Effects Of HCY At Different Concentrations On ENOS And ET-1 RT-PCR In Cultured HUVEC

PrefaceHHcy has been identified as an independent risk factor for AS. Up to now , the mechanism in atherogenesis of Hey is not clear . Endothelial dysfunction appears to play a major role in HHcy - induced vascular injury . So it has important clinical significance to investigate the mechanism in atherogensis of Hcy.As we all know , EC can induce both platelet antiaggregation and vasodilation by its production of NO and ET - 1 . When the endothelium was injured , NO decreased and ET - 1 increased . So the platelet antiaggregation became weaker . But the impaired endothelial function was the earliest phenomenon of AS . Some studies showed that NO is closely related with eNOS and ET - 1 is the major member of ET system . Other experiments showed that Hcy can reduce eNOS mRNA expression and NO level , but it can increase ET - 1 mRNA expression and ET - 1 level . So we suppose that HHcy can affect eNOS mRNA and ET - 1 mRNA expression . In this study , we tested eNOS mRNA and ET -1 mRNA in HUVEC cultured with different Hcy concentrations to investigate the possible molecular mechanisms of Hcy on atherogenesis .Materials and Methods(一) Materials1,Cells : HUVEC ECV.304 strain2, Reagent : Hcy , RPMI1640 medium , Trizol RNA extracted regent , RT - PCR kit , eNOS primer , ET - 1 primer , β - actin primer , PBS admoy juice , penicillin , stymcin .3, Apparatus : spectrophotometer , cell culture super - clean bench ,CO2 incubated trunk , low temperature super centrifuge , PCR meter , violet outside analysator , agarose gel electrophoresis system , horizontal strip electrophoresis apparatus , profound hypothermia box .(二) Methods1, Culture of cells : HUVEC (ECV. 304) were secondary cultured regularly in 1640 supplemented 10% FBS , in 5% CO2, at 37℃ and saturate humidity2, Groups of experiment ; HUVEC were cultured in culture benchs . When cells were at subconfluence , they were preincubated in fresh medium without FBS for 24 h , and then were devided into following groups at random on Hcy different concentrations . Such as 0. 00 , 0. 01 , 0. 10 , 0. 50 , 1. 00 , 5. 00 mmol/L . The group of 0. 00 mmol/L Hcy is the presence . Each group has five samples . All the cells were incubated for 24h.3,eNOS mRNA and ET -1 mRNA were assessed by RT -PCR..(1) Total RNA were extracted by Trizol ; (2) RNA were reverse transcribed into cDNA ; (3) Reversed - transcribed products were amplified with Tap DNA polymerase ; (4) PCR products were separated by agarose gel electrophoresis ; ( 5 ) The density of the amplified PCR fragments were analyzed by the gel documentation system and were expressed as ratios of eNOS and ET - 1 to theβ - actin band .4 ,statistical analysis ; All data were analyzed by software package of SPSS 12. 0 . Results were summarized with mean ± standard deviation . Statistical comparisons between groups were performed by the t — test and one - way ANO-VA . Values of P <0, 05 were considered significant .Results1, After HUVEC were exposed to Hcy at different concentrations for 24 h , eNOS mRNA expression was reduced significantly ( P < 0.05) , and it showed a dose — dependent manner .2, After HUVEC were exposed to Hcy at different concentrations for 24h ,ET - 1 mRNA expression was reduced significantly (P < 0.05) , and it showed a dose - dependent manner .DiscussionIt is known, normally, the vascular endothelium induces both platelet antiag-gregation and vasodilation by its production of NO and ET - 1. But a lot of experiments showed that Hcy impaired endothelial function through increasing ET -1 production and decreasing NO production.However, the half-life of NO is shorter , which is only 3-5 seconds. It is difficult to identify. And NO is synthesized from L - arginine by eNOS. So in this study we made eNOS mRNA expression instead of testing NO. Our experiment showed that incubating HUVEC with medium of different Hcy concentrations for 24h led to a dose - dependent decrease in eNOS mRNA expression . It proved that Hcy can inhibit eNOS mRNA expression. Other studies had the same results. In addition, various studies demonstrated that Hcy has oxidative stress mechanisms, as Hcy autooxidation leads to the formation of reactive oxygen species such as H2O2 and O2 . Those factors may inactivate NO,which leads to decrease eNOS bioactivity. But long - time inhibition of eNOS may induce AS. It suggested that low level of NO and eNOS is related to AS. At present, ET - 1 is the major member of ET system. Our study showed that, ET - 1 mRNA expression was reduced significantly with a dose - dependent manner, which is consistent with the results of recent researches in other countries.As we all know,there are two types of ET - Receptor-. ETA and ETB. ETA is mainly expressed in VSMC, mediates vessal contraction. And ETB is involved in the releasing of NO from endothelial cells, which results in vasodilation. Masaki pointed that under normal conditions , acting in an autocrine mechanism, ET - 1 is likely to bind with ETB - R in the EC membrane rather than ETA - R in the VSMC,which results in vasodialation instead of vaso - contraction as a whole. On the coutrary, studies in humane body had no such results. But the mechanisms of Hcy on ET in EC remain to be elucidated, and further investigations are urgently needed.