心脉通颗粒对血管内皮细胞氧化损伤保护作用的实验研究

心脉通颗粒对血管内皮细胞氧化损伤保护作用的实验研究 心脉通颗粒对血管内皮细胞氧化损伤保护作用的实验 研究 Abstract 1.1 1.1..1 1.2..3 1.3..4 1.4..6 2 HE..8 2.1 .8 2.2..8 2.3.10 3 H OECV-304.12 2 2 3.1..12 3.2..12 3.3..13 3.4..15 4 H O ECV-304MDA SOD17 2 2 4.1.17 4.2.17 4.3.17 4.4.19 5 H OECV-304 TPA PAI-121 2 2 5.1.21 5.2.21 5.3.23 5.4.24 6 H O ECV-304ET-1 TMmRNA27 2 2 I 6.1.27 6.2.27 6.3.28 6.4.30 7..33 7.1.33 7.2.33..34..39 .41. 42 IIXMT Xinmaitong granulaVEC vascular endothelial cell H O Hydrogen peroxide 2 2 MDA malondialdehyde SOD superoxide dismutase MTT monotetrazolium tPA tissue plasminogen activator PAI-1 plasminogen activator inhibitor 1 1 TM thrombomoduline ET-1 endothelin 1 1 mRNA Message ribonucleic acid Bp Base pair OD Optical density PCR Polymerase chain reaction SPSS Statistical package of social science ICC immunocytochemistry III , , -1 200μmol?L4 : ? 15% DMEM 28h ? H O 2 2 - 1 : 15% DMEM24h 200μmol?L H O2 2 4h ?15% DMEM 30μg/ml -1 24h, H O 200μmol?L 4h ?15% 2 2 -1 DMEM24h 200μmol?L H O4h 2 2HE M TTS OD M DA TPA PAI-1 RT-PCR 1ET-1 TMmRNA , , -1200μmol?L 4hHE 2 5 H OECV-304 MTT 2 2 -1 0.716?0.016 200μmol?L H O4h , 0.321?0.015 2 2 ,0.571?0.013 0.545?0.011 H O ECV-304 2 2H O P0.01, P0.01 , 2 2P 0.05 H O ECV-304MDA SOD 2 2 IV -1 MDA SOD 1.092?0.089 nmol/ml 18.722?1.430 U/ml 200μmol?L H O2 2 ECV-3044h ,MDA 2.319?0.230 nmol/ml P0.01 , SOD 7.859?0.496 U/ml P 0.01 24h , H O4h, 2 2 MDA 1.337?0.135 nmol/ml 1.447?0.104 nmol/ml SOD 12.418?1.065 U/ml 16.395?1.046U/ml P 0.01, P 0.01 , MDA P 0.05 SOD P 0.01 H O ECV-304TPA PAI-1TPA PAI-1 2 2141.19?5.71 142.27?5.51 TPA PAI-1 132.24?3.08 91.61?1.80 TPA PAI-1 P0.01, P0.01 TPA 128.77?2.72 104.35?2.33 PAI-1 107.06?2.44 105.18?3.64TPA PAI-1P 0.01, P0.01 TPA P0.01 PAI-1 P0.05 H O ECV-304 ET-1 TMmRNA 2 2 ET-1mRNA , ET-1mRNA TMmRNA TMmRNA ET-1/β-actin 0.289?0.023 ET-1/β-actin 0.751?0.075P 0.01 0.431?0.0410.423?0.025 P 0.01 ,P 0.01 TM/ GAPDH 0.820?0.057 TM/ GAPDH 0.315?0.016P 0.01 0.714?0.027 0.722?0.021 P 0.01, P 0.01 ET-1/β-actin TM/ GAPDH P 0.051 2V3 MDASODMDA,SOD MDASOD 4 TPA PAI-1 PAI-1TPA PAI-1PAI-1 TPA 5 ET-1mRNATMmRNAET-1mRNATMmRNA : ; ; VI ABSTRACT Objective: The oxidative damage of vascular endothelial cell is considered to be the initial factor of atherosclerosis. It is tightly close related with coronary disease , hypertension and so on. The research of the prevention to vascular oxidative damage disease in Chinese medicine is gradually becoming the research hot spot in the field o f cardiovascular in integrated traditional Chinese and Western medicine. In this experiment, we observed the protective function of serum containing Xinmaitong granule to the umbilical vein endothelial cell oxidative stress injury from cellular level and molecular biology level, and discussed its possible functional mechanism. Method Prepared the serum containing the medicine of Xinmaitong granule and created the model -1 of endothelial cell oxidative stress injury by 200μmolL H OThe cells were divided into 4 2 2 groups: ? Control group: Raised 28h in DMEM nutrient fluid including 15% normal mouse serum.? Model group: Raised 24h in DMEM nutrient fluid includi ng 15% normal mouse serum, - 1 then gives 200μmolL H O ,observes 4h.? Danshensu group: Joins the density of 30μg/ml 2 2 Danshensu in DMEM nutrient fluid including 15% normal mouse serum to continue to raise 24h, -1 then joins H O f inal density is 200μmolL to irritate cell for 4h.? The Xinmaitong group: 2 2 Raised 24h in DMEM nutrient fluid including 15% mouse serum containing the medicine of -1 Xinmaitong granule, then joins 200μmolL H O for 4h. The cellular form, and density was 2 2 observed through inverted microscope and HE dyeing. The cellular viability by MTT method; the activity of superoxide dismutase S OD in cell nutrient fluid and malondialdehydeM DA content in the cell by colorimetry method, the content of TPA, PAI-1 by immunocytochemistry method; the expression of mRNA of element 1 E T-1 and thrombus adjustment protein TM in cell endothelial by RT-PCR methodResults: The morphologic change of vascular endothelial cell: the a rrangement of the cell is close in control group, and in the paving stone shape observed under the inverted microscopethe cell turnes to be round , falls off and the intercellular space broadens, the density reduces -1 obviously, the cell looks like spin dle-shaped, nucleus diffraction enhances after 200μmolL hydrogen peroxide affects 4h .With HE dyes the normal vascular endothelial cellular volume is VII obviously big, the arrangement is close, obviously 2 -5 nucleoluses. after oxidative damage, the cellular form is anomalous, the density reduces obviously, it is accompanied by the karyopyknosis, the nucleus disruption, when damaged seriously, the structure of the nucleus and cytoblaste cannot be distinguished clearly The influence of Xinmaitong granule that H O suppress the activity of ECV-304: The MTT 2 2 of cell extinction value in control group is 0.716?0.016, the cell extinction value is 0.321?0.015 -1 when the model group joins 200μmolL H O after 4h, it is indicated that the survival 2 2 percentage obviously drops, compared with control group. But the extinction values are respectively 0.571?0.013, 0.545?0.011 in the medicated serum with the Danshensu and Xinmaitong granule in advance. Tt is indicated that they can reduce the inhibitory action of H O 2 2 to the ECV-304. And the Danshensu group and the Xinmaitong medicine group are remarkably different from the H O model group P0.01, P0.01, but in the Danshensu group and the 2 2 Xinmaitong medicine group cells’ activities are so close P0.05. The influence of Xinmaitong granule to the quality of ECV -304MD and the SOD damaged by H O : the expression of MDA and SOD are respectively 1.092?0.089 nmol/ml, 2 2 -1 18.722?1.430 U/ml in control group ,when 200μmolL H O stimulates ECV-304 cell for 4h, 2 2 the MDA is 2.319?0.230 nmol/ml in model group”s nutrient fluid , the release is obviously higher than the controlled group P0.01, the SOD content is 7.859?0.496 U/ml and lower than the control group obviously P0.01Joins the Danshensu in advance and the medicated serum with Xinmaitong attentively for 24h , then joins H O for 4h, both the Danshensu group and the 2 2 medicine group with Xinmaitong cause the MDA content to drop obviously , the contents are respectively 1.337?0.135 nmol/ml, 1.447?0.104 nmol/ml; The SOD content increases obviously, the contents are respectively 12.418?1.065 U/ml, 16.395?1.046 U/ml P0.01, P0.01, the MDA content in both the Danshensu group and the Xinmaiton g group have no statistic difference P0.05. But in the Xinmaitong granule group the SOD content is higher than the Danshensu group obviously P0.01. The influence of Xinmaitong granule to ECV -304TPA and the PAI-1 damaged by H O : In 2 2 the control group, TPA and PAI-1 have low expression, the levels are respectively 141.19?5.71,142.27?5.51; In the model group, TPA and the PAI -1 expression increases, TPA and the PAI-1 expression quite remarkably increase compared with the control group P VIII 0.01, P0.01, in Danshensu group and the medicine group with Xinmaitong the expression of TPA and PAI-1 have a remarkable difference compared with the control group P 0.01, P0.01; also the TPA expression in Xinmaitong group are obviously more than that of the Danshensu group P0.01, the PAI-1 expression in Xinmaitong group and the Danshensu group are quite the same P0.05. The influence of Xinmaitong granule to the expression of the ET -1, TMmRNA in ECV-304 damaged by H OCompared with the control group for ET-1mRNA product band, the 2 2 expression increases obviously and be bright obviously, in the Danshensu group and Xinmaitong group the brightness of ET-1mRNA product band is situated between the model group and the control group; its expression in the mod el group reduces obviously. Compared with the control group, the band tunes to be dark obviously, the brightness of TMmRNA product band in the Danshensu group and the Xinmaitong group is situated between the model group a nd the control group; analysised by the gelatin photograph system scanning, the ratio of integral value of light density ET-1/β-actin in the control group is 0.289?0.023, compared with the control group, the ratio of integral value of light density ET -1/β-actin in the model group is 0.751?0.075, it is obviously increases P0.01. It is 0.431?0.041in the Danshensu group and 0.423?0.025in Xinmaitong group , they have remarkable difference compared with the model group P 0.01 P 0.01 in the control group the ratio of integral value of light density TM/ GAPDH is 0.820?0.057, the ratio of integral value of light density TM/ GAPDH in model group is 0.315?0.016, compared with the control group ,and obviously reduces P0.01 in the Danshensu group and the Xinmaitong group they are respectively 0.714?0.027,0.722?0.021, it has remarkable difference compared with the model group P 0.01 P 0.01In the Xinmaitong group and the Danshensu group, both the ratio of integral value of light density ET-1/β-actin and the ratio of integral value of light density TM/ GAPDH do not have remarkable difference P 0.05. Conclusions: 1 The H O may lead the vascular endothelial cellular form to injury, the cellular density 2 2 reduces. 2 The medicated serum with Xinmaitong granule may be poss ible to suppress the reduce of the activity of cell led by H O , to enhance the activity, and have no statistic difference with 2 2 IX Danshensu. 3 The H O may cause to increase MDA in the vascular endothelial cell and reduce the 2 2 SOD activity, the medicated serum with Xinmaitong granule may enhance the SOD activity by reducing the MDA. Thus it has the function of xida tion resistance and anti-injury. Compared with the Danshensu there is no statistic difference that the Xinmaitong granule reduce the expression of MDA, but Xinmaiton g granule is more superor to the salvia miltiorrhiza monomer in enhancing the SOD activity. 4 After vascular endothelial cell oxidative damage, the TPA and PAI -1 expression increases, but the PAI-1 increase is more remarkable, the Xinmaitong granule and the salvia miltiorrhiza monomer can display fibrinolysis by increasing the TPA expression and reducing the PAI-1 expression, the Xinmaitong granule do not have remarkable difference in reducing the PAI-1 expression compared to Danshensu , but the Xinmaitong granule is superor to salvia miltiorrhiza monomer in increasing the TPA expression. 5 Af ter vascular endothelial cell oxidative damage, the expression of ET -1mRNA increases, the expression of TMmRNA reduces, the Xinmaitong granule and the Danshensu ca n reduce the expression of ET-1mRNA and increase the expression of TMmRNA to protect the vascular endothelial cell, also among them there are no remarkable differences. Key Words: The Xinmaitong granule; Danshensu; Endothelial cell; Hydrogen peroxide X 1 ,, [1] 1.1 1.1.1, , VEC, , [2] ,VEC ,, , , VEC,; VEC,,, , , , , ET NOET 50nm ET-l Ang ? A VP ET-l; NO PGIET-l ET-1, , 2 [3]EH ,ET , ,ET-1 [4] , ET NO VEC, , - NO VEC , NO, ,NON OS L- L-Arg, ,NOS, ,,NO , , [5] NO, , , , 1,NO ,,NOS VEC ET NOEH : 1 ,; 2 ET , ; 3 , ; 4 5-HT; 5 ,ET NO PGI , 2ACh EDRF NO ,ET-1 , NO ,, [6] 1.1.2eNOS ,C HF, , NO,, eNOS [7] M I , eNOS MI [8] ,eNOS MI eNOSeNOS [9] ,Cattaruzza eNOS eNOS,eNOS ,eNOS [10] CHFVEC, C ECVEC , ETVEC, ,CHF CEC ET [11] ,CHF CEC ET 35 CHF NO ,ET,ACE,NO ET , ETNO ,NO/ ETNO ET NO/ ET , 1.1.3 ASVEC, EDRF AS,,AS , C- C RP ET C2, VS MC c-fos ,c-myc ,VSMC DNA,VSMC , AS ,,NO , eNOS NO , TNF L DL ,eNOS[12] NOS ;NOSL- NONO 1.2 1. 2. 1TM 49600TM sTMTM, 1? 1, , TM ,TM [13] , , pmol , , , ?, ET-1 , -1 ,[14],, ,AS [15], ,ET-1AS 1. 2. 2, ,, , caspase-3 ,3 , , ,“ ” , [16] , , , , 1. 2. 3 ,VEC, ,, VEC, ,, [17] VEC ,,, PA I t-PA t-PA PAI, t-PA , , , [18] ; PAIt-PA VEC, t-PAPAI ,t-PA PAI,,, [19] [20]1. 3 ? :“ , , , ” , 1. 3. 1 [21][22] 1 1P- , 4 , , , ,, , 1. 3. 2, oxi dized low density lipoprotein ,ox-LDL ,ox-LDL , ,, , ,, , ,, [23] ? ox-LDL PKC , ,PKC, [24]ECV304, Fenton , [25],,NOS, , ox-LDL1. 3. 3 ,, [26] , , ,,,, [27]8 ,, , [28] , , ox-LDL , 5 , MTT , , [29] ox-LDL H O 2 2 H O, 2 2 , 1 , [30]H O CR-1740, 2 2 , , , ? a H O, 2 21.4 6 7 2 HE 2.1 2.1.1 BIO-RADFRANCE S.A FORMA Heraeus NIKON2.1.2 DMEM GIBCOHEPES GIBCOAmesico2.1.3ECV-304 ,“” 2.2 2.2.1 DMEM DMEM 850ml 100U/ml 100?g/m l 8 NaHCO 3.7g PH7.0-7.2 1000ml 0.22?m320?D-Hanks KCl 0.4g KH PO 0.06g NaCl 8g NaHCO 0.35g Na HPO 2 4 3 2 4 0.12g 0.01g 1000ml 0.22?m 20?0.10.1g 100ml D-Hanks2h 4? 0.22?m 20? 1 ml99 ml 75 400 ml 2 HE PBSNaCl8.5g NaH PO ?2H O 0.52g NaHPO ?12H O 2 4 2 4 2 7g800ml ,1000ml1000mlPBS 0.02M001mol/L 2.2.2ECV-304 ,15%DMEM 37? 5%C0 95% 2 6 3 5 ,64×10 cells 2?: 10%DMEM28h - 1 ? H O : 24h ,200μmol?L H O4hH O 2 2 2 2 2 2 ;NIKON 2.2.3 HE HE1 2 PBS 3 ×5/ 3 . 4% 604 PBS 3 ×5/ 5 106 3 ×5/ 7 3 8 3 ×5/ 9 159 10 51 11 151 2PBS 3 ×2/ 1 37080%,90%1 ×2/ 95100 2 ×1/ 1 42 ×30/ 15, ,16 2.3 , , ,, , , ,, , , , ,“ ”, , ,, 1-2 HE He matoxylin Heosine E HE , , 2-53-4, ,, , [32], ET NO,, 10 ,[33]2+, Ca - ATP ,, , , , , 11 3 H OECV-3042 2 3.1 3.1.1 23.1.2 DMEM HEPES2DMSO sigma MTT sigma 3.1.323.1.3SD : 2005A031SCXK 2003 -0001 , 280?20 g 20? -25? 40%-60% 10h 3.2 3.2.1 DMEM D-Hanks 0.1 2MTT0.5gMTT100ml PBS30min0.5mg/ml 0.22?m 4? 23.2.2 50 SD 2 ××100.162g/ml SD 100g 1ml 2 11-2h 4?2h 3500rpm/min 15min56? 30min 0.22?m 20?12 3.2.3 ECV-304 ,15%DMEM 37? 5%C0 95% 2 3 5 15%, 5 962×10 cells 4?: 15%- 1DMEM28h ? H O : 24h ,200μmol?L 2 2 H O4h ?: 30μg/ml 24h, 2 2 -1 H O 200μmol?L 4h ?: 15% 2 2 -1 DMEM 24h H O 200μmol?L 4h 2 23.2.4 M TT 52×10 cells 96 20μlMTT 5mg/m l 37?4h DMSO150μl 10570nm OD ODOD OD 6 % 1- OD / OD×100% 3.2.5x s ? SPSS13.0α 0.05 3.3 MTT 570nmOA570 OD0.716?0.016 H O2 2 0200μmol?L -1 H O4h , ECV-304 OD0.321?0.0152 2, H O 55.125?2.144 2 2 OD 0.571?0.013 0.545?0.011 H O ECV-3042 2 13 20.185?1.948 20.437?1.018H O 2 2 P 0.01, P 0.01 , P 0.051 1 2 x s 1 H OECV-304 n 6, 2 2OD% 0.716?0.016 - ? 0.321?0.015 55.125?2.144 H O 2 2 ? ? 0.571?0.013 20.185?1.948 ? ? ? ? 0.545?0.011 20.437?1.018 ? ? ?P 0.01P 0.05P 0.01H O2 2 14 3.43. 4. 1 1 2 34 “ ”1 pH 2 15 1988“” 3. 4. 2 MTT MTT MTT [34],, MTT , , , , , ,, MTT ,15% , MTT , , , MTT , , MTT OD , , ,, H O ECV-304, 2 2, H O ECV-3042 2 16 4 H O ECV-304MDA SOD 2 2 4.1 4.1.1 24.1.2 DMEM HEPES2M DAS OD4.1.324.2 4.2.1 DMEM D-Hanks 0.1 24.2.2 24.2.3 24.2.434.2.5 MDA SODM DA, SOD, ,4.2.6x s ? SPSS13.0α 0.05 4 3 MDA,SOD 1.092?0.089nmol/ml 18.722?1.430 U/ml -1 200μmol?L H OECV-3044h ,MDA 2.319?0.230 2 2 17 nmol/ml P 0.01 , SOD 7.859?0.496 U/ml P 0.01 24h , H O4h, 2 2 MDA 1.337?0.135 nmol/ml 1.447?0.104 nmol/ml SOD P 0.01, P 0.01 , 12.418?1.065U/ml 16.395?1.046 U/ml MDA P 0.05 SODP 0.012 3 4 x s 2 H O ECV-304MDA SOD n 10, 2 2MDA nmol/ml SODU/ml 1.092?0.089 18.722?1.430 ? ? H O 2.319?0.230 7.859?0.496 2 2 ? ? 1.337?0.135 12.418?1.065 ? ? ? ? 1.447?0.104 16.395?1.046 ? ? ? ?P 0.01P 0.01H O P 0.05, P 0.012 2__ 18 4.44. 4. 1 react ive oxygen species, ROS[35] , ROS ,H O ROS, , Fenton2 2 , ,, ,,,, H O 2 2 [36] ROS, ROS , ; ,, , , , ,19 4. 4. 2 MDA,SODMDA , , MDA , [37] ,,MDA , SOD , SOD , -2- , O , ,MDA [38] SOD , , H O ECV-304MDA, SOD H O 2 2 2 2 ECV-304 , , , , A C B[37], ,,[38]-,ADP/O ,RCR, ?OH , [39] , , , [40] , LDL, ?? , , ,,H O, MDASOD 2 2 SOD, 20 5 H O ECV304TPA,PAI-1 2 2 5.1 5.1.1 25.1.2 DMEM HEPES2 TPA,PAI-1 SABC DAB 5.1.325.2 5.2.1 DMEM D-Hanks 0.1 PBS 25.2.2 25.2.3 25.2.435.2.41 2 PBS 3 ×5/ 3 . 4% 604 PBS 3 ×5/ 5 . 3% H O 302 2 6 PBS 3 ×5/ 7 . 0.5%Triton-100 821 8 PBS 3 ×5/ 9 . 5%BSA 2010 ,TPA PAI-11: 100- 10ul 1.5 ml 1000ul PBS150ul 4? 1 1PBS 3 ×2/ 12 37? 2013 PBS 3 ×2/ 14 SABC 37? 2015 PBS 4 ×5/ 16 DAB A B C 1.5ml 1000ul , 150 ul/ ,DAB2517 18 7 19 ,10 20 ,95%2 100%2 100%2 21 22 , 5.2.5 20 , 2 40×1048um 20 1-20 Leica ,DAB HE, , ,, 2010 SPSS 20 , 10 ? 22 10 : NAc / 2 Fc;Ac1/ NvD?RSE [Nv]Nv4265; D 4 2 2 ;RSE[Nv] 5%Fc ?[10 / ],512×512;40×10 N10 5.2.6x s ? SPSS13.0α 0.05 5.3 TPA PAI-1141.19?5.71 142.27?5.51 5 9 TPA PAI-1 132.24?3.08 91.61?1.806 10 TPAP 0.05 PAI-1 P0.01 TPA 128.77?2.72 104.35?2.33 PAI-1 107.06?2.44 105.18?3.647 8 11 12 TPA PAI-1P 0.01 PAI-1 P 0.05 TPA P 0.013 5 x s 3 H O ECV-304TPA PAI-1 n 10, 2 2TPA PAI-1 141.19?5.71 142.27?5.51 ? ? H O 132.24?3.08 91.61?1.80 2 2 ? ? 128.77?2.72 107.06?2.44 ? ? ? ? 104.35?2.33 105.18?3.64 ? ? ? ?P 0.01P 0.01H O P 0.05, P 0.01 2 2 235.45. 4. 1 PGI ,2Ang? VECPGI PGI, 2 2 c AMP, cAMP ,[41] PGI TXA , 2 2 , AT? ,AT? ,-AT?,VEC,AT? ,vWF VEC ,Weibel-Palade vWF VEC, VEC [42] ,vWF ,VEC TFC EPCRTM PIATTM t -PA1P AI-1 [43] NO PGI , 2 24 , 5- , , ? A T ? ,AT ?Xa ,,, /, vWF [44],5. 4. 2 TPA PAI-1 , , ,,tPA PAI,tPA ,tPA ,,, PAI-1379, PAI tPA, PAI tPA1? 1 ,tPA , , PAI , ,PAI tPA ,, H O tPA PAI-1 , , 2 2 [45] ,,[46] , “ ” “ ” , ,,[47] ,, ,“ , , ”“ ”,, TF vWF , NO , 25 [48] , tPA ,PAI , [49]PAI, / , [50]ECV304 ,,, [51] , , , , ; , , ;, , [52] TPA PAI-1PAI-1TPA/PAI-1 1PAI-1TPA “ ” “ ” PAI-1 TPA,,, 26 6 H O ECV-304 ET-1 TMmRNA2 2 6.1 6.1.1 2 PC 808ASTEC SCT6.1.2 DMEM HEPES2TrizolPCR TaKaRa6.1.326.2 6.2.1 DMEM D-Hanks 0.1 PBS 26.2.2 26.2.3 26.2.436.2.5 RT-PCR ECV-304 ET-1 TMmRNATrizol RNA ET-1 TM β-actin GAPDH primer premier5.0 Genebank4 , RNA cDNA -20? PCR, 50μL TM :94? 5min ,94?40s 61?40s 72?40s30 1 72? 5min ET-127 94? 5 min ,94? 30s ,62?45s ,72?45s , 40