The Effects Of Safflor Yellow On Proliferation Of The Vascular Smooth Muscle Cells And Expression Of Bcl-2/bax Protein

bjective: Smooth muscle cells are under endothelial cells of the intima.They Play a role in synthesis and secretion of Collagen and other extracellular matrix . They maintain the tension through slow and slight contraction of the vessel wall in the adult body with good health.Vascular smooth muscle cell proliferation is an indispensable condition for the hardening of the arteries.In recent years a lot of research about inhibiting VSMC proliferation in development of drug had been done. With in-depth study of cell and molecular biology,the maturity of vitro cell culture techniques and drug serum,a wide range of application in clinical intervention of coronary heart disease,many researchers had studied that Chinese medicine inhibites VSMCs proliferation mechanism from the level of cellular and molecular.But it hasn't found the exact method of control.We observed the effects of safflor yellow (SY) on angiotensinⅡ-induced vascular smooth muscle cells (VSMCs) proliferation, cell cycle and expression of bcl-2/bax protein,to explore safflor yellow on of the mechanism of inhibiting the proliferation of VSMCs. Thereby this provides a new method for the treatment of cardiovascular disease such as hypertension,atherosclerosis,restenosis after coronary intervention,it also provides a new way to develop safflor yellow.Methods : The primary culture was done by modified tissue-piece inoculation and identification:1,Clean class of SD rats,without limiting the male and female , weighing 120-150g(Luzhou Medical College Center for animal experiments), rats were killed by dislocating neck cone,removed for the thoracic artery segment under sterile conditions, access to clean and smooth vessel,endometrium and the outer membrane were removed, cut into the size of the middle membrane about 1mm3,tissue was placed in the bottom of glass bottle with a straw,the distance of tissue between 2 and 3 mm.after 4~6h cultured tissue adhesion bottle,added 20%fetal bovine serum of DMEM/F12 medium training,put it into 37℃,5%CO2 incubator to cultivate.After one week,a small number of cells from the edge of tissue growed,When cells were covering 80% of bottom ,they were passaged.Morphology of the cells was observed by inverted microscope and identified smooth muscle-α-actin (α-SM-actin) by immunohisto- chemical methods .2,We did the experiment with the fourth to eighth generation of cells,(1)The cell proliferation ability was measured by MTT assay,selected the cells growing well,made of cell suspension, adjusted concentration of 4.0×106/ml,Inoculated in cell culture plates with 96 holes,at 37℃,5%CO2 incubator,cultured 24 hours, abandoned to the medium,holes with cells in were randomly divided into five groups,each has the same eight holes,added the intervention factors as follow:①normal control group (10%FBS medium),②angiotensinⅡgroup(with a concentration of 1.0×10-6mol/lAngⅡmedium),③angiotensinⅡ(1.0×10-6mol/l) + low concentration of SY(0.2g/l),④angiotensinⅡ(1.0×10-6mol/l) + middle concentration of SY(0.6g/l) ,⑤angiotensinⅡ(1.0×10 - 6mol/l)+high concentration of SY(1.0g/l).Cultivated 72 hours,added methyl thiazolyl tetrazolium (MTT), continued to incubate 4 hours, terminated culture ,disposabled carefully supernatant in each hole, added dimethyl sulfoxide (DMSO) 150ul per hole,colorded,the enzyme-linked immunosorbent assay detected the Optical density of cell in each hole.(2)Flow cytometer measured the cell generation cycle:selected the cells growing well,inoculated into five training battles of 75ml,a bottle as a group,at 37℃,5%CO2 incubator,cultured 24 hours, abandoned to the medium,added serum-free DMEM/F12 medium,incubated 24 hours,went disposable medium.Added intervention factors with the same on,continued to cultivate,after 72 hours went disposable medium, washed twice with PBS,0.25% trypsin digested,inhaled cell suspension into Centrifuge tube with a straw , two centrifugation 1000prm×10min , the supernatant wes discarded,added PBS blending into a single cell suspension,adjustied the concentration of 2~5×106/ml,added RNASE digestion,PBS washing,added propidium iodide(PI) response around 30min,filtered,measured cell cycle by flow cytometry ,analysised the proportion of various stages cells in the detected cells.(3)We used immunohistochemistry to detecte expression of bax/bcl-2 protein:Cell suspension inoculated into the six-well plates which the slide had put in,at 37℃,5%CO2 incubator,incubated 24 hours,went disposable medium,added intervention factors with the same on,continued to cultivate,after 72 hours went disposable medium,washed twice with PBS,With 4% paraformaldehyde fixed,PBS washed,hydration,added A solution (H2O2) 10min,washed with PBS, added B fluid 15min,dumped,added anti-1 (bax 1:100; bcl-2 1:100),put in incubators,after 30min,put into 4℃refrigerator overnight,washed with PBS,Cooled to room temperature, added anti-two,put into incubators in 20min,added D fluid,put into incubators in 20min,added DAB reagent,timely termination,Hematoxylin-stained 5min,Gradient alcohol dehydration,xylene transparent,neutral gum mounting.Five vision of each group was selected randomly under microscope at 400 times.Pictures were analyzed by Image-Pro image analysis system.Relative content of bcl-2 and bax was expressed by average integra optical density (AIOD).We used SPSS11.5 packages,the data was showed by mean±standard deviation,used one-way ANOVA and Least-significance difference test (LSD) to analysis,P<0.05 as statistically significance.Results:From the experiment,we could observe that the passaged VSMC developed typically"peak and valley"growth pattern through the inverted microscope and expressed the smooth muscle specific differentiation markerα-SM-actin.OD value of AngⅡgroup than the one of normal control group was significantly higher (P <0.05),OD value of SY group than the AngⅡgroup was significantly decreased (P <0.05).Compared with normal control group,the percentage of AngⅡgroup in G0/G1 phase decreased significantly (P <0.05),the percentage of the one in the G2/M and S phase significantly increased (P<0.05),AngⅡcan induce vascular smooth muscle cells proliferation;compared with the AngⅡgroup,the percentage of VSMCs of each SY group significantly increased in the G0/G1 phase (P <0.05),the percentage of VSMCs significantly reduced in the S phase (P <0.05),the percentage of the middle-dose and high-dose group of VSMCs significantly reduced in the G2/M phase (P <0.05),the low-dose group of VSMCs was no significant difference (P> 0.05);Expression of bax/bcl-2 protein was measured by Immunohistochemical method: compared with AngⅡgroup,bax protein expression of SY Group increased significantly , expression of bcl-2 protein decreased significantly(P< 0.05).Conclusion:AngⅡcan promote the proliferation of vascular smooth muscle in vitro;Safflor yellow can inhibit AngⅡ-induced proliferation of vascular smooth muscle cells,furthermore it had a concentration-dependent;This may be completed by regulating the expression of bax/bcl-2 protein.