Anti-oxidative Effect of Ligustrazine on Treatment and Prevention of Atherosclerosis

Purpose: To investigate the protective effects of ligustrazine on oxidative stress-induced atherosclerosis. Methods: The indicators related to oxidative stress were determined using commercially available assay kits. MTT assay was used to assess the survival rate of human umbilical vein endothelial cells (HUVECs). HUVECs apoptosis was analyzed using fluorescence staining and flow cytometry. mRNA expression level and activity of caspases 3, 8, and 9 were determined via quantitative real-time polymerase chain reaction (PCR) and caspase 3, 8, and 9 assay kits. Results: Ligustrazine concentration of < 80 µmol/L had negligible inhibitory effect on HUVECs viability and protected HUVECs against oxygen stress damage by regulating the indicators related to oxidative stress. Flow cytometry results show that ligustrazine ameliorated H 2 O 2 -induced apoptosis, while the proportion of cells that stepped into early apoptosis and late apoptosis or necrosis were 52.7 and 0.6 %, respectively, in the H 2 O 2 group, and 38.2 and 1.3 %, respectively, in the ligustrazine group. In addition, ligustrazine attenuated the up-regulation of caspase 3, 8, and 9 mRNA expression levels and activity. Conclusion: Ligustrazine can protect HUVECs against H 2 O 2 -induced injuries by regulating the indicators related to oxidative stress and suppressing the overexpression of caspases 3, 8, and 9. The protective mechanism of ligustrazine on H 2 O 2 -induced injury in HUVECs may be a caspase-dependent anti-apoptotic mechanism which provide important information for treating and preventing oxidative stress-induced atherosclerosis.


INTRODUCTION
Atherosclerosis (AS) is the primary cause of cardiovascular and cerebrovascular diseases [1].Substantial evidence indicates that oxidative stress contributes to the progression of AS [2].Oxidative stress can result in endothelial damage [3].Injury to endothelial cells is the initiating factor of AS [4].
Cells usually have three model systems for oxidative stress: extracellular sources of superoxide anion (O 2-), hydroxyl radical (H 2 O 2 ), and normobaric hyperoxia (elevated ambient oxygen) [5].Among the three, H 2 O 2 has been extensively used to induce endothelial cell-injury models in vitro because it can easily penetrate the plasma membrane and does not play a role in initiating lipid pre-oxidation and oxidizing DNA and amino acids [4].
Recently, many studies have suggested that natural bioactive compounds from plants can protect endothelial cells against oxidative damage [6].Ligustrazine (tetramethy-pyrazine) is the major active ingredient extracted from Ligusticum chuanxiong and is widely applied in the treatment of vascular diseases in China [7].Previous studies have reported that ligustrazine can effectively scavenge cytotoxic oxygen free radicals that can alleviate hepatic and kidney cell damage [8,9].However, the potential mechanism of ligustrazine involved in AS was still obscure.Therefore, in the present study, the H 2 O 2induced oxidative stress model was established using human umbilical vein endothelial cells (HUVECs) to explore the anti-oxidative effects of ligustrazine on oxidative damaged endothelial cells and the underlying mechanism involved in the pathogenesis of AS.

EXPERIMENTAL Materials
The HUVECs were provided by Nanchang University Medical School.The current study was approved by the Ethics Committee of Nanchang University (China).Trypsin and 3-(4, 5-dimethylthiazal-z-yl)-2, 5-diphenylterazolium (MTT) were purchased from Sigma (St. Louis, USA), and Dulbecco's Modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO (Carlsbad, USA).Ligustrazine was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).The LDH, MDA, GSH, NOS, nitric oxide (NO), and SOD assay kits were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Cell culture
The HUVECs were cultured in 25 cm 2 plastic flasks at 37 °C in a humidified CO 2 incubator (95 % air and 5 % CO 2 ).The complete medium for cell maintenance was 4.5 g/L glucose DMEM containing 10% FBS, L-glutamine, 100 IU/mL penicillin, and 100 IU/mL streptomycin.When cells were 80% confluent, they were subcultured using 0.25 % trypsin and 0.02 % EDTA, and the medium was changed every two days.Cells between passages 3 and 10 were used in the present study.

Determination of H 2 O 2 concentration in oxidative stress model (MTT assay)
The concentration-dependent studies of HUVECs induced by H 2 O 2 were conducted using MTT assay.HUVECs were counted and seeded into 96-well culture plates at a density of 5×10 3 cells/well.The cells were washed twice with PBS after incubation with various H 2 O 2 concentrations (0, 50, 100, 200, and 400 µM) for 8 h.For each well, the cells were then incubated with 100 μL MTT (final concentration of 0.5 mg/mL) for 4 h.After MTT removal, the colored formazan was dissolved in 100 μL of DMSO.The absorption values were measured at 490 nm using a Thermo Scientific Multiskan MK3 Microplate Reader (Thermo Fisher, USA).The viability of HUVECs in each well was presented as percentage of control cells.Six independent replicates were performed for each group.

Confocal laser scanning microscopy (CLSM)
The HUVECs were cultured as described above and were stained with Hoechst 33258 for 30 min at 37 °C.Cell shape and nuclear morphology with apoptotic characteristics were observed immediately using Zeiss LSM 710 confocal laser scanning microscope (Carl Zeiss MicroImaging GmbH, Germany).

Evaluation of ligustrazine cytotoxicity
The effect of ligustrazine on HUVEC viability was evaluated via MTT assay in 96-well plates at a cell density of 5 × 10 3 cells per well.The cells were washed twice with PBS, after being pretreated with ligustrazine (10,20,40,80,160, and 320 μmol/L) for 24 h.The MTT assay was done under the conditions described earlier.

Evaluation of effect of ligustrazine on the viability of H 2 O 2 -induced HUVECs
The cells were cultured as described above.The HUVECs were randomly divided into the control, H 2 O 2 , and five ligustrazine groups (5,10,20

Evaluation of oxidative stress parameters
The percentage of LDH release, NO production, SOD, GSH-Px, and NOS activities, and MDA concentration were determined using commercially available assay kits (Jiancheng Bioengineering Research Institute, Nanjing, China).All procedures complied with the manufacturer's instructions.In addition, the percentage of LDH release was defined as the release of LDH in the supernatant/(release of LDH in the supernatant + release of LDH from the cell lysate) × 100.

Flow cytometry
Here, HUVECs were harvested, washed, and double-stained with an Annexin V-fluorescein isothiocyanate apoptosis detection kit (BD Biosciences, USA).The cells were incubated in the dark at 4 °C for 10 min to 15 min and analyzed using a BD FACS Calibur TM flow cytometry system (Becton Dickinson, USA).All tests were done in triplicate.

Determination of the mitochondrial membrane potential (ΔΨ m )
Rhodamine 123, a cationic fluorescent dye whose mitochondrial fluorescence intensity decreases quantitatively with the dissipation of the mitochondrial membrane potential, was used to evaluate perturbations in mitochondrial membrane potential [10].The HUVECs were cultured as described above and divided into different groups (control, H 2 O 2 , and ligustrazine groups).After 24 h treatment, cells were harvested and washed twice with cold PBS and then incubated in the dark with rhodamine 123 (1 µmol/L) for 30 min at 37 °C.Fluorescence was measured using flow cytometry with an excitation wavelength of 485 nm.

Detection of caspase activity
The cells were cultured and treated as aforementioned.The fluorometric specific detection kits (KeyGEN, China) containing fluorescent substrates were used to analyze the activities of caspase 3, 8, and 9.The protocol for detecting caspase activity was conducted according to the manufacturer's directions.Experiments were performed in triplicate.

Quantitative real-time (qRT)-PCR analysis of mRNA expression
The total RNA was extracted from cultured cells using Trizol reagent (Invitrogen, USA).The RNA concentrations were determined at 260 nm, and the samples were then stored in a freezer.Firststrand cDNA was synthesized from 1 μg of total RNA using PrimerScript TM RT-PCR kit (TaKaRa Code: DRR041A) according to the manufacturer's instructions.The expression levels of genes (β-actin, caspase-3, caspase-8, and caspase-9) in each sample were determined via qRT-PCR in the ABI 7900HT Real-Time PCR system (Applied Biosystems, USA).The florescence signals were detected with the ABI 7900HT Version 2.3 sequence detection system (Applied Biosystems, USA).The PCR conditions were as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s, and 60 °C for 1 min.The gene expression data were normalized to the endogenous control β-actin, and the relative mRNA expression was calculated using the comparative cycle threshold (∆Ct) method.∆Ct is the difference between the Ct values of the target gene and β-actin.The primers used for qRT-PCR are listed in Table 1.

Statistical analysis
Statistical analysis was performed using the SPSS 17.0 package.The values are presented as mean ± standard deviation (SD, n ≥ 3).Oneway ANOVA and Student's t-test were conducted to determine statistical significance.Differences between groups were considered significant at p < 0.05.

Concentration-dependent survival rate of H 2 O 2 -induced HUVECs
Studies on the concentration-dependent survival rate of H 2 O 2 -induced HUVECs were performed in the present study.As shown in Figure 1

Apoptosis of HUVECs detected via Hoechst 33258 fluorescence staining
The CLSM results indicate that normal cells display weak fluorescence, whereas apoptotic cells show increasing bright fluorescence and typical apoptotic bodies.As shown in Figure 2, the chromatin in nucleus seemed to be condensed and marginalized in the H 2 O 2 group, suggesting that H 2 O 2 can result in HUVECs apoptosis.
A B

Cytotoxicity of ligustrazine on HUVECs
The effect of ligustrazine on the viability of normal HUVECs was concentration dependent (Figure 3).When the concentration was more than 160 µmol/L, the survival rate was 81.29 ±3.18 %, which is significantly less than that of the control group (p < 0.05).However, ligustrazine at concentrations ≤ 80 µmol/L had negligible inhibitory effect on HUVECs survival.Therefore, ligustrazine concentrations less than 80 µmol/L were used for the subsequent experiments.

Protective effect of ligustrazine on the viability of H 2 O 2 -induced HUVECs
The protective effects of ligustrazine on the viability of H 2 O 2 -induced HUVECs were evaluated via MTT assay.As shown in Figure 4 2).However, pre-incubation with ligustrazine (5, 10, 20, 40, and 80 µmol/L) for 24 h attenuated the changes in SOD and GSH-Px activities.

Influence of ligustrazine on oxidative stress indicators of H
All of these results suggest that ligustrazine can be an anti-oxidative agent protecting HUVECs against oxidative stress.

Effect of ligustrazine on apoptosis
The percentages of cells that stepped into early apoptosis and late apoptosis or necrosis were 52.7% and 0.6%, respectively, in the H 2 O 2 group, and 38.2% and 1.3%, respectively, in the ligustrazine group (80 µmol/L) (Figure 5).These results suggest that ligustrazine has a protective effect on HUVECs against oxidative damage, which is particularly important in maintaining normal physiological function and preventing the formation of atherosclerotic plaques.

Effect of ligustrazine on t mitochondrial membrane potential (ΔΨ m )
The changes in mitochondrial membrane potential reflect the initial cell apoptotic phenomenon.Here, ΔΨ m was measured using flow cytometry.A substantial decrease in ΔΨ m was observed in HUVECs upon exposure to H 2 O 2 .However, after pre-treatment with different concentrations of ligustrazine (20 and 80 μmol/L), ΔΨ m increased to a value higher than that in the H 2 O 2 group (Figure 6).These data indicate that ligustrazine could protect HUVECs against oxidative stress via mitochondrial induction pathways.

Effect of ligustrazine on caspase 3, 8, and 9 activities
Caspase 3, 8, and 9 activities are associated with specific intracellular polypeptide degradation during apoptosis.Caspase 8 and 9 are initiator caspases, and caspase 3 is considered as the main executor of apoptosis.The results in Figure 7 indicate that caspase 3, 8, and 9 activities are lower in the ligustrazine group than those in the H 2 O 2 group, suggesting that ligustrazine can protect HUVECs against oxidative stress and, hence, apoptosis.

Effect of ligustrazine on mRNA expression of caspase 3, 8, and 9
The mRNA expression levels of caspase 3, 8, and 9 were measured via qRT-PCR.The results reveal that the mRNA levels of caspase 3, 8, and 9 are significantly up-regulated in the H 2 O 2 group compared with those in the control group (p < 0.05).Pre-treatment with ligustrazine in endothelial cells led to a decrease in the mRNA expression levels of caspase 3, 8, and 9 that coincide with the enzyme changes (Figure 8), suggesting that the anti-apoptotic mechanism of ligustrazine may be caspase dependent.

DISCUSSION
A variety of factors, such as inflammatory cytokines, reactive oxygen species (ROS), and lipid oxidation enzymes, could result in vascular endothelial cell damage, of which the damage caused by ROS is the most crucial [12].Oxygen free radicals, the main oxidizing substances, participate in the body's normal physiological activity.Oxyradicals are closely related to the body's metabolism and signal transduction and play an important role in cell physiological function.Normally, a small amount of oxyradicals are by-products of cellular metabolism.However, under certain conditions, large amounts of oxyradicals are generated from the intracellular system, which may cause irreversible oxidative damages.H 2 O 2 has been suggested as inducer of apoptosis in several types of cells [13]

Figure 2 :
Figure 2: The micrograph of HUVECs in the control group (A) and H2O2 group (B) by CLSM.The results show that H2O2 can result in HUVECs apoptosis.

Figure 3 :
Figure 3:The effect of ligustrazine on the survival rates of HUVECs.

Figure 4 :
Figure 4: The protective effects of ligustrazine on HUVECs against oxidative injury.Pentagram (☆) indicates statistically significant differences between the H2O2 group and control group.

Figure 5 :
Figure 5: The flow cytometry of HUVECs apoptotic cells after treatment with the H2O2 and ligustrazine.Quadrant analysis of fluorescence intensity of gated cells in Annexin V-FITC and PI channels was from 16,000 events.A = control; B = H2O2 group; C = ligustrazine group (80 µmol/L).

Table 1 :
, the survival rates gradually decreased with the increase in H 2 O 2 concentration.When H 2 O 2 Primer sequences used for qRT-PCR

Table 2 ,
LDH release was 16.28 ± 1.71 % in the control group, and a dramatic increase (34.89 ± 0.98 %) was observed after exposure to 100 µmol/L of H 2 O 2 for 8 h.However, pre-treatment with the different concentrations of ligustrazine (> 5 µmol/L) for 24 h attenuated the H 2 O 2 -induced increase in LDH release (p < 0.05, Table2).
2 O 2 -induced injury LDH release, MDA and NO concentrations, and SOD, GSH-Px, and NOS activities were investigated to further confirm the protective effects of ligustrazine on H 2 O 2 -induced injury in HUVECs.As shown in

Table 2 :
Effect of ligustrazine on oxidative stress in HUVECs c 38.66±4.91 bcd 2.60±0.35bc 53.60±8.02ab 74.00±3.63b 90.44±4.06cd 40 µM of Ligustrazine 27.42±0.83d 36.52±2.68 Once the pro-apoptotic factors move from the mitochondria into the cytosol, these apoptogenic proteins activate caspase proteases, amplifying apoptosis.Caspases are family members of cysteine proteases that mediate cell death and are critical regulators of apoptosis[20].Studies have indicated that at least 11 kinds of caspase exist.Caspases 8 and 9 participate in the beginning of apoptosis, whereas caspase 3 is involved in the implementation of apoptosis.Our results show that the addition of ligustrazine attenuated the decrease in ΔΨ m and the up-regulation of caspase 3, 8, and 9 mRNA and activity, suggesting that the anti-apoptotic mechanism of ligustrazine against oxidative stress is a In summary, ligustrazine can protect HUVECs against H 2 O 2 -induced injury by regulating the indicators related to oxidative stress, enhancing anti-oxidant enzyme activity, and suppressing the overexpression of caspases 3, 8, and 9.The protective mechanism of ligustrazine on H 2 O 2induced injury in HUVECs may be a caspasedependent, anti-apoptotic mechanism involved in mitochondrial induction pathways.The results of the present study provide important information for the treatment and prevention of oxidative stress-induced AS. cell death induced by lysophosphatidylcholine. J Physiol Pharmacol 2010; 61: 375-381.18. Bustamante J, Caldas Lopes E, Garcia M, Di Libero E, Alvarez E, Hajos SE.Disruption of mitochondrial membrane potential during apoptosis induced by PSC 833 and CsA in multidrug-resistant lymphoid leukemia.Toxicol Appl Pharmacol 2004; 199: 44-51.19.Ouyang YB, Carriedo SG, Giffard RG.Effect of Bcl-XL overexpression on reactive oxygen species, intracellular calcium, and mitochondrial membrane potential following injury in astrocytes.Free Radical Bio Med 2002; 33: 544-551.20.Lago J, Santaclara F, Vieites JM, Cabado AG.Collapse of mitochondrial membrane potential and caspases activation are early events in okadaic acid-treated Caco-2 cells.Toxicon 2005; 46: 579-586. CONCLUSION