| Objective: 1.To establish a high performance liquid chromatography(HPLC)method for simultaneous determination of four phenolic components danshensu(DSS),protocatechuic acid(PCC),protocatechuic aldehyde(PCL)and salvianolic acid B(Sal B)in salvia decoction.2.To study the effect of these four phenolic components on the expression of intercellular adhesion molecule(ICAM-1)in human umbilical vein cells(EA.hy926)stimulated by sodium palmitate(SPA)and its mechanism.Methods: 1.Analysis was performed on a Waters Symmetry C18(4.6nm × 250 nm,5?m)column with gradient elution(0~7min,2%A?10%A;7~20min,10% A?23% A;20~35min,23%A?27%A;35~38min,27% A?38%A;38~42min,38%A;42~43min,38%A?2%A;43~56min,2%A)of acetonitrile(A)and 0.05% trifluoroacetic acid(B)at the flow rate of 0.8 mL/min.Detection wavelength was 288 nm,Column temperature was set at 25?.Then evaluated this methodology and measured the content of these four phenolic components in salvia decoction.2.Study the effects of four phenolic components on the expression of ICAM-1 induced by SPA in EA.hy926: The control group,the model group,the drug group and the positive drug control group were set up.The control group was treated with bovine serum albumin(BSA)(without SPA stimulation).The model group and the drug group were stimulated with SPA.At the same time,the four groups of phenolic components were given high,Medium and low dose,the positive drug control group was treated with probucol(Pro),each group were treated for 24 h.(1)The effects of SPA and four phenolic components on the survival rate,apoptosis and the formation of foam cells in EA.hy926 were studied by MTT assay,flow cytometry(FCM)and oil red staining.(2)The relationship of dose-course and time-course between SPA and ICAM-1 was studied by ELISA and western blot(WB).(3)The effects of four phenolic components on the expression of ICAM-1 in EA.hy926 stimulated by SPA were investigated by ELISA and WB.3.Explore the mechanism of EA.hy926 expressing ICAM-1 induced by SPA: The control group,the model group,TLR2 and TLR4 agonist(Lipopolysaccharide,LPS)group,TLR4 inhibitor(TAK-242)group and MyD88 inhibitor(ST2825)group were set up.The control group was treated with BSA,and in the model group,agonist group and inhibitor group were stimulated with SPA.At the same time,the agonist group and the inhibitor group were given high dose,medium dose and low dose of LPS,TAK-242 and ST2825,each group were treated for 24 h.(1)The effects of LPS,TAK-242 and ST2825 on the survival rate of EA.hy926 were studied by MTT assay.(2)The effects of LPS,TAK-242 and ST2825 on the expression of ICAM-1 in EA.hy926 induced by SPA were studied by ELISA and WB.Results: 1.In fourty minutes,the four phenolic components of DSS,PCC,PCL and Sal B in salvia decoction could be completely separated,and the linearity of the concentration and peak area was within a certain range,and the correlation coefficient r was over 0.9998.2.Increasing the concentration and time of stimulation of SPA,SPA reduced the survival rate of EA.hy926,promoted EA.hy926 apoptosis and promoted significantly EA.hy926 formation of foam cells.Four phenolic components could improve the survival rate of EA.hy926 and lower the formation of foam cell in EA.hy926 induced by SPA,Sal B and DSS lower the apoptosis of EA.hy926.Increasing the concentration and time of stimulation of SPA,SPA promoted the expression of ICAM-1 in EA.hy926.Four phenolic components reduced sharply the expression of ICAM-1,P65,MyD88,TLR1,TLR2,TLR4 and TLR6 in SPA stimulation group.Compared to SPA stimulation group,LPS increased the expression of ICAM-1,P65,MyD88,TLR2 and TLR4.TAK-242 could reduced the expression of ICAM-1,P65,MyD88,TLR4.ST2825 could reduced the expression of ICAM-1,P65,MyD88.Conclusion:1.the HPLC method is specific,quantitative and accurate,which is able to determine the content of four phenolic components in salvia decoction.2.SPA promotes significantly EA.hy926 formation of foam cells and increases EA.hy926 expression ICAM-1 which is possibly related with TLRs / MyD88 / NF-?B Pathway.Four phenolic components can improve the survival rate of EA.hy926 and lower the formation of foam cell in EA.hy926,Sal B and DSS inhibit the apoptosis of EA.hy926.These four phenolic components can inhibit the formation of foam cell in EA.hy926 induced by SPA,reduce the expression of ICAM-1,P65,MyD88,TLR1,TLR2,TLR4 and TLR6,which is probably associated with TLRs / MyD88 / NF-?B pathway. |
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