nitric oxide (NO) biological activity damage [29,30] and endothelial dysfunction and vascular remodeling [31][32][33] and finally leading to atherosclerosis, thrombosis, and other vascular-related diseases [34]. For a long time, the abnormality of ROS generation and the subsequent decrease of NO bioavailability in blood vessels are considered a copathogenic mechanism of endothelial dysfunction leading to various cardiovascular risk factors [35]. Researches indicated that long-term excessive ROS exposure might cause mitochondrial structure and function changes, which may induce endothelial dysfunction like senescence, apoptosis, and permeability changes and finally lead to thrombosis [36,37]. Meanwhile, endothelial cell apoptosis can induce mitochondria apoptosis and stimulate ROS production, further aggravating endothelial cell damage, blocking microvascular circulation, and inducing cardiovascular embolic diseases, such as atherosclerosis and thrombosis [38][39][40]. Therefore, ROS is essential for vascular endothelial cell survival and cardiovascular function in health and disease [40,41]; regulating oxidative stress and inhibiting cell apoptosis are effective treatments for thrombosis and cardiovascular diseases.
As one of the main forms of reactive oxygen species, H 2 O 2 induces ROS production in different ways, resulting in typical vascular endothelial cell injury. Therefore, the H 2 O 2 -induced oxidative stress model is usually used to research drugs that are beneficial to blood vessels [42][43][44].

Oxidative Medicine and Cellular Longevity
Our results showed that the activity of HUVECs was significantly decreased (P < 0:001), and the content of ROS was significantly increased (P < 0:001) in the model group (H 2 O 2 treatment group), while treatment with different concentrations of LEO significantly improved the activity of HUVECs and downregulated the increase of ROS induced by H 2 O 2 in a dose-dependent manner.
NO is known to have the strongest vasodilating effect, which can dilate blood vessels, improve microcirculation, regulate platelet activity, and promote angiogenesis and endothelial cell proliferation [45,46]. Therefore, the decrease of its bioavailability is one of the important characteristics of vascular endothelial cell injury. Lactate dehydrogenase (LDH) and malondialdehyde production (MDA) are two stable enzymes in the cytoplasm and are rapidly released into the culture medium when the plasma membrane is damaged, so they were usually used as indicators of oxidative stress of cell membrane damage. The combined action of superoxide dismutase (SOD) and other endogenous antioxidants can effectively scavenge intracellular ROS and play an important role in preventing cellular damage caused by oxidative stress. In this study, in the H 2 O 2 treatment group, the content of LDH and MDA increased significantly. The content of NO and SOD decreased particularly compared with the control group (P < 0:001), indicating that H 2 O 2 treatment induced the imbalance of antioxidant capacity and HUVEC injury. However, treatment with LEO (2.5, 5, and 10 μM) decreased the content of LDH and MDA and increased the NO content and the SOD activity significantly, indicating that LEO can improve the oxidative damage of HUVECs induced by H 2 O 2 .
The formation of new blood vessels is a complex process, which is a manifestation of the normal function of endothelial cell proliferation and migration. The formation of new blood vessels around the thrombus can restore the blood flow of some occlusive veins and relieve the thrombus symptoms. Therefore, the promotion of angiogenesis is a potential treatment for diseases such as acute myocardial infarction and ischemic heart failure [47]. Our results suggested that treatment with different concentrations of LEO significantly improved the ability of cell migration and tube formation (P < 0:001) induced by H 2 O 2 .
To sum up, our research indicated that LEO can efficiently reduce the production of ROS, repair vascular endothelial injury, and promote angiogenesis via the PI3K/Akt-eNOS pathway, as shown in Figure 9. Therefore, LEO might be a potential candidate in preventing oxidative stressinduced vascular-related diseases. These findings may provide an important scientific basis for further study of the effect of Leonurus japonicus Houtt on promoting blood circulation and removing blood clots.

Data Availability
The underlying data of the study can be obtained by contacting the authors if it is reasonable.

Conflicts of Interest
The authors have declared no other competing interests.  Figure 9: H 2 O 2 can induce the expression of apoptotic protein Bax and inhibit the phosphorylation of PI3K/Akt by increasing the content of ROS, while LEO can promote the phosphorylation of PI3K/Akt and further promote the expression of eNOS, thus promoting the survival, proliferation, migration, and NO release of endothelial cells. At the same time, phosphorylated-Akt can also inhibit the expression of apoptotic proteins such as Bcl2 and Bax, thus inhibiting endothelial cell apoptosis induced by ROS. "←" indicates activation, and "⟝" indicates inhibition. 10 Oxidative Medicine and Cellular Longevity