1072 Regular Article Biol. Pharm. Bull. 34(7) 1072—1077 (2011) Vol. 34, No. 7 Rb1 Protects Endothelial Cells from Hydrogen Peroxide-Induced Cell Senescence by Modulating Redox Status a b a a a a a Ding-Hui LIU, Yan-Ming CHEN, Yong LIU, Bao-Shun HAO, Bin ZHOU, Lin WU, Min WANG, a c ,a,c Lin CHEN, Wei-Kang WU, and Xiao-Xian QIAN* a Department of Cardiology, The Third Affiliated Hospital of Sun Yat-sen University; b Department of Endocrinology, The Third Affiliated Hospital of Sun Yat-sen University; Guangzhou, Guangdong 510630, China: and c Institute Integrated Traditional Chinese and Western Medicine, Sun Yat-sen University; Guangzhou, Guangdong 510630, China. Received February 12, 2011; accepted April 12, 2011; published online April 15, 2011 Senescence of endothelial cells has been proposed to play an important role in endothelial dysfunction and atherogenesis. In the present study we aimed to investigate whether ginsenoside Rb1, a major constituent of gin- seng, protects endothelial cells from H2O2-induced endothelial senescence. While H2O2 induced premature senes- cent-like phenotype of human umbilical vein endothelial cells (HUVECs), as judged by increased senescence- associated b-galactosidase (SA-b-gal) activity, enlarged, flattened cell morphology and sustained growth arrest, our results demonstrated that Rb1 protected endothelial cells from oxidative stress induced senescence. Mechan- istically, we found that Rb1 could markedly increase intracellular superoxide dismutase (Cu/Zn SOD/SOD1) activity and decrease the malondialdehyde (MDA) level in H2O2-treated HUVECs, and suppress the generation of intracellular reactive oxygen species (ROS). Consistent with these findings, Rb1 could effectively restore the protein expression of Cu/Zn SOD, which was down-regulated in H2O2 treated cells. Taken together, our data demonstrate that Rb1 exhibits antioxidant effects and antagonizes H2O2-induced cellular senescence. Key words senescence; human umbilical vein endothelial cell; reactive oxygen species; antioxidant Aging is considered as a high risk factor for cardiovascular antioxidant enzyme, is constitutively expressed in all mam- diseases, especially for atherosclerosis.1,2) Vascular aging is malian cells and is localized in the cell cytosol and the nu- believed to be associated with pro-oxidant and pro-inflamma- cleus.14) MDA is a major and stable end product formed dur- tory phenotypic and functional changes, which promote the ing the peroxidation of lipids and is regarded as marker of progression of cardiovascular diseases.3) lipid peroxidation in tissues.15) At the cellular level, senescence is a state of permanent Here, we reported that Rb1 antagonizes H2O2-induced growth arrest in which cells are alive and metabolically ac- HUVECs senescence through upregulating endogenous anti- tive for months, but are unresponsive to mitogenic stimuli.4) oxidants SOD and decreasing vascular lipid peroxidation. Free radical-dependent cellular damage theory was devel- oped since 1956 and remains the theory basis of aging.5) Ac- MATERIALS AND METHODS cumulating evidence supports the link between oxidative stress and aging by showing that oxidative stress induces cel- Chemicals and Reagents Rb1 was purchased from the lular senescence.6—9) Hydrogen peroxide is widely used to Division of Chinese Materia Medica and Natural Products, achieve oxidative stress-induced premature senescence National Institute for the Control of Pharmaceutical and Bio- within a short time period. logical Products, Ministry of Public Health (Beijing, China). Ginsenoside Rb1 is one of 40 bioactive extracts from gin- Hydrogen peroxide (H2O2) and dimethylsulfoxide (DMSO) seng which has been used as tonic remedy in traditional Chi- were obtained from Sigma (St. Louis, MO, U.S.A.). The nese Medicine for two thousand years.10,11) Previous studies reagent kits for the measurement of the levels of MDA and have demonstrated that increased generation of reactive oxy- SOD were purchased from Nanjing Institute of Jiancheng gen species (ROS) in the injured vascular can be reversed by Bioengineering (Nanjing, China). 2Ј,7Ј-Dichlorofluorescein Rb1 treatment which provides a protective effect on the vas- diacetate (H2DCFDA) and TRIzol reagent were obtained cular injury.12) Therefore, we postulate that ginsenoside Rb1 from Invitrogen (Carlsbad, CA, U.S.A.). PrimeScriptTM RT may play a role in protecting vascular cells from senescence reagent Kit and SYBR Premix Ex TaqTM Kit were obtained through modulating the balance of the antioxidant related en- from TaKaRa (TaKaRa, Dalian, China). All other reagents zyme and pro-oxidant related enzyme. To evaluate the pro- used were of analytical grade. tective effects of Rb1, we employed human umbilical vascu- Cell Culture Primary HUVECs were prepared from lar endothelial cells (HUVECs) as a model and determined newborn human umbilical cords within 6 h by 0.1% collage- the superoxide dismutase (SOD) and malondialdehyde nase (Gibco) perfusion as described previously.16) HUVECs (MDA) levels in the cells treated by Rb1. were plated into a 0.2% gelatin-coated cell culture flask and SOD, along with other enzymatic and non-enzymatic anti- grown in Medium 199 (M199) (Gibco) supplemented with oxidants, plays a pivotal role in preventing cellular damage 20% Fetal Bovine Serum (FBS) (Gibco), 20% Serum-Free caused by ROS. It exerts its role through reducing the super- Medium (SFM) (Gibco), 2 mmol/l glutamine, 60 mg/ml En- oxide radical to form hydrogen peroxide and oxygen. There dothelial Cell Growth Supplement (ECGS) (BD) and 5 U/ml are three forms of SODs which are designated as Cu/Zn- heparin, kept in a humid incubator at 37 °C in 5% CO2. Sub- SOD (SOD1), mitochondrial Mn-SOD (SOD2), and extracel- cultures were obtained by treating cells with 0.05% trypsin- lular SOD (SOD3).13) SOD1, the most extensively studied ethylenediaminetetraacetic acid (EDTA) solution (Gibco). ∗ To whom correspondence should be addressed. e-mail: [email protected] © 2011 Pharmaceutical Society of Japan July 2011 1073 Experiments were conducted on cells with a passage number time PCR reaction. b-Actin, a housekeeping gene, was used between passages 2 and 3. The cell type was determined by as internal control to account for variations in mRNA load- “cobblestone” cell morphology and by flow cytometric ing. The b-actin (GenBank No. NM_001101.3) primer se- analysis with a monoclonal anti-human CD31 antibody (BD, quences were: forward 5Ј-AGC GGG AAA TCG TGC GTG U.S.A.). More than 99% of the cells were positive for CD31. AC-3Ј and reverse 5Ј-TCC ATG CCC AGG AAG GAA GG- The protocol was approved by the Research Committee of 3Ј. The SOD1 (GenBank No. NM_000454.4) primer se- the Third Affiliated Hospital of Sun Yat-sen University and quences were: forward 5Ј-CAA AGA TGG TGT GGC CGA conformed to the principles outlined in the Declaration of TG-3Ј and reverse 5Ј-TTT CCA CCT TTG CCC AAG TCA- Helsinki. The parturients enrolled in the current protocol 3Ј. Real-time PCR was performed in an ABI 7000 PCR de- were negative for Human Immunodeficiency Viruses (HIV), tection system. The thermal cycle condition used for reverse Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) and transcription was as follows: 15 min at 37 °C and 5 s at 85 °C. gave written informed consent to providing their umbilical The condition used for real-time PCR was as follows: 30 s at cord. 95 °C, 40 repeats of 5 s at 95 °C and 31 s at 60 °C, and 1 re- Cell Treatment The exponentially growing HUVECs peat of 15 s at 95 °C, 1 min at 60 °C and 15 s at 95 °C. were plated at a cell density of 1ϫ105/well in 6-well plate Western Blotting The cells were lysed with whole cell and cultured overnight at 37 °C. The next day, the medium lysis buffer (KeyGen BioTechonology, Nanjing, China) ac- was changed with M199 supplemented with 2% FBS for at cording to the manufacturer’s instructions. Cells lysates were least 8 h to starve the cells. Then the cells were exposed to quantified by BCA methods according to the manufacture’s various concentrations of H2O2 to induce premature senes- instructions (Sangon BioTechonology, Shanghai, China), cence. After 1 h, the medium was replaced with normal then resuspended in the sample buffer and boiled for 5 min at medium. Different concentration of Rb1 (10, 20, 40 mmol/l) 100 °C. The same amount of protein was electrophoresed on was added in the media 30 min before the induction of senes- 4—12% gradient sodium dodecyl sulfate-polyacrylamide gel cence by addition of H2O2. Finally the cells were harvested in electrophoresis (SDS-PAGE) gels and transferred onto indicated time for Western blot analysis and real-time poly- polyvinylidene difluoride membranes (Millipore, U.S.A.). merase chain reaction (PCR). Membranes were blocked using 5% nonfat dried milk in Senescence-Associated b-Galactosidase (SA-b-Gal) TBST for 1 h at room temperature with gentle shaking, then Assay To determine the number of senescent cells, senes- probed with a rabbit antihuman SOD1 monoclonal antibody cence-associated b-galactosidase staining was performed as (EPITOMICS, U.S.A.) at the dilution of 1 : 100000 or glycer- described previously.17) Briefly, cells were washed twice in aldehyde-3-phosphate dehydrogenase (GAPDH) primary an- phosphate buffered saline (PBS) and fixated in 2% formalde- tibody followed by horseradish peroxidase (HRP)-conjugated hyde and 0.2% glutaraldehyde for 5 min at room temperature, goat anti-rabbit immunoglobulin G secondary antibody at the washed twice and incubated for 18 h at 37 °C (no CO2) with dilution of 1 : 10000. Membranes were developed using the fresh SA-b-gal staining solution (1 mg/ml 5-bromo-4-chloro- enhanced chemiluminescence detection method (Millipore, 3-indolyl-D-galactoside, 40 mmol/l citric acid/sodium phos- U.S.A.), and the signals were quantified using Quantity One phate dibasic at pH 6.0, 150 mmol/l NaCl, 2 mmol/l MgCl2, Software (Bio-Rad, U.S.A.).