Effects Of Berberine On The Proliferation, Migration And Apoptosis Of Vascular Smooth Muscle Cells In Vitro

Objective: Percutaneous coronary intervention(PCI) with balloon or stenting angioplasty is an important and effective treatment for symptomatic coronary artery diseases.It is long-term efficacy,however it is limited by restenosis. Proliferation and migration of vascular smooth muscle cell(VSMC) play an important role in the pathogenesis of atherosclerosis and post-angioplasty restenosis.Remarkable progress has been achieved with the introduction of drug eluting stents.With the polymer-based slow releasing of the therapeutic agents into the vessel wall,they inhibit the smooth muscle cells overgrowth and migration after injury and could decrease neointimal formation and restenosis.The current clinical trials showed that the drug eluting stenting could achieve a 5–10% angiographic restenosis rate in comparison with the 20–25% for traditional bare metal stenting[1,2].Berberine is a well-known component of the Chinese herb medicine Huanglian(Coptis chinensis).We investigated the effects of berberine on the preliferation, migration and apoptosis of vascular smooth muscle cells(VSMCs) in vitro.Methods: We cultivate the vascular smooth muscle cells quiescent with 10% FBS in DMEM.They grow from 8th to 11th generation.The vascular smooth muscle cells are devided into two groups,the 24 hours group and 72 hours group.And there are four different groups in every time group according to various concentration of berberine:0μmol/L,25μmol/L, 50μmol/L,100μmol/L and 200μmol/L.Cell proliferation was determined by direct cell counting with trypan blue and methylthiazoletrazolium(MTT) staining.We use Boyden chamber to count the amount of migratory cells to detect effect of berberine on the migration of vascular smooth muscle cells.We detect the effect of berberine on the apoptosis of vascular smooth muscle cells by the terminal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL) and the flow-cytometer(FCM) technique.Results: Effect of berberine on the proliferation of vascular smooth muscle cell with trypan blue staining is:the amount of cells dealed with 0μmol/L,25μmol/L,50μmol/L,100μmol/L, 200μmol/L concentration of berberine for 24 hours is 3.51±0.54×108/L,2.65±0.03×108/L,2.17±0.04×108/L, 1.76±0.01×108/L and 1.68±0.05×108/L And the amount of cells dealed with 0μmol/L,25μmol/L,50μmol/L, 100μmol/L and 200μmol/L concentration of berberine for 72 hours is 5.39±0.94×108/L,3.64±0.03×108/L,2.40±0.06×108/L, 2.16±0.04×108/L and 2.01±0.07×108/L.It has demonstrated that:The quantites of cells of unique density of berberine group after 24 hours and 72 hours decrease vs control group with trypan blue staining(P<0.05).Effect of berberine on the proliferation of vascular smooth muscle cell with methylthiazoletrazolium staining is:the result of MTT in 0μmol/L, 25μmol/L, 50μmol/L, 100μmol/L, 200μmol/L concentration of berberine groups for 24 hours is 0.139±0.024, 0.061±0.014,0.058±0.005,0.057±0.005 and 0.056±0.005.The result of MTT in 0μmol/L, 25μmol/L, 50μmol/L,100μmol/L, 200μmol/L oncentration of berberine groups for 72 hours is 0.196±0.118, 0.060±0.013,0.057±0.007, 0.054±0.002 and 0.054±0.005.It can be concluded that the growth of vascular smooth muscle cells of every concentrations of berberine group is inhibited by both in 24 hours and 72 hours group vs control group(P<0.01). Effect of berberine on the migration of vascular smooth muscle cell using Boyden chamber technique is:the amount of cells that cutting through the cellulose nitrate membranes(CN) dealed with 0μmol/L,25μmol/L,50μmol/L, 100μmol/L,200μmol/L berberine for 48 hours is 50.61±12.11 cells/HP,31.23±2.61 cells/ HP,24.50±1.67 cells/ HP,20.39±2.25 cell/ HP and 19.52±1.64 cells/ HP.Therefore the migration of vascular smooth muscle cells of every concentration of berberine group is inhibited vs control group(P<0.05). The rate of TUNEL positive cells dealed with 0μmol/L,50μmol/L, 100μmol/L concentration of berberine for 48 hours is 13.15±1.83%,12.04±1.36%,10.62±1.21%,9.73±1.68% and 9.72±1.16%.It do not increase with the growth of the level of berberine(P>0.05) vs control group.The apoptosis ratio of 0μmol/L,50μmol/L and 100μmol/L concentration of berberine group for 48 hours is 20.4 %,18.7 % and 17.2 %.Conclusion: Berberine decreases the activity of proliferation and migration of vascular smooth muscle cells contrast to the control group.Berberine affect the proliferation and migration of vascular smooth muscle cells.But it have not significantly encouraged the apoptosis of vascular smooth muscle cells.