Research On The Inhibitory Effects And Mechanisms Of Flavonoids From The Leaves Of Carya Cathayensis Sarg.on Vascular Smooth Muscle Cells

Objective:To screen a monomer compound which has an inhibitory effect on the proliferation and migration in vascular smooth muscle cells from total flavonoids from the leaves of Carya cathayensis Sarg.and their five monomer compounds,namely cardomonin,wogonin,chrysin,pinostrobin chalcone and pinocembrin.To investigate the effect and mechanism of this compound on rat vascular smooth muscle cells.This experiment provides an experimental basis for developing an effective cardiovascular drug.Methods:(1)Rat vascular smooth muscle cells(rVSMCs)were primary cultured by tissue sticking method.The inhibitory effects of total flavonoids from the leaves of Carya cathayensis Sarg.and their five monomer compounds on the proliferation and migration in rVSMCs were investigated.Cell proliferation was measured by direct counting the cells and cell migration was determined using the wound healing assay.(2)The proliferation and migration in rat vascular smooth muscle cells(rVSMCs)were induced by angiotensin ?(Ang ?).The best concentration of Ang ? was confirmed by stimulating rVSMCs with 10,100,1000 nM Ang ? for 24 h.The best timing of Ang ? was confirmed by stimulating rVSMCs with the confirmed concentration of Ang ? for 12,24,48 h.Then the most effective monomer compound was screened by investigating the inhibitory effects of total flavonoids from the leaves of Carya cathayensis Sarg.and their five monomer compounds on the proliferation and migration in Ang ?-induced rVSMCs.Cell proliferation was measured by direct counting the cells and cell migration was determined using the wound healing assay.(3)The influence of the most effective monomer compound on expressions of p-Akt?Akt?p-p38MAPK?p38MAPK?p-ERK?ERK in Ang ?-induced of rVSMCs was measured by Western-blot and the underlying mechanism was investigated.The inhibitory effects of Akt?p38MAPK?ERK were analyzed by using selective inhibitors MK-2206,SB203 5 80 and PD98059,respectively.Results:(1)Total flavonoids and cardomonin could effectively inhibit proliferation in rVSMCs,with IC50 38.7 ?g/mL and 23.4 ?g/mL,respectively;total flavonoids,cardomonin,wogonin and chrysin could effectively inhibit migration in rVSMCs,with IC50 17.60 ?g/mL?17.18?g/mL?23.20 ?g/mL and 22.39 ?g/mL,respectively.(2)The proliferation and migration in rVSMCs were significantly induced by 100 nM Ang ?for 12 h(P<0.01).12.5 ?g/mL total flavonoids and 10 ?g/mL cardomonin for 12 h significantly inhibited Ang ?-induced rVSMCs proliferation(P<0.01)in a does dependent manner while 50 ?g/mL wogonin,chrysin and pinostrobin chalcone for 12 h slightly inhibited Ang ?-induced rVSMCs proliferation(P<0.05).12.5 ?g/mL total flavonoids,10 ?g/mL cardomonin,wogonin,chrysin,pinostrobin chalcone and pinocembrin for 12 h significantly inhibited Ang ?-induced rVSMCs migration(P<0.01)in a does dependent manner.(3)The expressions of p-Akt,p-p38MAP and p-ERK in rVSMCs were significantly induced by 100 nM Ang ? for 12 h(P<0.01)while those of Akt,p38MAPK and ERK had no obvious changes.37 ?M cardomonin for 12 h significantly inhibited the expressions of p-Akt,p-p38MAP and p-ERK in Ang ?-induced rVSMCs(P<0.01)in a does dependent manner while those of Akt,p38MAPK and ERK had no obvious changes.100 nM MK-2206,10 ?M SB203580 and 30 ?M PD98059 for 12 h significantly inhibited Angll-induced rVSMCs proliferation and migration(P<0.01)Conclusion:Among total flavonoids from the leaves of Carya cathayensis Sarg.and their five monomer compounds,total flavonoids and cardomonin can effectively inhibit proliferation in rVSMCs;total flavonoids,cardomonin,wogonin and chrysin can effectively inhibit migration in rVSMCs while cardomonin can most effectively inhibit proliferation and migration in rVSMCs.At the same time,total flavonoids and cardomonin can effectively inhibit proliferation in Ang ?-induced rVSMCs;total flavonoids and their five monomer compounds all can effectively inhibit migration in Ang ?-induced rVSMCs while cardomonin can most effectively inhibit proliferation and migration in Ang ?-induced rVSMCs.Cardomonin can significantly inhibited the expressions of p-Akt,p-p38MAP and p-ERK in Ang ?-induced rVSMCs in a does dependent manner while those of Akt,p38MAPK and ERK have no obvious changes,which indicates that the down-regulation of p38 MAPK,Akt and ERK phosphorylation might be,at least in part,involved in cardamonin-suppressed proliferation and migration induced by Ang ? in rVSMCs.MK-2206,SB203580 and PD98059 can all effectively inhibit proliferation and migration in Ang ?-induced rVSMCs,which further clarify the mechanisms.