| Objective1.Screening active ingredient of plasminogen activator inhibitor-1(PAI-1)in five traditional Chinese medicines including,Salvia,Panax,safflower,Chuanxiong and Zedoary.2.Exploring the possible mechanism of Panax,Salvia,safflower,Chuanxiong,Zedoary by analyzing the interactions of each extract and protein PAI-1 column.3.Exploring the possible effect of the target component on the activity of PAI-1 protein and its poteicial mechanism.4.Exploring the possible role of the target component on PAI-1 protein,and observing the effect of the target compositions on secretory protein PAI-1 in normal human mesangial cells(HMC)and TGF-?1-induced HMC.Methods1.Frontal affinity chromatography with mass spectrometry(FAC-MS)was used to screen active ingredient of plasminogen activator inhibitor-1(PAI-1)in five traditional Chinese medicines.The extracts and five polar parts for petroleum ether,chloroform,ethyl acetate,butanol,water of the five traditional Chinese medicines as the sample to screen.Finding out target groups and determine their dissociation constant which are retained longer than corilagin and establishing the target component by analysising and comparing with control articles.2.To observe the target component' s effects on the activity of PAI-1 proteins by the direct reaction of target components and PAI-1.The PAI-1 protein standard solution were incubated for 45min at 37? with different concentrations of the target component.PAI-1 protein activity in solutions was detected by enzyme-linked immunosorbent assay(ELISA).3.The effect of target compositions on the activity of PAI-1 in Human mesangial cells was studyed.Four groups including blank control,TGF-b group(model group),TGF-?1 plus target compound and target compound group was set up.HMC were cultured with TGF-?1(5ng/mL)along(in model group),target compound of 5?g/mL,10?g/mL,20 ? g/mL,40 ? g/mL,80 ? g/mL,or TGF-?1 plus target compound.Cell proliferation was measured by MTT after treatment for 48h.4.PAI-1 protein activity and amount in cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA).5.Expression of PAI-1 mRNA in HMC treated as discribed above was detected RT-PCR.Results1.The results of FAC-MS screening show that FAC-MS can screen out a series target compounds which have longer retention time than corilagin using the binding affinities of components and affinity column.From more to less the order of the quantity of components which have strong bonding force with PAI-1 is Salvia,Chuanxiong,Zedoary,safflower,Panax.The most of the components of Salvia,Chuanxiong,safflower exist in ethyl acetate extract while the most of the components of Zedoary and Panax exist in petroleum ether extract.By comparison with the reference,two compounds have been recognized:Salvianolic acid B(Kd=14.3819 ?moL/L)and ?-linolenic acid(Kd=65.44 ?moL/L).2.Compared with the blank group,low concentration of salvianolic acid B and ?-Linolenic acid can enhance the activity of the PAI-1 protein.At 40 ? g/mL and 80 ? g/mL,salvianolic acid B while ?-Linolenic acid can inhibited the activity of the PAI-1 protein in dosage dependent manner.3.TGF-? 1(5ng/mL)protein can singnificantly inhibit the proliferation of HMC,increased activity,amount and express of mRNA of PAI-1 protein,indicating the selected HMC pathological model is feasible.4.Low concentrations of Salvianolic acid B and ?-linolenic acid could significantly inhibit the proliferation of cells while high concentration can significantly promote cell proliferation.5.Low concentration of Salvianolic acid B can significantly increase PAI-1 activity and the trendency is basically consistent with the direct action,with no significant differences in high concentrations;?-linolenic acid was significantly increased the activity of PAI-1 at low concentrations,a certain decreased at high concentration,but at 40 ? g/mL and 80 ? g/mL showed no strong inhibition effect.6.Compound with blank control,target compound group of two compents on PAI-1 protein has no effect markedly;compared with model group,the Salvianolic acid B of TGF-?1 plus target compound group have a significant difference at high concentrations by increasing PAI-1 protein content with concentration-dependent,?-linolenic acid also can increase the protein content,10 ? g/mL,40 ? g/mL,80 ? g/mL have significant differences.7.Compared with the control group,target compound group of two compents group have little change in expression of PAI-1mRNA,while ?-linolenic acid 80 ?g/mL showing upward trend.Compared with the model group,5 ? g/mL and 80?g/mL of Salvianolic acid B on PAI-1mRNA showed a upward trend,10 ? g/mL,20 ? g/mL,40 ? g/mL showed a downward trend,but both have no significant differences;?-linolenic acid 5 ? g/mL,10 ? g/mL,20 ? g/mL,80 ? g/mL both have upward trends,40 ? g/mL showed a downward trend,but also have no significant differences.Conclusion:1.FAC-MS can be quickly and effectively filter out the role of medicine in the target components.Chinese medicine is complex,which filter out specific targets for the role of the composition is very difficult,and FAC-MS method using strength between biological macromolecules and small molecules interacting with the sample specifically target the composition and role of the pro-screened out,greatly improving the speed of screening,for screening of active ingredients in traditional Chinese medicine provides an effective approach.2.It may be one mechanism to affect the activity of PAI-1 protein in blood circulation category of Salvia,safflower,Chuanxiong,Zedoary,safflower ethyl.Acetate fraction is the main active part in its role of PAI-1 of Salvia,safflower,Chuanxiong;while the main part of Zedoary is effective petroleum.Panax have the lest number of target compounds,suggesting a possible role of blood circulation of Panax is not primarily by affecting the activity of PAI-1 protein to achieve.FAC-MS results show that screening of the active ingredient,the efficacy of the medicine is worked by various components.3.Salvianolic acid B and ?-linolenic acid can act on PAI-1 protein directly and affect its activity in solution of PAI-1 protein.In cytopathology model of TGF-?1(5ng/mL)intervention,Salvianolic acid B and ?-linolenic acid can affect mRNA expression,content and activity of PAI-1. |
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