This study was designed to investigate the action of timosaponin B-II, a main bioactive compound in Anemarrhena asphodeloides Bunge, on the prevention from high glucose-induced apoptosis in human umbilical vein endothelial cells (HUVECs) and the potential mechanisms involved. The results showed that compared with the normal control group, exposure of HUVECs to high glucose media for 72 h resulted in a significant increase in reactive oxygen species production, Caspase-3 activity and the percentage of apoptotic cells (p < 0.01). However, pretreatment with TB-II significantly increased the viability of HUVECs and decreased Caspase-3 activity and the apoptosis rate in a concentration-dependent manner (p < 0.05). In addition, TB-II notably decreased the amount of reactive oxygen species and malondialdehyde, as well as promoted glutathione peroxidase activity, endothelial nitric oxide synthase activity and nitric oxide release (p < 0.05). These results suggest that TB-II has the antiapoptotic effect in endothelial cells through inhibition of high glucose-induced oxidative stress and has the potential for preventing diabetic cardiovascular complications.
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Key wordsï¼šTimosaponin B-II, Glucose, Endothelial cells, Reactive oxygen species, Caspase-3, Apoptosis
1. Introduction 
Diabetes mellitus is an important risk factor for the development of cardiovascular disease, and endothelial cells are considered candidates involved in the pathogenesis of vascular complications in diabetes mellitus . A growing body of research has demonstrated that chronic elevation of blood glucose levels induces vascular endothelial dysfunction . Among them, high glucose-induced endothelial cells apoptosis appears to play a critical role in the pathogenesis of diabetic cardiovascular disease . Oxidative stress, which is mainly caused by the excessive accumulation of reactive oxygen species (ROS), is believed to be one of the critical pathogenic factors in endothelial cell apoptosis , because oxidative stress can induce activation of Caspase-3 which is a critical mediator of endothelial cells apoptosis . In endothelial cells, high glucose increase the generation of ROS principally via the mitochondrial electron transport chain and the polyol pathway . It is, therefore, thought prevention of oxidative stress and endothelial cells apoptosis induced by high glucose might be important tools to develop novel therapeutic strategies for diabetic cardiovascular complications.
Rhizoma Anemarrhenae (Zhi-Mu in Chinese), the dried rhizome of Anemarrhena asphodeloides Bunge (AA, family Asparagaceae), has been used for antidiabetic therapy as traditional medicine in China, Japan, and Korea for centuries . Anemarrhena asphodeloides Bunge, primarily containing steroidal saponins, xantones, and polysaccharides, has multiple pharmacological actions including antidiabetic, antioxidative, antiinï¬‚ammatory, and antidepressive in modern medicine [7-10]. Timosaponin B-II (TB-II, Fig. 1), a main bioactive compound in this herbal medicine, has various kinds of pharmacological activities, such as antioxidative, antidementia, antiinflammatory [8, 11, 12]. Under the above backgrounds, this study was aimed to examine the effect of TB-II on high glucose-induced apoptosis in human umbilical vein endothelial cells (HUVECs) and investigated the potential mechanisms involved.
2. Materials and methods
Fetal bovine serum (FBS) and Dulbecco's modiï¬ed Eagle's medium (DMEM, low glucose) were purchased from Gibco (NY, USA). D-glucose, dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), endothelial cells growth supplement (ECGS) and heparin were obtained from Sigma-Aldrich (St. Louis, MO.USA). The reagent kits for measurement of the level of malondialdehyde (MDA), lactate dehydrogenise (LDH) and nitric oxide (NO) were purchased from Nanjing Institute of Jiancheng Biological Engineering (Jiangsu, China). ROS, glutathione peroxidase (GSH-Px), Caspase-3 activity assay kits and Annexin V -fluorescein isothiocyanate (FITC) apoptosis detection kit were produced by Beyotime Institute of Biotechnology (Jiangsu, China). Human endothelial nitric oxide synthase (eNOS) enzyme-linked immune-sorbent assay (ELISA) kit was produced by CUSABIO (Wuhan, China). TB-II (purity > 98.5 %) was purchased from National Institute for the Normal control of Pharmaceutical and Biological Products (Beijing, China).
2.2. Cell culture and treatment
HUVECs lines were obtained from the American Type Culture Collection (ATCC, CRL-1730) and cultured in DMEM (5.5 mM glucose) with 0.03 mg/mL ECGS, 0.1 mg/mL heparin, 10 % heat-inactivated FBS, 100 units/mL penicillin, and 100 Âµg/mL streptomycin. HUVECs were grown in a humidified atmosphere at 37 ËšC in 5 % CO2 and passed every 3-4 days.
In experiments, HUVECs were randomly divided into six groups: a normal control group, a DMSO control group (0.1 % DMSO), a high glucose (HG, 33 mM) group, and three TB-II groups (1 ÂµM, 10 ÂµM and 50 ÂµM). HUVECs in the normal control group were incubated under the normal growth conditions (5.5 mM glucose DMEM) for 90 h. Cells in DMSO control group were preincubated for 18 h with DMSO (final concentration 0.1 %) before incubation under the normal growth conditions for 72 h. Cells in high glucose group were preincubated for 18 h with he normal growth conditions before incubated with high glucose DMEM (33 mM) for 72 h. In the TB-II groups, cells were preincubated for 18 h with different concentrations of TB-II (1 ÂµM, 10 ÂµM and 50 ÂµM) before incubation for 72 h with high glucose [13, 14].
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2.3. Cell viability assay
Cell viability was evaluated by the MTT assay. In brief, cells were seeded in 96-well micro plates at 20 000 cells/well and treated with the test compounds for indicated period. After treatment with TB-II and high glucose, the culture supernatant was collected for LDH release assay, 20 Î¼L of MTT solution (5 mg/mL) and 180 Î¼L of fresh culture medium were added to each well, and the cells were further incubated for 4 h at 37 Â°C. After MTT removal, 150 Î¼L of DMSO was added to dissolve the formed crystals. The optical density was measured with a wavelength at 490 nm using a micro platereader (Molecular Devicesï¼ŒUSA) . The viability of each group was presented as percentage of the normal control group.
2.4. LDH release assay
LDH release was detected by a commercial assay kit (Jiancheng, China). At the end of incubation, the supernatant was collected and LDH release was measured according to the manufacturer's protocol and the absorbance was measured at a wavelength of 440 nm using a microplate reader.
2.5. Evaluation of ROS in HUVECs
The level of intracellular ROS was measured using the ROS-sensitive dye, 2â€², 7â€²-dichloro-ï¬‚uorescein diacetate (DCFH-DA, Beyotime, China), as a probe . To load the cells, HUVECs were incubated with final concentration of 20 Î¼M DCFH-DA for 30 min in the dark after incubation with TB-II and high glucose. Then the cells were washed with phosphate buffered saline (PBS) twice and harvested in DMEM. The fluorescence intensity of 2â€²,7â€²-dichloro-ï¬‚uorescein (DCF) was measured by using a microplate reader (Molecular Devicesï¼ŒUSA) at excitation wavelength of 488 nm and emission wavelength of 525 nm.
2.6. Preparation of cell lysates
HUVECs were seeded in 6-well plates at a density of 1Ã-105 cells/mL and allowed to attach for 24 h before treatment with drugs. After treatment with TB-II and high glucose, the cells were washed with ice-cold PBS twice, and scraped from the plates into ice-cold cell lysis buffer (20 mM Tris with pH 7.5, 150 mM NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, and 1mM phenylmethanesulfonyl fluoride). Next, lysed cells were centrifuged at 16 000Ã-g for 10 min at 4 Â°C to remove the cells debris, and protein concentration of the supernatant was determined at 570 nm by a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). The supernatant was stored at âˆ’80 Â°C until analysis of MDA, GSH-Px, and eNOS activity .
2.7. MDA assay
To assay lipid peroxidation, we quantified the generation of MDA using a commercial available kit, according to the manufacturer's instructions, as previously described [5, 17]. MDA levels were normalized as nanomoles per milligram of protein.
2.8. GSH-Px activity assay
The activity of GSH-Px was determined by using a commercially available kit according to the manufacturer's instructions. One unit of GSH-Px was defined as the amount that reduced the level of GSH by 1 ÂµM in 1 min per milligram of protein .
2.9. NO release and eNOS activity assay
NO release was detected by a Nitric Oxide Detection kit. Most of the NO is rapidly metabolized to nitrite and nitrate in physiological solutions. Because of the instable of NO in physiological solutions, NO release was determined through detecting the concentration of nitrite and nitrate in culture medium. In this kit, nitrate was reduced to nitrite specifically through nitrate reductase, and the concentration of nitrite was determined by colorimetric . In brief, at the end of incubation, culture medium was collected and the concentration of NO was determined according to the manufacturer's instructions. The absorbance was read on a micro platereader at a wavelength of 550 nm.
Activity of eNOS in the cell lysate was determined using an eNOS activity ELISA kit (Cusabio, China) according to the manufacturer's instructions. The absorbance was read on a microplate reader at a wavelength of 450 nm.
2.10. Apoptosis detection by ï¬‚ow cytometry
To evaluate the apoptosis of HUVECs, the Annexin V apoptosis detection kit (Beyotime, China) was used according to the manufacturer's instructions. In brief, after treatment with TB-II and high glucose, the cells were harvested and double stained with FITC-coupled Annexin V protein and propidium iodide for 10 min at room temperature in the dark . After incubation, the samples which counted more than 20 000 events were determined by the flow cytometer (Beckman, USA). Cells stained only with Annexin V were considered apoptotic, and cells stained with both Annexin V and propidium iodide were considered necrotic.
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2.11. Caspase-3 activity assay
Activity of Caspase-3 was determined according to literature [5, 20], with a slight modification. In brief, at the end of incubation, cells were washed with cold PBS, and resuspended in lysis buffer for 15 min. The lysate was centrifuged at 16 000Ã-g for 15 min at 4 Â°C to remove cells debris. Caspase-3 activity in cells lysate was measured by a Caspase-3 activity assay kit according to the manufacturer's instructions and the absorbance was measured using a microplate reader at a wavelength of 405 nm.
2.12. Statistical analysis
Data were expressed as mean Â± standard deviation (S.D). The groups were analyzed via t-tests or one-way analysis of variance (ANOVA) followed by Tukey-Kramer test using PASW statistics 18.0. In all cases probability values of p < 0.05 were taken as statistically significant.
3.1. Effects of TB-II on HUVECs viability
To evaluate the effects of TB-II on viability of high glucose-induced HUVECs, we performed the MTT assay. As shown in Fig. 2, cells were treated with high glucose for 72 h and cell viability decreased to 72.2 % of the normal control group. However, preincubation with various concentrations of TB-II (1, 10, and 50 ÂµM) significantly increased the viability of HUVECs which were treated with high glucose in a concentration-dependent manner (p < 0.05).
3.2. Effects of TB-II on LDH release
To further investigate the protective effects of TB-II, the level of LDH release was measured. As shown in Fig. 3, exposure to high glucose for 72 h markedly increased the LDH leakage in HUVECs (p < 0.01). On the contrary, pretreatment of cells with different concentrations of TB-II markedly attenuated high glucose-induced LDH release in a concentration- dependent manner (p < 0.01). The results were consistent with the MTT assay.
3.3. Effects of TB-II on ROS generation induced by high glucose
DCFH-DA was used to investigate the effect of TB-II on ROS generation in high glucose-induced HUVECs. As shown in Fig. 4, after incubation of HUVECs with 33 mM glucose for 72 h, ROS levels in HUVECs increased significantly (p < 0.01), which was significantly inhibited by various concentrations of TB-II (p < 0.05). We found that intracellular ROS of high glucose cultured HUVECs for 72h increased to 206.1 % compared with the normal control cells. However, pretreatment with TB-II at 1, 10, and 50 ÂµM significantly decreased intracellular ROS production induced by high glucose to 149.3 %, 138.4 % and 130.4 %, respectively.
3.4. Effects of TB-II on MDA generation induced by high glucose
As shown in Fig. 5, exposure to high glucose for 72 h markedly increased the MDA level in HUVECs (p < 0.01), while preincubation with TB-II decreased the rise of MDA markedly (p < 0.05). We found that MDA in HUVECs cultured with high glucose for 72 h increased to 134.6 % compared with the normal control cells. However, pretreatment with TB-II at 1, 10, and 50 ÂµM significantly decreased MDA generation induced by high glucose to 125.6 %, 118.1 % and 111.2 %, respectively.
3.5 .Effects of TB-II on GSH-Px viability
As shown in Fig. 6, treatment of HUVECs with high glucose for 72 h caused the GSH-Px activity to decrease by 29.2 % compared with the normal control group. However, preincubation with TB-II markedly attenuated the changes of GSH-Px viability induced by high glucose. At 1, 10, and 50 ÂµM of TB-II, the high glucose-induced decrease in GSH-Px activity was increased by 10.2 %, 18.3 % and 24.4 %, respectively.
3.6 .Effects of TB-II on NO release and eNOS viability
As illustrated in Fig. 7A, after treatment with high glucose for 72 h, NO production in HUVECs decreased by 45.4 % compared with the normal control group, while pretreatment with TB-II at 1, 10, and 50 ÂµM markedly inhibited high glucose-induced NO decrease (p < 0.01), in which NO production was restored to 62.5 %, 73.1 % and 81.1 % of the normal control group, respectively.
As illustrated in Fig. 7B, treatment with high glucose for 72 h inhibited eNOS activity (p < 0.01), while preincubation with different concentrations of TB-II increased the activity of eNOS markedly in a concentration- dependent manner (p < 0.05).
3.7 .Effects of TB-II on apoptosis of HUVECs induced by high glucose
To further evaluate the protective effect of TB-II against apoptosis induced by high glucose in HUVECs, cells apoptosis was determined by Annexin V-FITC and PI double staining methods (Fig. 8A). As shown in Fig. 8B, treatment with high glucose for 72 h significantly increased the percentage of apoptotic cells compared with the normal control groupï¼ˆp < 0.01ï¼‰, while preincubation with different concentrations of TB-II markedly decreased (p < 0.05) the percentage of apoptotic cells induced by high glucose in a concentration-dependent manner. The percentage of apoptotic cells was increased from 6.4 % (normal control group) to 34.0 % (high glucose group). However, pretreatment with TB-II at 1, 10 and 50 Î¼M for 18 h, the apoptotic cells represented 25.3 %, 17.5 % and 12.3 % of total cells, respectively.
3.8 .Effects of TB-II on Caspase-3 viability
To check whether HUVECs apoptosis induced by high glucose was mediated by Caspase-3 activation and whether TB-II can attenuate the increase of Caspase-3 activity in HUVECs induced by high glucose, activity of Caspase-3 was measured by a Caspase-3 assay kit. As shown in Fig. 9, high glucose strongly increasedCaspase-3 activity in HUVECs (p < 0.01), while pretreatment with TB-II at different concentrations for 18 h, markedly attenuated the increase of Caspase-3 activity induced by high glucose in a concentration- dependent manner (p < 0.05).
Evidences show that, high glucose is the important initiating factor and one of the major causes of diabetic vascular complications  and high glucose-induced endothelial cells apoptosis plays a critical role in this pathogenesis. Furthermore, overwhelming evidences prove that treatment with high glucose increased the number of apoptotic cells [15, 21]. In the current study, we also obtained similar results: treatment with high glucose for 72 h significantly increased the percentage of apoptotic cells and significantly decreased the cell viability compared with normal glucose treatment. Therefore, antiapoptotic agents are regarded as promising drugs for treatment of vascular complications in diabetic patients. The result of the current study also provided that pretreatment with TB-II decreased the number of apoptotic cells induced by high glucose.
Evidence has convincingly shown that the increase of glycemia is accompanied by oxidative stress generation that might instigate disease progression by oxidising biomolecules . For example, ROS induced by high glucose can result in endothelial dysfunction and cell apoptosis . In this study, our data shown that ROS levels in HUVECs increased significantly after cells incubated with high glucose, while TB-II markedly attenuated the increase in ROS levels. Therefore, we speculate that TB-II protect HUVECs from apoptosis may partly be due to the scavenging ROS and thereby preventing oxidative damage induced by high glucose. It has been shown that MDA can result in cell membrane breakdown and cells swelling , on the contrary, GSH-Px can decrease oxidative stress-induced endothelial cells apoptosis through dismutasing superoxide . In the current study, high glucose led to increase in intracellular MDA levels and decrease in GSH-Px activity, while TB-II could decrease MDA generation and inhibit the reduction of GSH-Px activity.
Endothelial NOï¼Œ produced by eNOS, plays an important role in the regulation of endothelial cell functions. It was reported that the NO synthesized from endothelium could act as an antioxidant and protect cells from oxidative stress-induced apoptosis [26, 27]. Studies have shown that high glucose resulted in decrease of eNOS activity and NO content . In the current study, we also obtained similar results: high glucose inhibited eNOS activity and NO releaseï¼Œwhile pretreatment of HUVECs with TB-II markedly inhibited high glucose-induced decrease in eNOS activity and NO release.
It is well known Caspase-3, as a member of the family of speciï¬c cysteine proteases, might result in DNA fragmentation, and activation of Caspase-3 is the center link of apoptosis . Previous data demonstrate that a glucose-induced rise in oxidative stress is associated with increased Caspase-3 in human endothelial cells . In our study, high glucose strongly increased activity of Caspase-3 in HUVECs; however, pretreatment of HUVECs with TB-II significantly attenuated the increase of Caspase-3 activity. The inhibitory effect of TB-II on Caspase-3 activity was consistent with the decrease of ROS production.
In conclusionï¼Œthe present study shown thatï¼ŒTB-II increased the viability of HUVECs and decreased the amount of LDH. In addition, TB-II markedly inhibited the endothelial cells apoptosis induced by high glucose. Furthermore, our data demonstrated that TB-II decreased the amount of ROS and MDA, as well as promoted the GSH-Px activity, eNOS activity and NO release. We also found TB-II attenuated the increase of Caspase-3 activity in HUVECs induced by high glucose. These results suggest that TB-II has the antiapoptotic effect in endothelial cells through inhibition of oxidative stress and has the potential for preventing diabetic cardiovascular complications. Further studies will focus on its detailed protective mechanism(s) in vivo.
Conflict of interest statement
Authors have no conflicts of interest.
This work was supported by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The assistance of the staff is gratefully acknowledged.