The Effective Of Proanthocyanidins In LPS-induced Vascular Endothelial Cell Injury

Atherosclerosis(AS) is a common disease which severely threatens human health. The incidence of atherosclerosis(AS) has increased in developed countries,even in China.The injury of vascular endothelial cells has been verified to play a key role in the early progression of AS.Lipopolysaccharides(LPS) are characteristic components of the cell wall of Gram negative bacteria,which stimulates VEC.Lipopolysaccharide is composed of three distinct domains,lipid A,a short core of oligosaccharide and the O-antigen polysaccharide.The lipid A domain is the bioactive component and is recognized during human infection.LPS was usually through two ways in vascular endothelial,direct or indirect.LPS can change cell morphology and function.The direct injury was that LPS promoted monocytes,neutrophils and T lymphocytes to combine vascular endothelial cells,through release of protease and oxygen free radicals directly damage VEC.LPS also indirectly injured VEC through humoral immune response,and it can accelerate the occurrence and development of AS.Proanthocyanidins(PC) which was extracted from grape seed was a kind of polyphenolic compounds with antioxidant,eliminating oxygen radicals activity.In our study,VEC stimulated with LPS was incubated with PC to detect if PC can inhibit the apoptosis of VEC which was caused by LPS and the change of several apoptosis genes.We can find a effective way to anti-atherogenic.Methods1.Cell cultureTissues from Human umbilical vein blood vessels was cultured and identified and passaged.Passages of 3-8 cells were utilized for vitro experiments.2.Cells were cultured in 4 groups: Control group:without any treatment;LPS group:Cells were transfected with LPS at the concentration of 1ug/ml for 24h in 37℃;low PC group:Cells were transfected with LPS at the concentration of 1ug/ml and 10mg/ml PC for 24h at 37℃;high PC group:Cells were transfected with LPS at the concentration of 1ug/ml and 20mg/ml PC for 24h in 37℃.3.MTTThe proliferation of VEC was assessed by MTT colorimetry.4.Flow cytometryFlow cytometry was used to detect the cell apoptosis.5.Total RNA was extracted from VEC with Trizol for reverse transcription and proliferation of cDNA according to the manuscriptures' instruction.Data were analyzed by computer and the results were expressed in RQ of Bcl-2 mRNA,BAX mRNA gene toβ-action.6.Total RNA was extracted from VEC with Trizol for reverse transcription and proliferation of cDNA according to the manuscriptures' instruction.Data were analyzed by computer and the results were expressed in RQ of Caspase-3 mRNA gene toβ-action.Results1.Primary culture of VEC was confirmed by immune histochemistry2.MTTThe proliferation of VEC was inhibited by LPS,but low and high PC can promote the proliferation of VEC.3.Flow Cytometry Detection show:Proanthocyanidins can effectively inhibit the apoptosis of VEC which is caused by LPS.4.PCR result indicate:Caspase-3 mRNA in LPS group was higher than control group.And,Caspase-3 mRNA in low and high groups were lower than LPS group.5.PCR result indicate:BCL-2 mRNA in PC group was higher than control group, which was much lower in LPS group.The BAX mRNA was opposite to BCL-2 mRNA.ConclusionsProanthocyanidins can effectively inhibit the apoptosis of VEC which is caused by LPS,which can protect blood vessel endothelium.