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1、Diosmetin Substituent in Galium verum L. Acting on Venous Thrombosis Lianrong Du Department of Food Science and Engineering Harbin Institute of Technology.HIT Harbin,China Weihong Lu* Department of Food Science and Engineering Harbin Institute of Technology.HIT Harbin,China Ge Wang The Fouth Clinic
2、al College of Harbin Medical University Harbin,China Haitian Zhao Department of Food Science and Engineering Harbin Institute of Technology.HIT Harbin,China AbstractGalium verum L. is a wild plant from Northeast China, this grass is well known as an excellent food source of vitamin, and it also cont
3、ains phytochemicals such as chlorogenic acid,salicylic acid and flavone. The present work evaluates the anti-venous thrombosis capacity of hydrophilic extracts of Galium. Anti-venous thrombosis activity values obtained for Galium were higher than those reported for other plant particularly rich in f
4、lavones. Diosmetin substituent compounds by high performance liquid chromatography (RPLC), as main factor responsible for anti-venous thrombosis activity, were determined. To investigation the proteome changes of the plasma, three groups of the wistar rats were selected, the controls group did not u
5、ndergo any processing, the model group and the prevention group were established thrombosis model in vitro and the difference between them was the prevention group had been given diosmetin substituent before which the model group hadnt. Plasma proteins were extracted by high velocity centrifugation
6、and separated by Sephadex column to remove the high-abundance proteins such as immunoglobulin. The low-abundance of proteins were separated by two-dimensional polyacrylamide gel electrophoresis and the images were analyzed by PDquest software.Comparative proteome was used in studying diosmetin subst
7、ituent of venous thrombosis, several differential expression of proteins were found, and the functions of these proteins and expression trends were analyzed. Clarify the thrombolytic mechanism of diosmetin substituent acting on the vein in order and find new substances with thrombolytic specificity.
8、 There may be providing a theoretical basis to find a new ideal thrombolytic substance and promote the treatment of venous thrombosis. Keywords-Venous thrombosis; Diosmetin substituent; Proteome; two-dimensional polyacrylamide gel electrophoresis I. INTRODUCTION Chinese doctors use Galium verum L. t
9、o treat veins of lower limb and it shows significant effectsPharmacological study indicated that it have anti-inflammatory, anti-thrombosis and anti-thrombosis activity in inferior vena cava.In our previous studies, we have obtained some components that have significant effects on treating phlebitis
10、 in Galium 1-2 . These components specially act on vein better than those acts on artery , such as rutin and Isoquercitrin. Five different flavones compounds were identified in the samples by means of HPLC and diode-array detection:diosmetin substituent,palustroside, diosmtin-D-glc (21)-L-ara, diosm
11、tin-7-o-D-glc and rutin. By means of solid phase extraction (SPE) three soluble polyphenolic fractions (phenolic acids, anthocyanins and flavonoids) were separated from the different sample extracts, and their respective anti-venous thrombosis activities calculated. Among them, diosmetin substituent
12、 and diosmtin-7-o-D-glc are the main contributors to the anti-venous thrombosis activity3-10. Hereditary anticoagulant protein deficiencies have important significance to the reasons of venous thromboembolism .According to the literature in recent years, PC, PS deficiencies, ant thrombin deficiencie
13、s (PROS1, PROC, SERPINC1) all result in the decrease or the loss of anticoagulant function of the body, and the incidence of venous thrombosis is 5-10%11.Studies from Japans situation have shown that defects in the formation of protein S is a major risk factor in venous thrombosis12. The study from
14、China Taiwans population also shows that the activities decreased of protein C and protein S is important risk factors in thrombosis. Meanwhile, activated protein C resistance has become the major risk factor of incidence of venous thrombosis in European ,and the molecular mechanism is Factor V Leid
15、en (R506Q) mutation, but the lower incidence in Asia 13. Researchers discover new anticoagulant and thrombolytic drugs constantly, our preliminary studies proved that the diosmetin substituent isolated from Galium verum L. is unique effective substance acting on venous thrombosis, which has a specif
16、ic affinity to the vein without affecting the arterial system. And there have not been reported Galium verum L.,acting on venous thrombosis abroad. The studies of Fibrinolytic drugs for the process and pathological significance at home and abroad sponsor acknowledgments：Innovation Fund of Harbin Ins
17、titute of Technology HIT.NSRIF.2008.18 The Program for Tackling Key Problems of Heilongjiangprovince GC07C360 978-1-4244-4713-8/10/$25.00 2010 IEEEwere related to protein C, protein S and antithrombin concentration, the generality of these studies is point in-depth and side limitations ,and proteomi
18、c techniques can discover functional proteins and drug targets in large-scale and high-throughput, change the drug target and discovery new drug model fundamentally. In recent years ,research in the cardiovascular proteome has identified more than 40 kinds of protein level changes, the literature on
19、ly focused on thrombin, pulmonary embolism diagnosis and the study of platelet fibrinogen receptor genotyping, whereas less of Proteome Research in venous thrombosis. Therefore, to explore the process and mechanism of Galium verum L. in the overall perspective of proteome ,in the comparative study o
20、f proteome of diosmetin substituent which acting on venous for a series of different expressed proteins, find a certain specific target protein which have function of antithrombotic and to analyze the function of these proteins and the trend of clear the effect and mechanism of diosmetin substituent
21、 acting on venous will probably look for a new ideal for the thrombolytic substances, and provide theoretical basis for enhance the effect and the specific of thrombolytic. For the development and utilization of Galium verum L., it provides a good application prospects. II. MATERIALS AND METHODS A.
22、Wistar rats selection and establishment of vein thrombosis model Three months healthy Wistar rats (20020g) were taken as experimental subjects, animals were randomly divided into 3 groups including control group, model group and prevention group, they were breed as the same condition. Previous studi
23、es have determined the best efficient injection dose of diosmetin substituent, 10mg/kg medicine were given to prevention group rat every day by intraperitoneal injection, for 2 weeks cautiously. Then establishment of Rats Deep Vein Thrombosis Model in model group and in prevention group by chandler
24、ligation methods in vitro and deal with nothing to the control group. Electron microscopic observation was used to determine if the vein thrombosis was established successfully in vascular wall. B. Total Protein Preparation Inferior vena cava blood was abtained from each group respectively, includin
25、g thrombosis ,and Blood was collected in Ep tube that containing sodium citrate solution (9:1), shaken up immediately and avoided foam formation. After 30 minutes in 4 refrigerator, plasma was centrifugation at 3000 rpm for 5min at room temperature. The supernatant was then centrifugation at 10000 r
26、pm for 8min at 4 ,collected this supernatant and packaging tags ,then frozen rapidly in a well-closed plastic container at 70C, do the same way to the three groups. C. Depletion of high-abundance proteins Plasma contains a large number of high-abundance proteins, in order to obtain clearly two-dimen
27、sional polyacrylamide gel electrophoresis atlas, the following program was used to removed high-abundance proteins: with the Sephadex G-75 column chromatography , the mobile phase was phosphate buffer, the detection wavelength was 280 nm, ,the flow rate was 0.2 ml/min. plasma protein was initial sep
28、arated by molecular weight, fraction of high-abundance protein was removed and the low-abundance protein was precipitated with precooled solution of 10% TCA for 1 h at -20. After washing with ice-cold acetone, the proteins were resolublized in the sample buffers of 2-DE,or the fractions was dialysis
29、ed, freeze-dried low-abundance protein and recovere samples. Protein concentration was estimated with the Coomassie brilliant blue method. D. Establishment of Plasma 2-DE and image analysis Protein Separation: first to the solid-phase pH gradient isoelectric focusing (IEF), 7cm strips was selected (
30、pH 3-10 NL, pH 4-7 L) and sample volume is 200ul that contain 350ug low- abundance protein .After 12 hours of active rehydrated at 18,the program has been set:250V for 30min,500V for 30min,1000V for 1 h,4000V for 3h and 4000V for 20000Vh.Then the strips was placed on the dry and thick filter papers,
31、 sip up the mineral oil and redundant samples with wet filter paper.Equilibrated two times 15min respectively and transfer the strips to the second vertical slab SDS-PAGE, confer upon the strips with low melting point agrose ,and 50V for 30min ,120V until the end. Stain: The gels were stained with b
32、lue silver to get significantly differences proteins. Image Analysis:the gels were scanned by a UVP scanner ,the changes of protein expression content and the differential expressions of protein spots were analyzed with Bio-Rads PDQuest software .Stained gel images collected by the scanner, PDQuest7
33、.4.0 software was used for analysis gels intensity correction, point measurement, background reduction, homogenization, matching and so on .Protein molecular weight, isoelectric point and the match rate of the spots between gels were determined by means of this method.The relative intensity of the p
34、ercentage of protein spots were used for comparison between groups.Increase or decrease 200% were defined as differential protein spots. Proteins Molecular weights were determined by standard protein markers and pI were determined by the protein spots on IPG strips. Statistical Analysis: data was ex
35、pressed by mean, data were analyzed by SPSS statistical software, when P 0.05 have statistically significant. III. RESULTS A. Establishment of Thrombosis Model Slit the white lines the abdominal peritoneum, separation of vein and inferior vena cava at the bottom of left renal, ligatured with the thi
36、ck silk, and sutured the abdominal wall. As the venous blood flow was blocked, venous thrombosis after 6h.The image is shown in Figure 1 as follows: Figure 1. The establishment of the Thrombosis Model. B. The images of electron microscopic observation Electron microscopic observation revealed that t
37、hrombus did not well formed in the control group and the prevention group , however the formation of thrombus in the model group are very well. This suggests that the establishment of Rats Deep Vein Thrombosis Model is successful. They are shown as follows: Figure 2. Electron microscopic observation
38、 of vascular wall of the control group Figure 3. Electron microscopic observation of vascular wall of the model group Figure 4. Electron microscopic observation of vascular wall of the prevention group The results of electron microscopic observation of vascular wall show the control group does not f
39、orm thrombosis (Figure 2), and thrombosis appears in the model group (Figure 3), while the prevention group does not form thrombosis (Figure 4). This indicates that protein C, protein S and antithrombin in endothelial cell protein C pathway had a certain influence on blood coagulation. PC, PS and an
40、tithrombin deficiency will result in decreased function of the body or loss of anticoagulant, which will lead to the formation of venous thrombosis. Thrombosis is formation in he model group indicates that the establishment of venous thrombosis model in vitro is successful, while the prevention grou
41、p does not formation of venous thrombosis, indicating diosmetin substituent plays an excellent role in preventing venous thrombosis, which are consistent with the results of previous studies. C. Comparison of PAGE-SDS imagines among different groups In order to determine the efficient of the removal
42、 high-abundance protein, and explore the differences between three groups of proteins express, PAGE-SDS electrophoresis is conducted and the result is shown in Figure 5 at below: Figure 5. PAGE-SDS imagines among different groups From the PAGE-SDS image of three groups we can see that the efficient
43、of removal high-abundance protein is well compare to the total plasma .Compare the model group with the prevention group, it can conclude that the model group has two protein bands which expressed differentially, and expressing quantity of protein is high ,while the prevention group does not have pr
44、otein expression, which is provided evidence for the next two-dimensional electrophoresis, and it is laid a foundation for treatment of venous thrombosis with diosmetin substituent. D. Comparison of 2-DE images among different groups After the treatment of remove high-abundance proteins, the separat
45、ion and enrichment of the low-abundance proteins better than before and improve the clarity of the spots .A stable 2-DE separation conditions is established, 350g of low-abundance proteins is used for 2-DE separation respectively. Figure 6 shows the controls sample of 2-DE gel. Figure 6. Example 2-D
46、E gel of the master Comparative and analysis 2-DE gel images of the control group, the model group and the prevention group, there are 204 protein spots in the Master gel ,and 33 expressed differential proteins protein spots were identified in the model group , where 18 differently protein spots inc
47、reased expression and 15 decreased expression; There are 26 differently proteins spots were identified in the prevention group , where 16 expressed differential proteins spots increased expression and 10 decreased expression. IV. DISCUSSION The composition of proteins possesses more than ten thousan
48、d forms at least, and most of them are existence of low-abundance. Disease markers in the disease process are the low-abundance proteins which were secreted into the plasma by the body .High-abundance proteins in plasma in the process of carrying out 2-DE will interfere with the low-abundance protei
49、n spots, for in this experiment Sephadex G-75 was used to separate high-abundance and low-abundance proteins, two peaks were obtained which were detected by UV analyzer for first time. All the low-abundance proteins extraction was collected and desalting, then concentration, then high concentration
50、of low-abundance proteins were obtained. 2-DE images analysis shows it improves the chances of identification of low-abundance proteins, particularly the chances of identifying biomarkers, which provides technical support for plasma proteome study. The establishment of venous thrombosis model in vit
51、ro is the key to the experiments, thrombosis model was established through the ligation of left renal vein of inferior vena, deal with nothing to the control group, the model group is establish thrombosis model directly. While the prevention group was administration for half month and then establish
52、 the thrombosis models, thrombosis were detected by electron microscopic observation of vascular wall .The time of ligation and the dose of drug is a key factor in thrombosis, the effect of thrombosis is well when ligation time is 4-6 hours and the dose of drug is 0.5g/kg.day.The reasons are protein
53、 C, protein S and antithrombin in endothelial cell protein C pathway had a certain influence on blood coagulation , PC and PS are vitamin K-dependent anticoagulant protein, which play an important role in the coagulation adjustment process.Protein C has been hydrolyzed to activated protein C by Thro
54、mbomodulin (TM) and the complex of thrombin. in the participation of PS. APC inactivation of activated coagulation factor V (Va), reduced pro-coagulant activity in the body when Ca2+ and phospholipids existed.While PS can also be directly reversible combination with pro-coagulant factors FVa and FXa
55、, thereby inhibiting the activity of prothrombin complex concentrate directly. With the development research of drug proteome, people will re-understanding the laws of life activities at the protein level, such as growth, development and metabolic regulation. It provides an important theoretical bas
56、is for the study of the mechanisms of major diseases, prevention and development of new drugs which accelerate the development of new treatments significantly. Therefore, this study selected the appropriate ways to deal with the plasma samples, use 2-DE strategies, study the control group ,model gro
57、up and prevention group of plasma samples of comparative proteome respectively. The experimental results obtained were analyzed with biological informatics databases ,access to Plasma Proteome differential expression quantity of proteins, type and expression of information. Present study, two-dimens
58、ional electrophoresis has been found differences expressed protein between the model group and the prevention group .The experiments will continue to study the changes of differences protein expression in-depth, high performance liquid chromatography and mass spectrometry will be used to determine t
59、he protein content and analysis the protein structure and function , and hope to find specific proteins which have the impact on venous thrombosis, which lay a certain foundation for identify plasma markers of venous thrombosis, provide theoretical basis and technical guidance for the early preventi
60、on of venous thrombosis. ACKNOWLEDGMENT We thank collaborators of the Life science and Engineering lab of Harbin Institute of Technology for important suggestions. Thanks Xin-Zhu Wang who working in Biochemistry Department, Imperial College. 1 Youchang Zhu ,Medicinal plants in the Northeast. Heilong
61、jiang Science and Technology Press,1989,pp.1055. 2 Gang Tan, Treatment of Deep Venous Thrombosis by Galium verum L.Injection in Clinical Research,China Science and Technology Information Institute, Chinese Dissertation Database ,2000. 3 Weihong Lu, Xiaomin Yu, Yingli Ma, Determination of Diosmin and
62、 Palustroside in Galium perum Lfrom Difierent Areas by HPLC, Vol.24 , Chinese Wild Plant Resources,2005,pp.62-64. 4 Weihong Lu,WANG Jing,Xiaomin Yu, Separation and Purification of Diosmin of Galium perum by Macroporous Resins, Vol.18 Nat Prod Res Dev, 2006,pp.896-899. 5 Weihong Lu ,Fan Yang,Xiaomin
63、Yu ,Guoyu Shi, Determination of Different Origin Galium perum L,Chlorogenic Acid use HPLC. Vol.24 ,Traditional Chinese Medicine Information,2004,pp.59-60. 6 Mingyu Cui ,Youliang Ji ,Lianjie Su,Weihong Lu,Chemical Composition and Pharmacological Effect of Galium verum L. , Vol.23 , Foreign Medical .
64、Plant Med,2008,pp.112-114. 7 Yingli Ma, Weihong Lu ,Xiaomin Yu,The Chemical Constituents of Galium verum L. , Vol.36,Herbs,2005,pp.1464-1466 8 Weihong Lu,Xiaomin Yu,Yingli Ma , Research The Effective Site, the ActiveIngredient and the Fingerprint of Medicinal Plants Galium verum L.,Natural Product R
65、esearch and Development,2006,PP.1-4. 9 Jing Ren , Weihong Lu, Weiming Wang ,The role of blood system Geranium Wood glycoside on tail-suspended rats, Vol. 34,Journal of Traditional Chinese Medicine,2006,pp.50-51 10 Weihong Lu,Xiaomin Yu, Study on Medicinal Herb-Galium verum L. Antibiotic,Anti-inflamm
66、atory Activity,Compounds and Fingerprint. Nat Prod Res Dev, 2006，pp. 1-4,8. 11 Tsuda H, Hattori S, Tanabe S, Iida H, Nakahara M, et al,Screening for aetiology of thrombophilia: a high prevalence of proteinS abnormality,vol.36, Ann Clin Biochem, 1999,pp.423- 432. 12 Shen MC, Lin JS, Tsay W, High prev
67、alence of antithrombin III,protein C and protein S deficiency, but no factor V Leiden mutation invenous thrombophilic Chinese patients in Taiwan.vol.87, Thromb Res ,1997,pp.377-85. 13 Shen MC,Tsay W, Lin JS ,Protein C and protein S deficiencies are the most important risk factors associated with thrombosis inChinese venous thrombophilic patients in Taiwan. Vol.99, Thromb Res,2000,pp.447-452.
