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Research Communication 2,3,5,40-Tetrahydroxystilbene-2-O-b-D-glucoside Protects Human Umbilical Vein Endothelial Cells Against Lysophosphatidylcholine-Induced Apoptosis by Upregulating Superoxide Dismutase and Glutathione Peroxidase Jing Zhao1 Shouzhu Xu1,2 Fan Song1 Lun Nian1 Xuanxuan Zhou1 Siwang Wang1* 1 Department of Natural Medicine, School of Pharmacy, Fourth Military Medical University, Xi’an, People’s Republic of China 2 Department of Pharmacology, Xi’an Jiaotong University School of Medicine, Xi’an, People’s Republic of China Abstract 2,3,5,40 -Tetrahydroxystilbene-2-O-b-D-glucoside (TSG) has been shown to protect human umbilical vein endothelial cells (HUVECs) from lysophosphatidylcholine (LPC)-induced injury; however, the underlying molecular mechanism remains to be determined. The aim of this study was to investigate the protective mechanism of TSG against LPC-induced injury in HUVECs. We established a stable LPC-induced cell model by treating HUVECs with various concentrations of LPC and found 10.0 mg/mL of LPC to be optimal for inducing HUVECs injury. The effects of TSG on LPC-induced cell injury were assessed by cell counting kit-8, apoptosis assay, transmission electron microscope, and measurement of malondialdehyde (MDA), the antioxidant enzymes superoxide dismutase (SOD), reactive oxygen species (ROS), glutathione peroxidase, and mitochondrial membrane potential. The mRNA and protein levels of caspase-3, Bax, Bcl-2, PARP-1, and cytochrome C were assayed by real-time reverse transcriptasepolymerase chain reaction and immunoblotting, respectively. TSG pretreatment was able to prevent LPC-induced HUVECs injury and restore cell viability in a concentration-dependent manner. LPC treated cells showed typical apoptotic morphological changes including cytoplasmic vacuolation, swollen mitochondria, and characteristic biochemical hallmarks of apoptosis including loss of mitochondrial membrane potential, activation of caspase-3, decrease of Bcl-2, increase of PARP-1, upregulation of Bax, and release of cytochrome C, all of which were apparently inhibited by TSG pretreatment. Treatment of HUVECs with LPC led to decrease of SOD and glutathione peroxidase in addition to rapid increase of MDA and ROS levels. Pretreatment with TSG restored SOD and glutathione peroxidase levels to that of normal levels, and significantly decreased ROS and MDA levels. Our data indicate that TSG inhibits apoptosis of HUVECs mediated by LPC through blocking the mitochondrial apoptotic pathway and suggest that the mechanisms underlying the protective effects of TSG are related to the activation of SOD and glutathione peroxidase, the clearance of intracellular ROS, and reduction of lipid peroxidation. C 2014 IUBMB Life, 66(10):711–722, 2014 V Keywords: 2,3,5,40 -tetrahydroxystilbene-2-O-b-D-glucoside; human umbilical vein endothelial cells; lysophosphatidylcholine; apoptosis; superoxide dismutase; reactive oxygen species; glutathione peroxidase; endothelial injury; mitochondrial membrane potential Introduction C 2014 International Union of Biochemistry and Molecular Biology V Volume 66, Number 10, October 2014, Pages 711–722 Address correspondence to: Siwang Wang, Department of Natural Medicine, School of Pharmacy, Fourth Military Medical University, 169 West Changle Road, Xi’an 710032, People’s Republic of China. E-mail: [email protected] Received 11 September 2014; Accepted 12 October 2014 DOI 10.1002/iub.1321 Published online 8 November 2014 in Wiley Online Library (wileyonlinelibrary.com) IUBMB Life Atherosclerosis (AS) is the leading cause for cardiovascular disease and hence one of the most common diseases affecting human health, but the underlying molecular mechanism remains to be determined. The response-to-injury hypothesis states that AS is a chronic inflammatory process followed by localized injury to the vessel wall, in particular to the endothelial layer lining the lumen of the vessel. Thereafter vascular endothelial injury is considered to be the initial step during development and progression of AS (1,2), suggesting that 711 IUBMB LIFE agents, which could protect against vascular endothelial injury, may reduce the incidence of cardiovascular disease. Accordingly, several such agents have been identified and tested in vitro and in vivo (3–5). Earlier report showed that 2,3,5,40 -tetrahydroxystilbene-2-O-b-D-glucoside (TSG) could prevent vascular endothelial dysfunction by regulating the expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in experimental atherosclerosis in atherogenic-diet rats (6), indicating that TSG is a potential drug for the prevention and treatment of cardiovascular disease. TSG is one of the major bioactive constituents extracted from Polygonum multiflorum Thunb and demonstrates various pharmacologic activities including antioxidant, antiinflammatory, and anti-atherosclerotic effects (7–9), improvement of memory and learning ability (10), neuroprotection (11), anti-aging (12), promotion of hair growth (13), and attenuation of human platelet aggregation (14). It was recently reported that TSG could protect human umbilical vein endothelial cells (HUVECs) from lysophosphatidylcholine (LPC)induced injury by attenuating the expression of asymmetric dimethylarginine (ADMA) and enhancing the production of nitric oxide (NO) (15); however, the underlying molecular mechanisms are largely unknown. HUVECs play an important role in the regulation of vascular physiological functions and the maintenance of vascular homeostasis (16), while LPC is closely related with AS (17). LPC is a major lipid constituent of oxidized LDL and plays an important role in oxidized LDL-induced endothelial dysfunction. LPC increases oxidative stress by generating reactive oxygen species (ROS) and decreasing NO release, thereby causing endothelial dysfunction (18–20). Previous studies demonstrated that TSG significantly reduces ROS (21) and protects HUVECs cells from the injury caused by hydrogen peroxide (H2O2) (22). As ROS such as H2O2 are potent cellular damaging agents, these observations led us to hypothesize that TSG might protect HUVECs from apoptosis induced by LPC. In this study, we investigated the protective mechanism of TSG in HUVECs against LPC by establishing a stable LPCinduced cell model, followed by treatment with various concentrations of LPC in parallel with Simvastatin, which is being widely used to treat AS in clinical and scientific research (23–25). Our results showed that TSG inhibited the apoptosis of HUVECs induced by LPC through blocking the mitochondrial apoptotic pathway. Our study may provide an experimental and theoretical basis for further development of TSG to protect against cardiovascular diseases such as AS. Materials and Methods The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration, and was approved by the Human Ethics Committee of Fourth Military Medical University, China. Written informed consent was obtained from individual participants. 712 Reagents Human umbilical vein endothelial cells (HUVECs) were cell line established by the Fourth Military Medical University; DMEM (5.6 mM or 25 mM glucose) was purchased from Hyclone Thermo Fisher Scientific; TSG (C20H22O9, molecular weight 406.4, purity 99.0%) was provided by the Fourth Military Medical University; Simvastatin (C25H38O5, molecular weight 418.6, purity 99.0%) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); LPC (C24H50NO7P, molecular weight 495.6, purity 99.0%) was purchased from Sigma (MO). Cell Culture and Treatment HUVECs were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 mg/mL penicillin, and 100 mg/mL streptomycin at 37 C in a 5% CO2 incubator. HUVEC cells between passages 3 and 10 were used for experiments. When cells were close to 70–80% confluence, new medium was added before drug treatment. The groups of experiment: 1) DMEM: cells were incubated with DMEM for 24 H; 2) LPC: cells were incubated with 10.0 mg/mL LPC for 24 H; 3) Simvastatin: cells were treated with 5.0 mmol/L Simvastatin for 1 H, and then exposed to a final concentration of 10.0 mg/mL LPC for 24 H; 4) TSG (0.1): cells were incubated with TSG 0.1 mmol/ L for 1 H, and then exposed to a final concentration of 10.0 mg/ mL LPC for 24 H; 5) TSG (1.0): cells were incubated with 1.0 mmol/L TSG for 1 H, and then exposed to a final concentration of 10.0 mg/mL LPC for 24 H; 6) TSG (10.0): cells were incubated with 10.0 mmol/L TSG for 1 H, followed by continued exposure to a final concentration of LPC 10.0 mg/mL for 24 H. MTT Assay Cells were cultured at a density of 2.0 3 104 cells per well in flat-bottom 96-well plates and incubated for 24 H. Then, cells were treated with 5.0, 10.0, 20.0, and 40.0 mg/mL LPC for 24 H, followed by addition of 3-(4,5-dimethylthiazol-2-yl)22,5-diphenyltetrazolium bromide reagent (MTT, Beijing, China). After incubation for 4 H, the optical density (OD) was measured at 490 nm using a Microplate Reader Model M680-UV Spectrophotometer (Bio-Rad Laboratories, Marnes La Coquette, France). CCK-8 Assay The CCK-8 reduction assay kits (Beyotime Institute of Biotechnology, Changsha, China) were used as a qualitative index of cell viability according to the manufacturer’s instructions. After 24 H incubation with different compounds as described for MTT assay, 20 mL CCK-8 was added and cells were cultured for additional 4 H. The optical density (OD) was measured at 450 nm using a Microplate Reader Model M680-UV Spectrophotometer (Bio-Rad Laboratories, Marnes La Coquette, France). Annexin V/PI Staining Apoptotic cells were analyzed with an Annexin V (Fluorescein isothiocyanate [FITC]-conjugated)/PI apoptosis kit (Keygen Biotech, Nanjing, Jiangsu, China) coupled with flow cytometry. After treatment with different drugs for 24 H, 0.5–1 3 106 cells TSG Protects Huvecs By Upregulating SOD and GSH-PX TABLE 1 The sequences of real-time PCR primers used in this study Gene Forward Reverse b-Actin 50 TAGTTGCGTTACACCCTTTCTTG 30 50 TCACCTTCACCGTTCCAGTTT 30 Bax 50 CCTTTTGCTTCAGGGTTTCAT 30 50 CTCCATGTTACTGTCCAGTTCGT 30 Bcl-2 50 TTCTTGAAGGTTTCCTCGTCC 30 50 GAATCTGCTGGTCATTTGCC 30 Caspase-3 50 GCACTGGAATGACATCTCGG 30 50 AAACTGCTCCTTTTGCTGTGA 30 PARP-1 50 CATCGAGGTGGCCTACAGTCT 30 50 ATTGTGTGTGGTTGCATGAGTG30 Cyt C 50 TCGTTGTGCCAGCGACTAA 30 50 TGCCTCCCTTTTCAACGG 30 were harvested and resuspended in 500 mL binding buffer. Subsequently, the cells were incubated with 5 mL of Annexin V-FITC (50 mg/mL) and 5 mL of PI (50 mg/mL) for 15 Min in the dark, followed by flow cytometer analysis (Becton Dickinson, San Jose, CA). For all the samples, 2 3 105 cells were acquired and analyzed with Cell Quest software (Becton Dickinson). The percentages of early apoptotic cells (the lower right in the scatter plot) and late apoptotic/necrotic cells (upper right in the scatter plot) were calculated for comparison. Transmission Electron Microscope Analysis The transmission electron microscope was used to observe the cell morphology. After 24 H incubation with different compounds as described above, cells were collected by centrifugation (1,000 rpm, 3 Min), washed twice with phosphatebuffered saline (PBS) and fixed in freshly prepared 1% paraformaldehyde with 2% glutaraldehyde for 24 H. Samples were further treated with 1% osmium tetroxide for 2 H, dehydrated in graded ethanol, and embedded in araldite. Ultrathin sections were cut and stained with uranyl acetate and lead citrate, and then observed under transmission electron microscope (JEM-101, Jeol Electron, Japan). Detection of MDA, Superoxide Dismutase, and Glutathione Peroxidase After incubation with different compounds for 24 H as described above, cells were harvested with 0.25% trypsin, and washed twice with PBS. Then, the contents of MDA, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were determined using the corresponding detection kits (Lipid Peroxidation MDA Assay Kit, Total Superoxide Dismutase Assay Kit with WST-8, Total Glutathione Peroxidase Assay Kit, Beyotime Institute of Biotechnology, Changsha, China) according to the manufacturer’s instructions. At the same time, the concentrations of MDA, SOD, and GSH-Px were determined using ELISA kits (Elabscience, Wuhan, China). Detection of ROS Levels ROS generation of HUVECs induced by LPC was measured by staining DCFH-DA (Beyotime Institute of Biotechnology, Changsha, China). After incubation in six-well culture plates for 24 Zhao et al. H, cells were exposed to different compounds as indicated at 37 C for 24 H. Cells were washed twice with PBS. Then, the cells were resuspended in fresh medium containing 10 mM DCFH-DA and analyzed by flow cytometry. At the same time, the concentration of ROS was detailed analysis by ELISA kits (Elabscience, Wuhan, China). Determination of the Mitochondrial Membrane Potential After treatment, cells were washed twice with PBS and incubated in serum-free medium containing JC-1 dye in the dark at 37 C for 20 Min. After washing with JC-1 dyeing buffer twice, cells were harvested with 0.25% trypsin and washed twice with PBS. The cells were analyzed by flow cytometry with excitation and emission wavelengths of 480 and 530 nm, respectively. At the same time, parallel fluorescence intensity was detected using laser confocal microscopy (Olympus FV1000, Japan). The contents of mitochondrial membrane potential (MMP) were determined using detection kits (Beyotime Institute of Biotechnology, Changsha, China) according to the manufacturer’s instructions. Reverse Transcriptase-Polymerase Chain Reaction Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analyze mRNA expression of genes involved in apoptosis. Total RNA was prepared using Trizol reagent (TaKaRa, Japan) according to the manufacturer’s instructions. The quantity and purity of the RNA were assessed by measuring the absorbance at 260 and 280 nm. The cDNA was synthesized from total RNA (2 mg) with oligo (dT) primers using an M-MLV reverse transcriptase First Strand Kit (Invitrogen). All PCR conditions were optimized to produce a single product in the exponential range. Quantitative real-time PCR was performed using SYBR Green master mix and the detection of mRNA was analyzed using an ABI Step One real-time PCR System (Applied Biosystems, Foster City, CA). Primer sequences for the reference gene b-actin and the genes of interest are listed in Table 1. Typical PCR thermocycler profile was the initial step, 95 C for 10 Min followed by a second step at 95 C for 15 Sec and 60 C for 30 Sec for 40 cycles with a melting curve analysis. The level of 713 IUBMB LIFE actin (Cat No: AP0060, Bioworld). After incubation with second antibody, immune complexes were detected using ECL western blotting reagents (Thermoscientific, Thermo Fisher Scientific). Immunoreactive bands were quantified using the Gel Doc TM XR with Lab image 4.0.1 software (Bio-Rad Laboratories, Marnes La Coquette, France). Measured intensities were corrected with the internal control (b-actin). Statistical Analysis The differences were analyzed by ANOVA. All values were expressed as mean 6 SD from at least three independent experiments. Statistical significance was defined as P < 0.05. Results TSG Prevents the Loss of Cell Viability Following LPC Treatment FIG 1 TSG protected HUVECs cells from LPC in a concentration-dependent manner. (A) HUVECs were incubated with 5.0, 10.0, 20.0, and 40.0 mg/mL of LPC for 24 H and cell viability was analyzed using MTT assay in comparison to DMEM. (B) HUVECs were treated with LPC alone for 24 H or pretreated with Simvastatin, 0.1, 1, or 10 mmol/L TSG for 1 H, followed by treatment with LPC for 24 H. Cell viability was assessed by CCK-8 assay in comparison to DMEM only control group. Cells without drug addition served as control (DMEM). Data are mean 6 SD (n 5 8), *P < 0.05, **P < 0.01. target mRNA was normalized to the level of the b-actin and compared with the control. Data were analyzed using the DDCT method. Western Blot Total proteins were extracted by lysing cells with Radio Immunoprecipitation Assay (RIPA) Lysis Buffer (Beyotime Institute of Biotechnology, China). Protein concentrations were determined using the BCA method (Beyotime Institute of Biotechnology). Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. After being blocked with 5% skim milk in Tris-buffered saline (pH 7.6) (TBS) at room temperature, the membranes were incubated overnight at 4 C with primary antibodies against Bax (Abcam, ID:ab32503), Bcl2 (Abcam, ID:ab117115), Caspase-3 (Abcam, ID:ab2171), Cyt C (Abcam, ID:ab13575), PARP-1 (Abcam, ID:ab32378), and b- 714 To establish a suitable experimental model of HUVECs injury induced by LPC, we treated HUVECs with 5.0, 10.0, 20.0, and 40.0 mg/mL of LPC in DMEM for 24 H and assessed cell viability using MTT assay. The OD values of cells treated with 5 mg/mL LPC, 10.0 mg/mL LPC, 20 mg/mL LPC, and 40 mg/mL LPC were 0.76 6 0.18, 0.52 6 0.15, 0.35 6 0.11, and 0.25 6 0.11, respectively (Fig. 1A). The results showed that 10.0 mg/mL of LPC could decrease HUVECs viability to 50% relative to that of untreated group. The results suggest that LPC, a main active component of oxidized-LDL, has the ability to induce HUVECs injury in a concentration-dependent manner, and 10.0 mg/mL of LPC is the appropriate concentration to establish a HUVECs injury model. We next treated HUVECs with LPC alone for 24 H or pretreated with Simvastatin, 0.1, 1, or 10 mmol/L TSG for 1 H followed by treatment with LPC for 24 H. We assessed cell viability using CCK-8. 10.0 mg/mL of LPC for 24 H decreased the cell viability. In contrast, Simvastatin significantly abolished the loss of cell viability following treatment with LPC. The cell viability in the 10.0 mmol/L TSG1LPC group was significantly higher than that of LPC group; the cell viability in the 0.1 mmol/L TSG1LPC and 1.0 mmol/L TSG1LPC treated groups was lower than that of the Simvastatin1LPC treated group but higher than LPC group (Fig. 1B). These results clearly demonstrated that TSG could protect HUVECs from LPC in a concentration-dependent manner. TSG Protects HUVECs Against LPC-Induced Apoptosis To investigate the mechanism of the prevention of LPC-induced loss of cell viability by TSG, we first analyzed cell apoptosis by flow cytometry using Annexin-PI double staining of HUVECs treated with LPC alone for 24 H or pretreated with Simvastatin, 0.1, 1, or 10 mmol/L TSG for 1 H, followed by treatment with LPC for 24 H. In comparison to the control DMEM, LPC significantly induced cell apoptosis. As expected, Simvastatin significantly inhibited LPC-induced apoptosis. Interestingly, increasing the concentration of TSG significantly reduced the apoptosis rates in comparison to that of LPC treatment alone (Table 2). To support the above observation that LPC induced cell apoptosis is protected by TSG, we checked cell morphology after treatment of HUVECs with LPC alone or in combination TSG Protects Huvecs By Upregulating SOD and GSH-PX TABLE 2 Effects of TSG on cell apoptosis induced by LPC (x 6 SD, n 5 3) Group Apoptosis rate (%) DMEM 4.7 6 2.6 LPC 28.1 6 2.9c Simvastatin 1 LPC 11.2 6 2.5b TSG (0.1 mmol/L) 1 LPC 19.1 6 3.2a TSG (1.0 mmol/L) 1 LPC 13.4 6 5.8a TSG (10.0 mmol/L) 1 LPC a P < 0.05, P < 0.01 versus LPC group, c P < 0.01 versus DMEM group. b FIG 2 Zhao et al. b 6.3 6 3.9 with Simvastatin, or different concentrations of TSG by transmission electron microscope. As shown in Fig. 2, the HUVECs in DMEM alone displayed normal chromatin architecture and abundant mitochondria. In sharp contrast, LPC treatment caused significant apoptosis and cytoplasmic vacuolation. Simvastatin apparently rescued the apoptotic morphology change following LPC treatment. TSG obviously attenuated the loss of mitochondria and cytoplasmic vacuolation that was induced by LPC. These results indicate that TSG has a protective effect against LPC-induced apoptosis to HUVECs in vitro. TSG Inhibits LPC-Induced Upregulation of Caspase-3, Bax, PARP-1, and Cyt C and Downregulation of Bcl-2 To further investigate cell apoptosis signaling in HUVEC cells treated with LPC, we examined the effects of LPC on Caspase-3, Bax, Bcl-2, PARP-1, and Cyt C mRNA expression by RT-PCR and found that the Caspase-3, Bax, PARP-1, and Cyt C mRNA levels were significantly increased, while Bcl-2 level was obviously TSG abolished LPC-induced apoptosis to HUVECs in vitro. Transmission electron microscope micrographs of HUVECs treated with different compounds as indicated for 24 H. DMEM: Abundant mitochondria were seen in the cytoplasm. LPC: The cells exposed with LPC 10.0 mg/mL showed an abundance of cytoplasmic vacuolation and swollen mitochondria. Simvastatin1LPC: Reduced cytoplasmic vacuolation and swollen mitochondria were seen in the cytoplasm. TSG1LPC: With the increase of concentration of TSG, mitochondria condition was improved markedly (original magnification 330,000). In all treatment groups, swollen mitochondria were detected in comparison to that of DMEM control group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] 715 IUBMB LIFE FIG 3 TSG inhibits the upregulation of Caspase-3, Bax, PARP-1, and Cyt C in addition to the downregulation of Bcl-2 by LPC. HUVECs were treated with LPC alone for 24 H or pretreated with Simvastatin, 0.1, 1, or 10 mmol/L TSG for 1 H, and then treated with LPC for 24 H. mRNA levels of Bax, Bcl-2, Cyt C (A), Caspase-3, and PARP-1 (B) were determined by real-time PCR with b-actin as internal control. (C and D) Cells were treated as (A and B), the protein levels of Bax, Bcl-2, Caspase-3, and PARP-1 were determined by immunoblotting with b-actin as loading control. Data of quantitative analysis are listed below the corresponding western blots results. Data are the mean 6 SD (n 5 3), *P < 0.05, **P < 0.01 versus LPC group, #P < 0.05, ##P < 0.01 versus LPC group, &P < 0.05, &&P < 0.01 versus LPC group, $P < 0.05, $$P < 0.01 versus LPC group, qP < 0.05, qqP < 0.01 versus LPC group, $ $ P < 0.01 versus DMEM group. decreased following LPC treatment. 5.0 mmol/L of Simvastatin and TSG significantly reduced these mRNA level alterations. Moreover, increased concentration of TSG further inhibited the 716 effects of LPC. In addition, the effect of 10.0 mmol/L of TSG was superior to that of the 5.0 mmol/L of Simvastatin (Figs. 3A and 3B). Consistent with the results from RT-PCR, similar changes of TSG Protects Huvecs By Upregulating SOD and GSH-PX FIG 4 Zhao et al. TSG inhibits the decrease of the mitochondrial transmembrane potential (MMP) by LPC. HUVECs were treated with LPC alone or pretreated with Simvastatin, 0.1, 1, or 10 mmol/L TSG for 1 H, and then treated with LPC for 24 H. Images were captured under an inverted laser scanning confocal microscope after JC-1 staining. The first column picture was merged the second, and presented in the third column. In unhealthy mitochondria, JC-1 produced green fluorescence in the form of monomer. In normal mitochondria, JC-1 formed polymer in the mitochondrial matrix and produced red fluorescence. Compared with DMEM group, the red fluorescence in LPC group decreased significantly, thus red and green fluorescence ratio decreased. With the increase of concentration of TSG, red and green fluorescence ratio increased significantly (original magnification 3600). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] 717 IUBMB LIFE TABLE 3 TSG prevents the decrease the mitochondrial membrane potential by LPC (x 6 SD, n 5 3) Group Mitochondrial membrane potential (MFI) DMEM 12.0 6 1.7 LPC 1.3 6 0.6c b Simvastatin 1 LPC 5.1 6 0.9 TSG (0.1 mmol/L) 1 LPC 2.2 6 0.7a TSG (1.0 mmol/L) 1 LPC 3.7 6 1.2a TSG (10.0 mmol/L) 1 LPC 7.8 6 1.9b a P < 0.05, P < 0.05 versus LPC group, c P < 0.01 versus DMEM group. b the protein levels of Caspase-3, Bax, Bcl-2, and PARP-1 were found by Western blot analysis (Figs. 3C and 3D). These results indicated that the apoptosis induced by LPC might involve caspase regulation and mitochondrial apoptotic pathway. TSG Inhibits the Decrease of the MMP and the Increase Cyt C Release into the Cytoplasm from Mitochondria in HUVECs by LPC decrease of SOD and GSH-Px by LPC. Moreover, TSG inhibited the LPC-induced changes of these molecules in a concentrationdependent manner (Fig. 6). These results suggest that TSG might protect HUVECs against injury induced by LPC by increasing the expression of the antioxidant enzymes SOD and GSH-Px, scavenging ROS, and reducing lipid peroxidation. Discussion In this study, we established an LPC-induced cell model to investigate the protective mechanism of TSG in HUVECs by LPC. Our results showed that the treatment of HUVECs with LPC resulted in significant cell apoptosis, which could be restored by TSG pretreatment. Most importantly, we found that TSG inhibited the production of MDA and ROS and reduction of SOD and GSH-Px by LPC. Our results suggest that TSG might protect HUVECs against LPC-mediated injury by antagonizing LPC-induced apoptosis via upregulating the expression of the antioxidant enzymes SOD and GSH-Px, scavenging ROS, and reducing lipid peroxidation. Cell apoptosis is a self-regulating programmed cell death, which balances cell survival and death and maintains normal tissue homeostasis (27). Although apoptosis can be triggered by different kinds of stimuli, the downstream signaling pathways of cell apoptosis are common (28,29). Endothelial apoptosis can damage the physiological barrier of blood vessels and endothelial dysfunction, promote clotting, and reduce To verify the result of Cyt C as detected by RT-PCR, we further assayed the protein expression of Cyt C and MMP. As shown in Fig. 4 and Table 3, treatment with 10.0 mg/mL LPC significantly decreased the red florescence intensity in HUVECs. However, pretreatment of HUVECs with TSG was able to rescue the median fluorescence intensity (MFI) in a concentrationdependent manner. In addition, 10.0 mmol/L of TSG could recover the MMP of cells treated with LPC to a normal level. The change in the MMP is associated with the permeability of the mitochondrial membrane and the release of cytochrome C into the cytoplasm from the mitochondria (26). Indeed, treatment of HUVEC cells with LPC resulted in apparent increase of Cyt C, which was significantly inhibited by either Simvastatin or TSG (Fig. 5). These results demonstrated that TSG inhibited the LPCinduced decrease of MMP and the concomitant increase of Cyt C release into the cytoplasm from mitochondria in HUVECs. TSG Inhibits the Production of MDA and ROS and Decrease of SOD and GSH-Px by LPC To investigate the mechanism by which TSC inhibited the LPCinduced apoptosis in HUVECs, we determined the status of SOD, GSH-Px, MDA, and ROS of HUVECs treated with LPC alone or in combination with Simvastatin, or different concentrations of TSG for 24 H. Relative to normal DMEM group, the levels of MDA and ROS were significantly increased in HUVECs treated with 10.0 mg/mL of LPC, while the levels of SOD and GSP-Px were significantly decreased. In contrast, both Simvastatin and 10.0 mmol/L TSG inhibited the production of MDA and ROS and 718 FIG 5 TSG inhibits the increase Cyt C release into the cytoplasm from mitochondria in HUVECs by LPC. HUVECs were treated with LPC alone or pretreated with Simvastatin, 0.1, 1, or 10 mmol/L TSG for 1 H, and then treated with LPC for 24 H. The protein level of Cyt C was determined by western blots. Data are the mean 6 SD (n 5 3), *P < 0.05, **P < 0.01 versus LPC group, &&P < 0.01 versus DMEM group. TSG Protects Huvecs By Upregulating SOD and GSH-PX FIG 6 TSG inhibited the changes of MDA, ROS, SOD, and GSH-Px by LPC in a concentration-dependent manner. After incubation for 24 H with different compounds as indicated, cells were harvested, and the contents of MDA (A1 and A2), SOD (B1 and B2), ROS (C1 and C2), and GSH-Px (D1 and D2) were determined, respectively, by detection kits and ELISA kits. (E) One experiment representative of three is shown. The results were determined by flow cytometry analysis. Data are the mean 6 SD (n 5 3), *P < 0.05, **P < 0.01 versus LPC group, &P < 0.05, &&P < 0.01 versus DMEM group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] active material of vascular endothelial cells such as NO generation, causing AS in the early stage (30,31). Furthermore, endothelial apoptosis is closely associated with hyperlipidemia (32), hypertension (33), and diabetes (34). Therefore, to prevent cardiovascular disease especially AS, it is important to protect endothelial cells from apoptosis resulted from oxidative stress. Oxide-LDL, a stimulus of AS, is currently being widely used to induce oxidative stress in experimental models. Similar to oxide-LDL, LPC is also able to induce injury of vascularendothelium-dependent diastolic function. LPC is derived from polar surface phosphatidylcholine (PC) of lipoproteins or from Zhao et al. cell membrane-derived PC as a result of phospholipase A2 (PLA2). In this study, we used LPC to induce oxidative stress injury in HUVECs. Our results showed that LPC was able to induce HUVECs injury in a concentration-dependent manner and 10.0 mg/mL of LPC was the optimal concentration to induce HUVECs injury. Next, we investigated the protective effects of TSG against LPC-induced oxidative stress injury in HUVECs. The results demonstrated that TSG could inhibit the LPC-mediated HUVECs injury and could restore cell vitality in a concentration-dependent manner. Moreover, the protective effects of TSG (10.0 mmol/L) were stronger than those of Simvastatin (5.0 mmol/L) in HUVECs. 719 IUBMB LIFE FIG 6 720 (Continued) TSG Protects Huvecs By Upregulating SOD and GSH-PX Oxidative stress results from the imbalance between ROS and antioxidants and contributes to the pathogenesis of cardiovascular diseases and other diseases. In this study, treatment of HUVECs with 10.0 mg/mL LPC resulted in 6-fold increase of the ROS level and 1.92-fold upregulation of the concentration of the oxidative product MDA, indicating oxidative stress injury in HUVECs. These data suggest that accumulation of ROS causes lipid peroxidation. Indeed, treatment of HUVECs with LPC led to decrease of SOD and GSHPx in addition to rapid increase of MDA. Interestingly, pretreatment with 10.0 mmol/L TSG restored the SOD and GSHPx levels to that of the DMEM only group, and significantly decreased ROS and MDA levels. Moreover, the ability of TSG (10.0 mmol/L) to activate the antioxidant enzymes SOD and GSH-Px in HUVECs was significantly stronger than that of Simvastatin (5.0 mmol/L). These results suggest that the mechanism underlying TSG protection against LPC-induced HUVECs oxidative stress injury involves the elevation of the expression of the antioxidant enzymes SOD and GSH-Px and the clearance of ROS. One of the mechanisms of cellular injury by oxidative stress is the induction of cell apoptosis. Our results suggest that TSG may protect HUVECs against LPC-induced apoptosis by promoting the expression of the antioxidant enzymes SOD and GSH-Px and the clearance of ROS. There are three apoptotic pathways: cell surface death receptors-Caspase-8Caspase-3 (29); mitochondria/MMP-Cyt C-Caspase-9-Caspase-3 (35,36); endoplasmic reticulum/Ca21-Caspase-12-Caspase-3 (37). The three apoptotic cascades converge on the activation of caspase-3, which results in the cleavage of intracellular poly (ADP ribose) polymerase (PARP-1) and the activation of DNase, leading to DNA fragmentation (38–40). Bcl-2 family members play important roles in mitochondria-dependent activation of caspase cascades (41). Previous studies have demonstrated that ROS increases the membrane permeability of mitochondria, causing the depolarization of the MMP and the release of Cyt C from mitochondria into the cytosol, triggering the caspase cascade, and leading to cell apoptosis (42–44). Our data showed that LPC (10.0 mg/mL) significantly induced apoptosis in HUVECs, accompanied with the loss of the MMP, the release of Cyt C, increase of caspase-3, Bax, and PARP-1, and decrease of Bcl-2. Whereas, TSG apparently abolished these effects induced by LPC, such as inhibition of the expression of Caspase-3 by LPC. Taken together, we conclude that the underlying mechanism of the protection of TSG against LPCinduced injury in HUVECs involves eliminating ROS by upregulating SOD and GSH-Px, and inhibiting the mitochondriadependent apoptotic pathway through stabilizing MMP, preventing Cyt C release, increasing Bcl-2 expression, and decreasing Caspases-3 activation. In summary, we discovered a novel mechanism by which TSG protects endothelial cells from oxidative stress involved in LPC-induced injury in HUVECs. Our findings will be helpful to develop TSG as a candidate for the prevention and treatment of cardiovascular diseases including AS. Zhao et al. Acknowledgements This research was supported by the Major Science and Technology Projects in Shanxi province, China (2012KTCQ03-02). 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