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Waterlogging stress is a common limiting factor for winter rapeseed, which greatly affects the growth and potential production. The present study was conducted to investigate the effects of waterlogging with different durations (0day (D0), 6days (D6) and 9days (D9)) and supplemental nitrogen fertilization (N1, 0 kg ha−1; N2, 30 kg ha−1; N3, 60 kg ha−1 and N4, 90 kg ha−1) on the physiological characteristics, dry matter and nitrogen accumulation in winter rapeseed (Chuanyou36). The results showed that the supplementary application of nitrogen fertilizer could effectively improve the physiological indexes of winter rapeseed in both pot and field experiments. The supplemental nitrogen increased the chlorophyll content in leaves, enhanced the activities of SOD, CAT, and POD, and decreased the MDA content in leaves and roots of rapeseed. The chlorophyll contents, the antioxidant enzyme activity of leaves and roots significantly increased under D6N3 and D9N4 conditions in both (pot and field) experiments. However, MDA contents significantly decreased compared with waterlogging without nitrogen application. Moreover, the application of nitrogen fertilizer after waterlogging increased the accumulation of dry matter and nitrogen in rapeseed at different growth stages. Therefore, waterlogging stress significantly inhibited the growth and development of rapeseed, but the application of nitrogen fertilizer could effectively reduce the damage of waterlogging. The N-induced increase in waterlogging tolerance of rapeseed might be attributed to the strong antioxidant defense system, maintenance of photosynthetic pigments and the nutrient balance.

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 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 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.