Introduction
A monolayer of vascular endothelial cells (VECs) acts as a physiological barrier between blood vessels and vascular tissues; this monolayer maintains the integrity of the vascular wall and the function of blood circulation. Ischemia/reperfusion injury of important organs, including the heart, brain and kidneys, causes severe damage to VECs and increases the production of reactive oxygen species (ROS) (1). ROS act as important intracellular messengers, inhibiting the protein kinase B (AKT) and mitogen-activated protein kinase 1 signaling pathways, and directly inducing cell apoptosis (2,3). ROS also affect the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway, which activates or inhibits downstream target proteins, including B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax) and caspase-3, via phosphorylation, and regulates various biological functions, including cell growth, proliferation, adhesion and apoptosis (4). Additionally, the changes to cell metabolism and apoptosis caused by ROS are important factors in cardiovascular dysfunction (5). Thus, the regulation of ROS-associated pathways may be an important mechanism for the protection of VECs.
Radix Astragali is the dried root of the leguminous plant Astragalus membranaceus (Fischer) Bge. var. mongolicus (Bge.) Hsiao. According to traditional Chinese medicine, Radix Astragali demonstrates efficiency in tonifying Qi to reinforce Yang, strengthening superficial resistance, promoting urination to expel internal toxins/pus, promoting tissue regeneration and improving the healing of sores; therefore, it is an important and commonly used traditional Chinese medicine for strengthening healthy energy and tonifying Qi (6). Astragalus polysaccharide (APS) is the primary active ingredient of Radix Astragali and previous studies have demonstrated it to have a variety of pharmacological effects. APS reduces the damage to VECs caused by hypoxia/reoxygenation (HR) and reperfusion injury of human cardiac microvascular endothelial cells (HCMECs) (7,8). In particular, A-3, a component of APS, may protect the function of VECs from damage induced by paraoxon, which is associated with increased superoxide dismutase (SOD) and decreased malondialdehyde (MDA) levels (9). Additionally, the combined use of Radix Astragali and ligustrazine significantly protected VECs by elevating nitric oxide (NO) release (10). Radix Astragali also inhibited endothelial cell apoptosis induced by advanced glycation end products via the downregulation of ROS levels (11). A previous investigation indicated that APS may suppress HR-induced damage to HCMECs damage by alleviating the oxidative stress caused by ROS and increasing NO levels. Additionally, APS was demonstrated to activate the PI3K-AKT-endothelial NO synthase (eNOS) signaling pathway, thus promoting the proliferation and differentiation of endothelial progenitor cells in the peripheral blood of patients with type 2 diabetes (12). APS also inhibited the apoptosis of HCMECs induced by oxygen and glucose deprivation, with the effects potentially mediated by changes in AKT phosphorylation levels (13). Additionally, it was previously demonstrated that APS potentially protects HCMECs from HR injury via regulation of the PI3K-AKT signaling pathway.
Thus, the present study aimed to investigate whether APS protects HCMECs from HR-induced injury via inhibition of ROS-induced oxidative stress and cell apoptosis, and if APS alters the regulation of the PI3K-AKT pathway, using an HCMEC model of HR-induced injury.
Materials and methods
Materials and cell culture
APS was purchased from Nanjing Zelang Medical Technology Co., Ltd. (Nanjing, China). A bicinchoninic acid (BCA) assay kit, caspase-3 assay kit, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) probe, Fura-2/AM probe and Hoechst apoptosis kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Methylthiazolyl tetrazolium (MTT) was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Rabbit anti-human monoclonal antibodes: AKT (cat. no. 1063-1), Bax (cat. no. 1017-1), Bcl-2 (cat. no. 1080-1), phosphorylated-AKT (p-AKT; cat. no. 5508-1), PI3K (cat. no. 1683-1) and GAPDH (cat. no. 5632-1) were provided by Epitomics (Burlingame, CA, USA). SOD, MDA and NO assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China; cat. nos. A001-1, A003-1, and A012, respectively). Na2S2O4 was obtained from Aladdin Reagent (Shanghai) Co., Ltd. (Shanghai, China).
HCMECs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). The cells were incubated with rabbit anti-human polyclonal anti-factor VIII [cat. no. bs-0434R; Shanghai Kemin Biotech Co., Ltd. (Shanghai, China)] and anti-CD31 (cat. no. BA1346; Wuhan Boster Biological Technology, Ltd., Wuhan, China) antibodies, and Dil-acetylated low-density lipoprotein (Sciencell Research Laboratories) to confirm their endothelial phenotype. The protocol and characterization were performed according to a previous study (14). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.).
Cell grouping
HCMECs were divided into the following five treatment groups: Control, HR, and APS-low (-L), -medium (-M) and -high (-H). Cells in the control group were cultured without any treatment. In the HR group, 200 μl cells (1×105/ml) were incubated with 1 mM Na2S2O4 for 4 h and cultured in DMEM for a further 24 h. In the APS-L, -M and -H groups, cells were pretreated with 25, 50 or 100 μg/ml APS, respectively, for 12 h. Cells (200 μl; 1×105/ml) were then incubated with Na2S2O4 for 4 h and cultured in DMEM for a further 24 h. The concentration range of APS used was selected according to the results of a pilot study that used a wider concentration range (data not presented).
Cell viability and apoptosis
Cells in the exponential growth phase were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 h. Following treatment, 20 μl MTT (5 mg/ml) was added to each well. After 4 h, the culture media was discarded and replaced by 150 μl dimethyl sulfoxide. After 10 min incubation, the absorbance was determined at 570 nm using an Infinite F200 microplate reader (Tecan Group, Ltd., Männedorf Switzerland). For cell apoptosis measurements, 200 μl cells (1×105/ml) were cultured in 6-well plates, incubated with Hoechst and analyzed using a Hoechst apoptosis kit, according to the manufacturer's protocol.
Intracellular ROS levels
Cells were treated as described. Following treatment, 200 μl cells (1×105/ml) were washed with phosphate-buffered saline (PBS; Beyotime Institute of Biotechnology), then incubated with 20 μM DCFH-DA in PBS for 2 h. Subsequently, cells were examined with a fluorospectrophotometer (SPEX Fluorolog-2; Horiba, Ltd., Kyoto, Japan) at excitation and emission wavelengths of 340 and 520 nm, respectively to measure the levels of intracellular ROS.
Intracellular Ca<sup>2+</sup> measurements
Cells (200 μl; 8×103/ml) were seeded onto 20-mm coverslips in 6-well plates and cultured for 48 h. Cells were then treated as described and cultured for a further 48 h. Following incubation with 1 μl Fura-2/AM and 499 μl Ca2+ solution (2 mM) for 30 min, cells were washed three times with PBS. Cells were examined using the fluorospectrophotometer at excitation wavelengths of 340 and 380 nm. The fluorescence intensity ratios at 340 and 380 nm were analyzed to determine the intracellular Ca2+ levels.
Intracellular MDA, SOD and NO measurements
Cells in the logarithmic growth phase were seeded in 6-well plates and cultured for 48 h. Cells (200 μl; 8×103/ml) were then treated as described and cultured for a further 48 h. Cells were collected by centrifugation (1,000 × g for 10 min) and repeated freeze/thaw cycles were performed to effuse the cellular contents. The supernatant was collected and used to determine the expression of MDA, SOD and NO using assay kits, according to the manufacturer's protocols.
Expression levels of PI3K/p-AKT, Bcl-2 and Bax
Cells in the logarithmic growth phase were seeded in 6-well plates and cultured for 48 h. The cells (200 μl; 8×103/ml) were then treated as described and cultured for a further 48 h. The cells were collected and lysed (Beyotime Institute of Biotechnology), and the lysates were centrifuged at 1,000 × g for 10 min to obtain the cellular protein. Protein concentration was determined using the BCA kit. Equal protein samples were loaded onto 10% SDS-PAGE gels (40 V for 4 h). The separated proteins were transferred onto a nitrocellulose membrane (Beyotime Institute of Biotechnology) and blocked (Beyotime Institute of Biotechnology). The membrane was then incubated with the PI3K, p-AKT, Bcl-2 and Bax primary rabbit anti-human monoclonal antibodies (1:100) at 4°C overnight, followed by incubation with goat anti-rabbit horseradish peroxidase-conjugated secondary IgG(H+L) antibody [cat. no. A0208; Beyotime Institute of Biotechnology (dilution, 1:500)]. Reactive protein was detected with an enhanced chemiluminescence western blotting kit (Beyotime Institute of Biotechnology) and Quantity One software version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) on a ChemiDoc XRS gel imaging system (Bio-Rad Laboratories, Inc.).
Caspase-3 activity
Cells in the logarithmic growth phase were seeded in 6-well plates and cultured for 48 h. Cells (200 μl; 8×103/ml) were then treated as described and cultured for a further 48 h. Cells were collected via centrifugation (1,000 × g for 10 min), lysed and analyzed following the addition of the substrates from the caspase-3 assay kit, according to the manufacturer's protocol. Absorbance at 405 nm (Multiskan Spectrum; Thermo Fisher Scientific, Inc.) was used to determine caspase-3 activity.
Statistical analysis
The paired Student's t-test was performed to analyzed the data and SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA) was used. Data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.
Results
Effect of APS on cell viability and apoptosis
The current study investigated the effect of treatment with Na2S2O4 and APS on HCMEC viability (Fig. 1A) and apoptosis (Fig. 1B and C). Following Hoechst staining, apoptotic cells exhibited bright and white fluorescence (Fig. 1C). Compared with the control group (untreated HCMECs), treatment with Na2S2O4 resulted in a significant reduction in cell viability (P=0.003) and a significant increase in the number of apoptotic cells (P= 0.001). By contrast, APS treatment resulted in elevated cell viability and reduced apoptosis in a concentration-dependent manner. At the middle and high concentrations of APS, cell viability was significantly increased compared with the HR group (P=0.009 and P=0.002, respectively; Fig. 1A), possibly due to the reduced apoptosis levels.
Effect of APS on intracellular ROS activity and Ca<sup>2+</sup> concentration
Fig. 2 indicates the ROS and Ca2+ levels in HCMEC treated with Na2S2O4 (the HR group) and APS. ROS (Fig. 2A) and Ca2+ (Fig. 2B) levels in the HR group were significantly increased in comparison with the control group (P=0.001). When cells were treated with the middle and high doses of APS, the intracellular levels of ROS and Ca2+ were significantly reduced compared with the HR group (APS-M, P=0.016 and APS-H, P= 0.004, and APS-M, P=0.027 and APS-H, P= 0.005, respectively). Thus, APS effected ROS and Ca2+ levels in a concentration-dependent manner.
Effect of APS on intracellular NO content
Intracellular NO levels were measured following treatment with Na2S2O4 (the HR group) or APS (Fig. 3). NO levels in the HR group were observed to be reduced compared with the untreated cells (P=0.001). APS-L did not reverse the Na2S2O4-induced decrease in ROS levels in the HR group. However, following treatment with the middle and high doses of APS, the NO level was significantly elevated compared with the HR group (P=0.07 and P=0.002, respectively), suggesting that APS can increase NO levels following HR injury of HCMECs.
Effect of APS on intracellular MDA content and SOD activity
MDA and SOD levels in HCMECs treated with Na2S2O4 (the HR group) and various concentrations of APS are presented in Table I. Compared with the untreated cells, the MDA concentration in the HR group was significantly increased by ~1.4 fold (P=0.001). However, compared with the HR group, the level of MDA was significantly decreased when cells were pretreated with the three doses of APS (P=0.008, P=0.005 and P=0.003, respectively). As for SOD, the levels were significantly reduced in the HR group compared with the control group (P=0.001). Additionally, compared with the HR group, APS pretreatment significantly increased the levels of SOD in a concentration-dependent manner (P=0.008, P=0.002 and P=0.002, respectively).
Effect of APS on PI3K/p-AKT protein expression levels
Western blotting with qualitative and quantitative analysis was performed to evaluate the expression of PI3K/p-AKT in HCMECs following treatment with Na2S2O4 (the HR group) and various concentrations of APS. As presented in Fig. 4, similar levels of AKT were observed in all groups, thus, AKT was used as the internal control of quantification of p-AKT levels. Reduced levels of PI3K and p-AKT were detected in the HR group compared with the control group (P=0.001), suggesting the downregulation of PI3K levels and reduced phosphorylation of AKT. Following APS pretreatment at all three doses, the PI3K and p-AKT levels were significantly upregulated in a dose-dependent manner compared with the HR group (PI3K: APS-L, P=0.007; APS-M, P=0.001; and APS-H, P=0.001 and p-AKT: P=0.009, P=0.001 and P= 0.001).
Effect of APS on Bcl-2 and Bax protein expression levels
The expression levels of Bcl-2 and Bax in HCMECs were determined by western blotting with qualitative and quantitative analysis (Fig. 5). Compared with control cells, the expression of Bcl-2 in the HR group was significantly reduced (P=0.003). However, compared with the HR group, the middle and high doses of APS significantly increased the Bcl-2 expression levels (P=0.008 and P=0.002, respectively). As for Bax, its expression was significantly increased in the HR group (P=0.001). Following APS preconditioning, the high expression of Bax induced by odium dithionite was decreased by APS at all the three doses to a relatively low level (all P=0.001).
Effect of APS on caspase-3 activity
The activity of caspase-3 in HCMEC was determined using a caspase-3 assay kit and the results are presented in Fig. 6. The activity of caspase-3 was significantly increased by Na2S2O4 treatment (the HR group) compared with the control cells (P=0.001). Notably, the middle and high doses of APS significantly reduced the high activity of caspase-3 induced by Na2S2O4 compared with the HR group in a concentration-dependent manner (P=0.003 and P=0.001, respectively).
Discussion
The vascular endothelium is composed of a monolayer of endothelial cells that secrete a variety of vasoactive substances via autocrine and paracrine mechanisms, targeting various cell types, including vascular smooth muscle cells and peripheral white blood cells. Therefore, the vascular endothelium not only serves as a physiological barrier, it is also important in antithrombosis and inhibition of inflammation of the vascular wall. HR-induced injury damages the vascular endothelium and, thus, impairs the function of VECs. Using MTT and Hoechst assays, the current study demonstrated that HCMECs are protected by APS, particularly at high doses, following HR-induced injury.
Free radicals are important in the endothelial injury induced by HR. ROS produced by HR stimuli penetrate the cellular membrane causing lipid peroxidation and cellular damage. The stable internal and external environments of the vessels are disrupted by ROS, resulting in VEC injury. ROS-induced endothelial injury is associated with elevated levels of intracellular free Ca2+, an important second messenger in cells. An excess of Ca2+ promotes the hydrolysis of phospholipase into noxious substances, including fatty acid and leukotriene, which are harmful to cells and promote the decomposition of cytoskeletal components, leading to cellular damage. Dysregulation of Ca2+ levels in VECs changes the expression levels of eNOS and induces cell apoptosis (15). Additionally, increases of Ca2+ levels exceeding the normal threshold in cells facilitates the accumulation of ROS. ROS accumulation reduces the activity of eNOS, which catalyzes NO production under normal physiological conditions. NO is a vascular protective factor produced by endothelial cells. During oxidative stress, the majority of superoxide anions inhibit the biological activity of eNOS, thus reducing the production and activity of NO. The decreased NO level and biological activity initiates vasodilatation and damage to the function of VECs. The activation of AKT in VECs may increase the release of NO and maintain the integrity of the functional layer of VECs (16).
A previous report demonstrated that Radix Astragali protects endothelial cells from apoptosis via the inhibition of ROS (10). Zhu et al (17) observed that 10–50 μg/ml APS significantly inhibited ROS production induced by tumor necrosis factor-α in HCMECs. Additionally, APS was demonstrated to ameliorate diabetes in palmitate-induced KKAy diabetic mice via the ROS pathway (18). The current study investigated the detailed mechanisms involved in the protection of HCMECs from HR by APS. It was observed that APS protected HCMECs from HR-induced injury by significantly decreasing the levels of ROS and Ca2+, and enhancing the levels of NO. It was also demonstrated that APS was able to protect VECs from HR-induced injury via regulating the levels of vasoactive substances and oxidizing materials, including ROS.
Excessive accumulation of ROS directly results in lipid peroxidation of the cell membrane, with MDA produced as the typical by-product. The levels of MDA are associated with the severity of oxidative stress experienced by cells. SOD is an endogenous antioxidative enzyme that breaks down intracellular ROS when cells are exposed to an external stress. Thus, changes to the levels of intracellular MDA and SOD can be used to indirectly reflect the degree of cellular oxidative damage. It was previously reported that APS treatment reduced ROS and MDA levels, and increased the expression levels of SOD in EA.hy926 cells with bronchopulmonary dysplasia (19). Similarly, the present study observed that APS treatment decreased the MDA levels and increased the SOD levels in HCMECs with HR injury.
The PI3K/AKT signaling pathway regulates various biological functions of cells, including cell growth, proliferation and adhesion. Intracellular accumulation of ROS inhibits the PI3K/AKT signaling pathway and induces cell apoptosis (2,3). Specifically, the activation of PI3K results in the recruitment and phosphorylation of AKT, and affects the target proteins of this pathway, including Bcl-2, Bax and caspase-3, via a signaling cascade (4). The upregulation of PI3K/AKT signaling may inhibit the apoptosis of HCMECs and the endothelial dysfunction induced by HR (20). Activation of the PI3K/AKT pathway may also prevent HR-induced apoptosis of myocardium microvascular endothelial cells (21). It was previously indicated that the PI3K/AKT signaling pathway is important in the regulation of endothelial cell apoptosis, thus, APS may exert cytoprotective effects via regulation of PI3K/AKT signaling. Cao et al (22) observed that APS inhibits the apoptosis of myocardial cells and reduces heart failure in a doxorubicin-induced mouse model via the suppression of AKT activity and the reduction of ROS levels. Ye et al (23) demonstrated that the proliferation of MDA-MB-468 breast cancer cells was arrested by regulating AKT phosphorylation at Thr308 and Ser473. Additionally, extracts of Radix Astragali attenuated cytokine-induced keratinocyte damage via the intracellular ROS level and the PI3K/AKT pathway (24). Astragaloside was previously demonstrated to inhibit myocardial cell apoptosis induced by doxorubicin via a reduction in ROS levels, which was associated with the PI3K/AKT signaling pathway (25). In the current study, APS attenuated HR-induced HCMEC damage via upregulation of PI3K expression and increased phosphorylation of AKT. This suggests that APS protects HCMECs from HR injury through regulation of the PI3K/AKT signaling pathway.
The accumulation of ROS enhances apoptosis in endothelial cells (26). The Bcl-2 protein family is important in the process of apoptosis. In particular, Bcl-2 is the major anti-apoptotic protein. It binds to the pro-apoptotic protein, Bax, forming heterodimers in the outer mitochondrial membrane; this reduces the release of caspase from the mitochondria, leading to inhibition of cell apoptosis. Caspases are essential proteins in cell apoptosis; in particular, caspase-3 is the crucial effector and a focal point of the apoptosis pathway. The apoptosis of HCMECs induced by HR was associated with decreased expression levels of Bcl-2, and increased expression levels of Bax and activated caspase-3 (27). Xiao et al (28) reported that treatment with 100–200 μg/ml APS decreased the apoptosis of HL-60 cells by inhibiting the activity of caspase-3. In the current study, it was observed that APS protected HCMECs from HR-induced injury by upregulating Bcl-2 expression levels, downregulating Bax expression levels and inhibiting caspase-3 activity. Additionally, the current study demonstrated that HCMEC protection by APS was concentration-dependent. The higher dose of APS was associated with the greatest change in ROS, Ca2+, NO, MDA, SOD, PI3K/AKT, Bcl-2 and Bax levels, as well as caspase-3 activity.
Notably, the lack of antagonist or agonist use to intervene in key signaling mechanisms was a limitation of the present study, and should be taken into consideration when developing protocols for future studies.
In conclusion, APS protected HCMECs from HR-induced injury by reducing the levels of ROS, Ca2+, MDA and Bax, increasing the levels of NO, SOD, Bcl-2 and PI3K, enhancing the phosphorylation of AKT, and inhibiting the activity of caspase-3. Furthermore, APS acted in a concentration-dependent manner, providing greater protection at higher doses. These results may provide an insight into the mechanisms associated with HR-induced injury of HCMECs and the protective effect of APS. The findings of the current study may serve as a guideline for the clinical application of APS and the treatment of HR-induced injury.