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Objective To investigate the effect of Sini Decorction on preventing vascular restenosis after iliac artery balloon injury in rabbits, and the levels of serum cholesterol. Materials and methods Twenty four control group, model group and Sini Decorction group. Rabbits of control group were fed with common forage, but model group and Sini Decorction group were fed with high fat diet (1.0% cholesterol, 10% lard). Two weeks later, the iliac arteries of rabbits were injured by balloon for model group and Sini Decorction group, rabbits in control group were sham operated. After operation, rabbits in Sini Decorction group were intragastric administrated with Sini Decorction (Strobal: Dried Ginger: Radix Glycyrrhiza=5:3:2) 2 g/kg once a day. All rabbits were killed four weeks after operation and serum samples were stored to assay the levels of serum cholesterol, the microstructure of iliac arteries was observed by optical microscope, the ultrastructure of iliac arteries was investigated by transmission electron microscope and scanning electron microscope. Results Endotheliocytes lined up in order in iliac arteries of control group and Sini Decorction group, but the endotheliocytes desquamated in that of model group. Endothelium was completed and no foam cells were found in control group, but the endotheliocytes were desquamated and a great deal of foam cells were found in model group, endotheliocytes were normal and less foam cells were found in Sini Decorction group. The lumina was wider and intima was thinner in SND group than that of model group. The thickness of intima in model group exceeded that in control group and Sini Decorction group (1549.75±416.53 mm 2 vs 0.00 mm 2 vs 646.70±121.92 mm 2 , p 2 vs 526.43±155.85 mm 2 vs 334.55±129.63 mm 2 , p Conclusion Sini Decorction can lessen intimal hyperplasia and vascular stenosis, inhibit the development of artherosclerosis with iliac artery injury in rabbits, the mechanism may be connected with increasing the level of HDL-C, decreasing the serum levels of TC, TG and LDL-C.

Objectives To detect the effect of Sini Decoction on the expression of Sirt-1 and eNOS in EAhy926 cell injured by homocysteine. Methods Model of EAhy926 cell injured by homocysteine was made,the protection on the EAhy926 cell of Sini Decoction with different dosages were observed. NO concentration of cell culture fluid was detected by nitrate reductase method, effect of Sini Decoction on the expression of protein of Sirt-1 and eNOS in EAhy926 cell were observed by Western-blot, and effect of Sini Decoction on the expression of mRNA of Sirt-1 and eNOS in EAhy926 cell were observed by fluorescent quantitation PCR. Results After Model of EAhy926 cell injured by homocysteine was made, we found that cultured with 0.5,1.0,2.0,4.0,8.0 μmol/L homocysteine, cells grew less than cultured with normal culture medium, and with the increase of homocysteine concentration, the number of attached cell grew downwards obviously, as culturing with homocysteine 4.0 μmol/L for 24h did lower damage to cells and could induce effective cell injuring, it was made to be the model of injury. To detect the effect of Sini Decoction on EAhy926 cell injured by homocysteine, well growing EAhy926 cells were cultured in culture plate. 24 h later, cells were cultured with DMEM medium containing 2% fetal calf serum for 8 hours to make cells hungry, then cultured with medium containing Sini Decoction 0, 0.25, 0.5, 1.0g/ml respectively for 30 minutes,then cultured with medium containing homocysteine 4.0 μmol/L for 24 h. It was found that, compared with control group, attached cells in Sini Decoction groups grew better, and attached cells in Sini Decoction 1.0g/ml plus homocysteine 4.0 μmol/L group grew best. 4.0 μmol/L. Detected by nitrate reductase method, it was found that compared with control group, there was no obvious change of NO concentration of cell culture fluid in Sini Decoction 1.0 g/ml group, but in homocysteine 4.0 μmol/L medol group, NO concentration of cell culture fluid decreased oberviously, and in Sini Decoction groups, NO concentration of cell culture fluid increased, and in Sini Decoction 1.0g/ml plus homocysteine 4.0 μmol/L group it was the most obvious ( p p p Conclusions Homocysteine may injure EAhy926 cell by suppressing the expression of Sirt-1 then suppressing the expression of eNOS system, while Sini Decoction may protect EAhy926 cell by enhancing the expression of caveolin-1 then enhancing the expression of eNOS system.

Objectives To investigate the effects of Tongxinluo and Homocysteine (Hcy) on proliferation of human umbilical vein endothelial cells (Huvecs) in vitro . Methods Isolation,culture and identification of Huvecs:With the informed consents of puerperants, the normal fetal umbilical cords were obtained through uterine-incision delivery in Third Affiliated Hospital, Sun Yat-sun University. Huvecs were isolated by Percoll density gradient centrifugation from fetal umbilical cords with digestion of collagenase type perfusion, and then suspended in Medium 199 supplemented with 20% FBS, 2 mM L-glutamine, 50 μg/ml endothelial cell growth factor, 100 μg/ml heparin and 100μg/ml streptomycin cultured in 0.05 volume fraction of CO2 incubator at 37°. The culture solution was changed after 24 hours, Huvecs cultured to 80% confluence, the passage was developed with 0.25% trypsinase and 0.02% EDTA. After the Huvecs were identified by flow cytometry with the cell marker CD34, The second or third passage was used for study. MTT assay was used to detect cell viability: Huvecs during the logarithmic phase were inoculated in 96-well plates with Medium 199 in 0.05 volume fraction of CO2 incubator at 37° for 48h, at 80% confluence, incubated in serum-free medium,and divided into two groups, eachgroup including eight parallel wells, blank control group was untreated, he latter cultured and added with Hcy of the concentrations of 0.1, 0.25, 0.5, and 1.0 mmol/L respectively,or Hcy 0.5 mmol/L+Tongxinluo of the concentrations of 0.1, 0.5, 1.0, and 2.0mg/ml. After the cells were cultured for 24 hr, 5 g/L MTT solution (20μL) was added, then dimethyl sulfoxide was used for vibration and dissolvement. Optical density of each hole was detected by using enzyme-linked immunosorbent assay to analyse different concentrations of Hcy and Tongxinluo on the proliferation of human mammary stemscells. Results Hcy of different doses inhibited the viability of Huvecs dose-dependently, the Optical density were 0.526 ± 0.017, 0.439 ± 0.010, 0.258 ± 0.008, and 0.209 ± 0.004 respectively, lower than that (0.578 ± 0.023) of blank control group (all P P P Conclusions Hcy can inhibited the viability of Huvecs dose-dependently, and this effect is most prominent at the concentrations of 0.5 mmol/L for 24 hours; Tongxinluo can enhance the proliferation activity of Huvecs, and this effect is most prominent at 1.0mg/ml for 24 hours.

Objectives Senescence is involved in many age-related diseases, such as atherosclerosis, hypertension and diabetes. So how to delay or solve the problems caused by ageing has become a hot topic in the past decade. To explore the best stress-induced premature senescence (SIPS) model in vitro , we applied different dosages of H 2 O 2 to the primary human umbilical vein endothelial cells (HUVECs). Methods SIPS was induced by H 2 O 2 with different concentration (0, 20, 40, 60, 80, 100 μM) in HUVECs . Senescence related gene was evaluated with western blot or real-time PCR. Senescence-associated beta-galactosidase (SA-β-gal) staining was used to examine the phenotype of senescence. Apoptosis was analysed by flow cytometry. Results The SA-β-gal staining result showed that the senescent cell was detected more significantly with H 2 O 2 ≥40 μM compared with the control group. Also the mRNA of p21 was upregulated significantly with 40μM H 2 O 2 and more. The PAI-1 protein was increased significantly with 60 μM H 2 O 2 and more. In the apoptosis aspect, there was a significant increased number of apoptotic cells in 80 and 100 μM group. Conclusions H 2 O 2 can induce senescence of the primary HUVECs. In consideration of the its apoptotic effect, the 60 μM H 2 O 2 could be the best concentration to induce senescence. Because of the high relevance with human, the primary HUVECs is a better candidate applied to explore the mechanism and the improved treatment of senescence in vitro . Besides H 2 O 2 , there are some other ways to induce senescence, such as UV and gene knock down. Among these methods, H 2 O 2 induced senescence via oxidative stress is the most relavent to the enviroment in the aged population.

Objectives Senescence of endothelial cells has been proposed to play an important role in endothelial dysfunction andatherogenesis. In the present study we aimed to investigate whether Tongxinluo (TXL) capsule protects human umbilical vein endothelial cells (HUVECs) from H 2 O 2 -induced endothelial senescence. Methods Solation, culture and identification of HUVECs: With the informed consents of puerperants, the normal fetal umbilical cords were obtained through uterine-incision delivery in Third Affiliated Hospital, Sun Yat-sun University. HUVECs were isolated by Percoll density gradient centrifugation from fetal umbilical cords with digestion of collagenase type I perfusion, and then suspended in Medium 199 cultured in 0.05 volume fraction of CO 2 incubator at 37°C. After the HUVECs were identified by flow cytometry with the cell marker CD31, The second or third passage was used for study. The exponentially growing HUVECs were plated at a cell density of 1×10 5 /well in 6-well plate and cultured overnight at 37 °C. The next day, the medium was changed with M199 supplemented with 2% FBS for at least 8 h to starve the cells. Then the cells were exposed to various concentrations of H2O2 to induce premature senescence. After 1 h, the medium was replaced with normal medium. Different concentration of TXL (0.1, 0.5, 1.0, and 2.0 mg/ml) was added in the media 30 min before the induction of senescence by addition of H2O2. Finally the cells were harvested in indicated time for Western blot analysis and real-time polymerase chain reaction (PCR). The MDA level and SOD activity were determined using commercially available kits following the manufacturer9s instructions. Intracellular ROS generation was monitored by flow cytometry using peroxide sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate (H2DCFDA, Invitrogen). Results (1) Treatment with H2O2 caused significant increase in intracellular thiobarbituric acid reactive substances (TBARS) level (p 2 O 2 decreased the activity of SOD to 50.4±6.9%. However, pre-incubation with TXL (0.1, 0.5, 1.0, and 2.0 mg/ml) significantly increased SOD activity compared with H 2 O 2 alone treated group (p (2) Real-time PCR analysis showed that at the time point of 24 h, SOD1 mRNA in the 60mmol/l H 2 O 2 treated group decreased by 1.99 fold, compared with untreated group (p (3) Western blotting results demonstrated that the level of SOD1 protein reduced in H 2 O 2 treated group compared to untreated group after 24, 48 and 72 h incubation. However, TXL reversed the decreased expression of SOD1 protein in HUVECs treated with H 2 O 2 in a dose-dependent manner As the control, TXL alone did not significantly change the expression of SOD1 at either mRNA or protein level. (4) TXL alone did not change the ROS generation. However, compared to the nontreated control group, 60 mmol/l H 2 O 2 significantly increased DCF fluorescence whereas pre-treatment with 1.0 mg/ml TXL markedly inhibited the production of ROS induced by H 2 O 2 . Conclusions our data demonstrate that ginseno side TXL modulates redox status such as upregulating SOD1 expression, scavenging ROS, and decreasing the peroxidation to prevent the cellular senescence in HUVECs.

Objectives To investigate the change of serum level of Pigment epithelium-derived factor (PEDF) in coronary heart disease patients. Methods 40 coronary heart disease patients, 29 males and 11 females were recruited into this study, which contains acute myocardial infarction 6 cases, unstable angina pectoris 15 cases, stable angina 19 cases, according to the New York Heart Association (NYHA) diagnostic criteria, ruling out infectious diseases, tumour, renal inadequacy, diabetes, Hepatic function damage, cardiac inadequacy, hyperthyrea, anaemia and hyperthermy. Control group contains 35 healthy people with similar gender, age and social background. Venous blood on an empty stomach were drawn next day after admission and were put in tubes, and were centrifuge in 1000 rpm for 10 min, then blood serum were imbibed and stored under −80°C. The serum of normal control group were taken in similar ways. Serum level of PEDF and IL-6 were determined by ELISA Kits (Bought from USCN life science Company, Wuhan, China and KeyGEN Biotech Company, Nanjing China). Meanwhile, serum level of total cholesterol, triglyeride and lipoprotein cholesterol were determined. Degree of stegnosis of coronary by Gensini scoring system based on the result of coronary arteriongraphy. Results The serum level of PEDF (8.39–2.64 ug/ ml) and Il-6 (7.40–4.41 pg/ml) in coronary heart disease group were higher than PEDF (6.952.92 µg/ml) and Il-6 (4.09–3.70 pg/ml) in control group (p 0.05), but there was correlativity between serum level of PEDF and IL-6 (r=0.241, p Conclusions The results suggest that Pigment epithelium-derived factor increases greatly in CHD patients, Pigment epithelium-derived factor may be an important factor of atherosclerosis.

Objectives To investigate the effects of Jiawei Buyang Huanwu Decoction on vascular stenosis and transforming growth factor -β1 (TGF-β1) after iliac artery were injured by balloon in diet-induced atherosclerotic rabbits. Methods 24 male New Zealand albino rabbits were equally randomized into control group, model group and drug group. The iliac arteries of the rabbits in the latter two groups were subjected to balloon injury. Four weeks later, serum TGF-β1 level was assayed, Endothelial hyperplasia, eNOS Protein and mRNA expression were observed in injured iliac artery. Results Optical microscope revealed narrowed vascular lumen, thicken intima and numerous arteriosclerotic plaques in the model group compared with the control group, whereas the vascular lumen and intima thickness remained basically normal in drug group. The serum TGF-β1 level was lower in drug group than that of model group, Immunohistochemistry and RT-PCR results showed that eNOS protein and mRNA expression was lower in rabbit iliac artery of drug group than that in model group. Conclusions Jiawei Buyang Huanwu Decoction can lessen intimal hyperplasia and vascular stenosis in iliac artery injury rabbits, and the mechanism of which may be related to decrease in TGF-β1 protein and gene expression.

Objectives To detect the effect of Sini Decoction on the expression of Caveolin-1 and eNOS in EAhy926 cell injured by homocysteine. Methods Model of EAhy926 cell injured by homocysteine was made, the protection on the EAhy926 cell of Sini Decoction with different dosages and different durations were observed. The effection of Sini Decoction on the expression of protein of Caveolin-1 and eNOS in EAhy926 cell were observed by Western-blot, and effection of Sini Decoction on the expression of mRNA of Caveolin-1 and eNOS in EAhy926 cell were observed by fluorescent quantitation PCR. Results After model of EAhy926 cell injured by homocysteine was made, we found that cultured with 0.5, 1.0, 2.0, 4.0, 8.0 µmol/l homocysteine, cells grew less than cultured with normal culture medium, and with the increase of homocysteine concentration, the number of attached cell grew downwards obviously, as culturing with homocysteine 4.0 µmol/l for 24 h did lower damage to cells and could induce effective cell injuring, it was made to be the model of injury. To detect the effection of Sini Decoction on EAhy926 cell injured by homocysteine, well growing EAhy926 cells were cultured in culture plate. 24 h later, cells were cultured with DMEM medium containing 2% fetal calf serum for 8 h to make cells hungry, then cultured with medium containing Sini Decoction 0, 0.25, 0.5, 1.0 g/ml respectively for 30 min, then cultured with medium containing homocysteine 4.0 µmol/l for 24 h. It was found that, compared with control group, attached cells in Sini Decoction groups grew better, and attached cells in Sini Decoction 1.0 g/ml plus homocysteine 4.0 µmol/l group grew best. Detected by western-blot, it was found that, compared with control group, there was no obvious change of protein of Caveolin-1 and eNOS in Sini Decoction 1.0 g/ml group, but in homocysteine 4.0 µmol/l medol group, expression of Caveolin-1 protein enhanced obviously, expression of eNOS protein weakened obviously, and in Sini Decoction groups, expression of Caveolin-1 protein weakened, and expression of eNOS protein enhanced, and in Sini Decoction 1.0 g/ml plus homocysteine 4.0 µmol/l group it was the most obvious p Conclusions Homocysteine may injure EAhy926 cell by enhancing the expression of caveolin-1 then suppressing the expression of eNOS, while Sini Decoction may protect EAhy926 cell by suppressing the expression of caveolin-1 then enhancing the expression of eNOS.

Objectives To investigate the effect of Jiawei Buyang Huanwu Decoction in preventing vascular restenosis and on the nitric oxide (NO) system after rabbit iliac artery balloon injury. Methods 24 male New Zealand albino rabbits were equally randomised into control group, model group and drug group. The iliac arteries of the rabbits in the latter two groups were subjected to balloon injury. Four weeks later, serum NO synthases (NOS) activity and endothelial nitric oxide synthase (eNOS) level was assayed, Endothelial hyperplasia, eNOS Protein and mRNA expression were observed in injured iliac artery. Results Optical microscope revealed narrowed vascular lumen, thicken intima and numerous arteriosclerotic plaques in the model group compared with the control group, whereas the vascular lumen and intima thickness remained basically normal in drug group. The serum NO level and total NOS activity were lower in model group than that of control group and drug group, while there is no difference between the latter two. Immunohistochemistry and RT-PCR results showed that eNOS protein and mRNA expression was stronger in rabbit iliac artery of control group and drug group than that in model group. Conclusions Jiawei BHD can lessen intimal hyperplasia and vascular stenosis in iliac artery injury rabbits, this effect is possibly related with that Jiawei BHD activated the NO system.

Objective To investigate the effects of Tongxinluo (TXL) on Homocysteine (HCY)-induced endothelial dysfunction and inflammatory response. Methods Isolation, culture and identification of human umbilical vein endothelial cells (HUVECs): With the informed consent of puerperants, the normal fetal umbilical cords were obtained through uterine-incision delivery in Third Affiliated Hospital, Sun Yat-sun University. HUVECs were isolated by Percoll density gradient centrifugation from fetal umbilical cords with digestion of collagenase type I perfusion, and then suspended in Medium 199 cultured in 0.05 volume fraction of CO 2 incubator at 37°C. After the HUVECs were identified by flow cytometry with the cell marker CD34, The second or third passage was used for study. MTT assay was used to detect cell viability: HUVECs during the logarithmic phase were inoculated in 96-well plates with Medium 199 in 0.05 volume fraction of CO 2 incubator at 37°C for 48 h, at 80% confluence, incubated in serum-free medium, and divided into two groups, each group including eight parallel wells, blank control group was untreated, the latter cultured and added with HCY of the concentrations of 0.1, 0.25, 0.5, and 1.0 mmol/l, respectively, or HCY 0.5 mmol/l+TXL of the concentrations of 0.1, 0.5, 1.0, and 2.0 mg/ml. After the cells were cultured for 24 h, 5 g/l MTT solution (20 μl) was added, then dimethyl sulfoxide was used for vibration and dissolvement. Optical density of each hole was detected by using enzyme-linked immunosorbent assay to analyse different concentrations of HCY and TXL on the proliferation of human mammary stems cells. Western blot was used to examine the protein expression of the cell inflammatory factors, tumour necrosis factor (TNF)α, interleukin (IL)-6, macrophage chemoattractant protein (MCP)-1, and intercellular adhesion molecule (ICAM)-1, ELISA was used to detect the contents of the cell inflammatory factors. Results HCY of different doses inhibited the viability of HUVECs dose-dependently, the inhibition rates of HCY were 0.099±0.079, 0.239±0.046, 0.637±0.007, and 0.553±0.029, respectively (all p 0.01). Conclusion Tongxinluo inhibits homocysteine-induced endothelial impairment and inflammatory response, the definite mechanism of which requires further research.

Objective To investigate the change of vascular endothelium in coronary heart disease patients by measuring the function of brachial artery endothelium with high definition power ultrasound. Method Thirty one coronary heart disease patients with heart failure, 21 males and 10 females were recruited into this study, According to the New York Heart Association (NYHA) classification, patients were divided into Class I (n=11), Class α (n=11), Class β (n=9), and all the patients were excluded from tumour and infection. Brachial artery endothelial function of all patients was measured next day after hospital admission. Normal control group consisted of 12 healthy people with age and gender matching. After measuring the inner diameter brachial artery with 7.0 MHz high definition power ultrasound linear array searching unit, reactive engorge test will be done. Distal end of target artery was pressed by pneumatic tourniquet up to 200 mm Hg (1 mm Hg=0.133 kPa), kept on 5 min, then deflated and decompressed. After 1 min later, inner diameter of same part of the brachial artery were measured. After when the inner diameter of brachial artery returned to fundamental numerous, patients took nitro glycerol 0.5 mg sublingually. In 2–5 min time, inner diameter of same part of the brachial artery were measured. Then percentage of changed numerous of inner diameter of brachial artery after pressing and taking nitro glycerol of fundamental numerous were calculated, named endothelium-depended vascular dilatation (Δ1%) and none endothelium-depended vascular dilatation (Δ2%) respectively. Result Endothelium-depended vascular dilatation (7.8–5.1%) and none endothelium-depended vascular dilatation (11.6–7.2%) in CHD patients decreased greatly than Normal control group (15.1–3.9%, 23.1–2.7%, p