Liu Jinlai

GW24-e2393 The effect of rosuvastatin on reendothelialisation of late endothelial outgrowth cell influenced by C-reactive protein

Objectives To observe whether CRP influenced expressions of eNOS/NO in late endothelial outgrowth cells (EOCs) though signalling pathway mediated by receptor for advanced glycation end products (RAGE), and whether rosuvastatin could improve EOCs’ reendothelialisation influenced by CRP though restraining RAGE. Methods The EOCs were treated with CRP of different concentrations and treating times, and EOCs were pre-incubated with rosuvastatin of different concentrations and then stimulated with 50ug/ml CRP. The eNOS of EPCs were measured by quantitative PCR and NO secretions were detected by NO nitrate reductive enzymatic. The RAGE protein expressions were checked by western blotting. MTT assay was used to test proliferation of EPCs, transwell assay for migration, and adhesiveness assay for adhesion. Results The eNOS mRNA expression and NO secretions decreased significantly with CRP-stimulated concentration and time, and down-regulated by rosuvastatin. Also, the RAGE protein expression rised in a dose-dependent and time-dependent manner. The RAGE was significantly decreased by rosuvastatin with different dosages. After treatment with different dosage rosuvastatin, the vitro functions (migration and adhesion) of CRP-influenced EOCs were dosage-dependently upregulated. However, the proliferation had no changes. Conclusions CRP could influence expressions of eNOS/NO though signalling pathway mediated by RAGE. Rosuvastatin could improve EOCs’ reendothelialisation influenced by CRP though restraining RAGE.

EFFECTS OF JIAWEI BUYANG HUANWU DECOCTION ON VASCULAR STENOSIS AND TGF-β1 AFTER BALLOON INJURY OF RABBIT ILIAC ARTERY

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.

THE EFFECT OF JIAWEI BUYANG HUANWU DECOCTION ON THE NO SYSTEM AFTER ILIAC ARTERY BALLOON INJURED IN RABBITS

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.

TONGXINLUO CAPSULE PROTECTS ENDOTHELIAL CELLS FROM HYDROGEN PEROXIDE-INDUCED CELL SENESCENCE BY MODULATING REDOX STATUS

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 H2O2-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 CO2 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 1×105/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 manufacturers 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<0.01 vs untreated control), while pre-incubation with TXL (0.5, 1.0 mg/ml) markedly attenuated the increase (p<0.05). Compared to control group, treatment with H2O2 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 H2O2 alone treated group (p<0.01). (2) Real-time PCR analysis showed that at the time point of 24 h, SOD1 mRNA in the 60mmol/l H2O2 treated group decreased by 1.99 fold, compared with untreated group (p<0.01). However, SOD1 mRNA increased by 1.54, 1.77, 1.75 fold in 0.1, 0.5, 1.0mg/ml TXL pretreated group respectively, compared with H2O2 alone treated group (p<0.01). (3) Western blotting results demonstrated that the level of SOD1 protein reduced in H2O2 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 H2O2 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 H2O2 significantly increased DCF fluorescence whereas pre-treatment with 1.0 mg/ml TXL markedly inhibited the production of ROS induced by H2O2. 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.

Outcome of transplanted mesenchymal stem cells labelled with superparamagnetic iron oxide after myocardial infarction in swine

Objectives To trace and evaluate intracoronary transplanted mesenchymal stem cells (MSCs) labelled with superparamagnetic iron oxide (SPIO) by using magnetic resonance imaging (MRI) in a swine model of myocardial infarction (MI). Methods After isolated by density gradient centrifugation, autologous MSCs were transfected with green fluorescent protein (GFP) by lentiviral vector and labelled by superparamagnetic iron oxide (SPIO). Two weeks after MI, swine were randomised to intracoronary transplantation of dual-labelled MSCs (n=10), MSCs-GFP (n=10) and saline (n=5). MRI examination was performed with a 1.5T clinical scanner at 24 h, three weeks and eight weeks after cells transplantation. Signal intensity (SI) changes, cardiac function and MI size were measured using MRI. Correlation between MR findings and histomorphologic findings was also investigated. Results It was demonstrated that when MSCs were labelled with SPIO (25 µg Fe/ml plus 0.8 µl/ml lipofectamine) the labeling efficiency reached 95%∼l00%, with no effects on GFP expression. Multipotentiality was not affected especially for cardiomyocyte-like cells differentiation. SI on T2*WI decreased substantially in the interventricular septum 24 h after injection of MSCs. Changes in SI at 24 h, three weeks and eight weeks were (52.98±10.74)%, (21.53±5.40)% and (6.23±2.01)%, respectively (p<0.05). Both dual-labelled and MSCs-GFP could dramatically reduce the size of MI and improve cardiac function. Histological data revealed that Prussian blue stain-positive cells were found mainly in the border zone which also showed green fluorescence but negative for macrophage marker (CD68). The number of iron-positive cells per HPF in the border zone and infarct zone were 36.2±3.8 and 9.7±2.1, respectively (p<0.05). Conclusions In vivo long-term tracing of dual-labelled MSCs can be achieved by MRI. Intracoronary transplantation of dual-labelled MSCs can increase cardiac function and reduce the size of MI.

Effects of Tongxinluo capsule on homocysteine-induced endothelial dysfuncion and inflammartoy response

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 CO2 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 CO2 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); the growth rates of HCY-induced HUVECs treatment by TXL of the concentrations of 0.1, 0.5, 1.0, and 2.0 mg/ml were 0.682±0.312, 0.710±0.040, 0.855±0.130, 0.784±0.031, respectively, higher than that of the control group (all p<0.01). Western blotting and ELISA showed that homocysteine significantly increased the expression of TNFα, IL-6, MCP-1 and ICAM-1, which are 0.71±0.15, 0.89±0.32, 0.80±0.27, 0.54±012, respectively, higher than that of 0 hour (all p<0.01). However, the expression levels of TNFα, IL-6, MCP-1 and ICAM-1 of the 0.50 mmol/l HCY-induced HUVECs treatment by TXL (1 mg/ml) were 0.24±0.04, 0.15±0.03, 0.13±0.04, 0.22±0.06, respectively, not significantly different from those at the of 0 hour (all p>0.01). Conclusion Tongxinluo inhibits homocysteine-induced endothelial impairment and inflammatory response, the definite mechanism of which requires further research.

Effects of Sini decoction on vascular stenosis of iliac artery in rabbits after injured by balloon and levels of serum cholesterol

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 mm2 vs 0.00 mm2 vs 646.70±121.92 mm2, p<0.05). The area of lumina was smaller than that of control group and Sini Decorction group (149.80±73.21 mm2 vs 526.43±155.85 mm2 vs 334.55±129.63 mm2, p<0.05). The serum levels of TC (31.48±3.71 mmol/L vs 22.42±7.76 mmol/L vs 2.5±1.55 mmol/l), TG (6.27±1.69 mmol/l vs 3.98±1.56 mmol/l vs 1.62±0.83 mmol/l) and LDL-C (22.14±8.48 mmol/l vs 13.14±5.95 mmol/l vs 1.33±1.03 mmol/l) were higher in rabbits of model group than that of SND group and control group (p<0.05). On the other hand, the serum level of HDL-C was higher in SND group than that in control and model group (1.07±0.77 vs 0.50±0.29, p<0.05). 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.

Effects of Sini decoction on vascular stenosis of iliac artery and levels of serum cholesterol in rabbits after injured by balloon

Objective To investigate the effect of Sini Decoration on preventing vascular restenosis after iliac artery balloon injury in rabbits, and the levels of serum cholesterol. Materials and methods Twenty four New Zealand albino rabbits were randomly divided into three groups, named control group, model group and Sina Decoration group. Rabbits of control group were fed with common forage, but model group and Sini Decoration 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 Decoration group, rabbits in control group were sham operated. After operation, rabbits in Sini Decoration group were intragastric administrated with Sini Decoration (Strobal: Dried Ginger: Radix Glycyrrhiza=5:3:2) 2 g/kg once a day. All rabbits were sacrificed 4 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 Decoration 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 Decoration 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 Decoration group (1549.75±416.53 mm2 vs 0.00 mm2 vs 646.70±121.92 mm2, p<0.05). The area of lumina was smaller than that of control group and Sini Decoration group (149.80±73.21 m2 vs 526.43±155.85 mm2 vs 334.55±129.63 mm2, p<0.05). The serum levels of TC (31.48±3.71 mmol/l vs 22.42±7.76 mmol/l vs 2.5±1.55 mmol/l), TG (6.27±1.69 mmol/l vs 3.98±1.56 mmol/l vs 1.62±0.83 mmol/l) and LDL-C (22.14±8.48 mmol/l vs 13.14±5.95 mmol/l vs 1.33±1.03 mmol/l) were higher in rabbits of model group than that of SND group and control group (p<0.05). On the other hand, the serum level of HDL-C was higher in SND group than that of in control and model group (1.07±0.77 vs 0.50±0.29 vs 0.27±0.12, p<0.05). Conclusions Sini Decorction can decrease intimal hyperplasia and vascular stenosis, inhibit the development of artherosclerosis with iliac artery injury in rabbits. The mechanism may be connected with the increased the level of HDL-C, along with the decreased serum levels of TC, TG and LDL-C.

The change of vascular endothelium in coronary heart disease

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<0.001). With the aggravation of heart failure from Class α to Class β, endothelium-depended vascular dilatation (11.6–3.4% vs 7.5–4.9% vs 3.4–3.7%, p<0.05) and none endothelium-depended vascular dilatation (18.1–4.8% vs 10.4–6.1% vs 5.2–3.7%, p<0.05) decrease markedly. Conclusion The results suggested that endothelium-depended vascular dilatation and none endothelium-depended vascular dilatation decreased greatly in CHD patients. The vascular endothelial function damaged significantly with the aggravation of heart failure.

The protective effects of Jiawei Buyang Huanwu decoction on vascular endothelia and mechanism after iliac artery balloon injured in rabbits

Aim To investigate the protective effects of Jiawei Buyang Huanwu Decoction on vascular endothelial function and mechanism after iliac artery were injured by balloon in diet-induced atherosclerotic rabbits. Methods (1) The experiment was conducted in the Laboratory of Institute for Integrated Traditional Chinese and Western Medicine, Sun Yat-sen University from May 2006 to June 2007. Twenty four male New Zealand albino rabbits, were randomised and divided into three groups, called control group, model group and drug group respectively. Each group contained eight rabbits. (2) Rabbits of control group were fed with common forage, but model group and drug group fed with high fat diet. Two weeks later, the iliac arteries were injured by balloon for model group and drug group. Meanwhile, drug group were fed with Jiawei Buyang Huanwu Decoction after the operation, 2 ml/kg/day, containing herbal drug 5 g/ml. (3) At the end of four weeks, the rabbits were killed. Serum samples were stored to assay the levels of cholesterol (total cholesterol (TC), high-density lipoprotein of cholesterol (HDL-C), low -density lipoprotein of cholesterol (LDL-C), and triglyceridemia (TG)), activity of superoxide dismutase (SOD) and levels of malondialdehyde (MDA). Injured iliac artery was fixed by neutral formalin to observe the endothelial hyperplasia by light microscope, and the results were analysed by picture analysis system. (4) Differences of measurement were compared with single factor analysis of variance as well as repetitive measurements analysis of variance. Results One rabbit died in every group during the trial, 21 rabbits were involved in the results analysis. (1) Light microscope showed that the vascular lumina were narrower, intima was thicker and there were more enormous arteriosclerosis plaques in model group rabbits than that of control group. The lumina were wider and intima was thinner in drug group than that of model group. Image analysis showed that the thickness of intima in model group exceeded that in control group and drug group (p<0.05). The area of lumina was smaller than that of control group and drug group (p<0.05). (2) The serum levels of TC, TG and LDL-C were higher in rabbits of drug group than that of control group (p<0.05), But lower than that of model group (p<0.05). the serum level of HDL-C was higher in drug group than that in control and model group (p<0.05). (3) The serum SOD activity in drug group was higher than that of model group (p<0.05). The serum MDA level in control group was lower than that of model group and drug group, and the serum MDA level in drug group is lower than that of model group (p<0.05). Conclusion Jiawei BHD has significant preventive effect on intimal hyperplasia and the development of atherosclerosis in rabbits with iliac artery injury, and the mechanism of which may be related to modify lipid metabolism, increase SOD activity and decrease lipid peroxidation to protect vascular endothelial function.