| Objective: animal experiments studies in earlier stage confirmed that the extract of Equisetum which obtained by water apozem and n-butanol had an effect on protecting vascular endothelium and improving atherosclerosis.To further confirm effective parts and anti-atherosclerosis mechanism of Equisetum, total flavone and phenolic acid of Equisetum was extracted through macroporous resin, and its effects on apoptosis and inflammatory reaction in ECV304 cells which induced by ox-LDL was observed.Methods:The First part1 Extraction and Isolation of EquisetumProper Equisetum pieces were weighed and its 12 times of 70% ethanol was added,which was heated to extract three times. It cost 2h every time. Then it was isolated and purified by distilled water,30% ethanol and 70% ethanol through the macroporous resin after filtration. To collect the eluate of 70% ethanol used as an effective part,which contents of total flavone and phenolic acid was detected.2 Detection of total flavone and phenolic acid in Equisetum.Quercetin and ferulic acid were diluted into different times of concentration and measured by ultraviolet absorption spectrophotometer. Then standard curve and regression equation were got. According to the regression equation, the contents of total flavone and phenolic acid were obtained. Extract of Equisetum that contained certain quality was quantified accuratly.Then dried it until it reached constant weight. The purity is the ratio of the quality of total flavone and phenolic acid to the quality of dry.3 preparation of culture fluid of total flavone and phenolic acid The concentrated solution of 70% ethanol extract was fully dissolved in DMSO (5%). Then DMEM was added to make its concentration were 1000μg/ml, 500μg/ml,250μg/ml respectively, sterilized by ultraviolet radiation,-20℃preservation. The Second part1 isolation, oxidation and identification of Low-density lipoproteinThe chemical precipitation was used to extract low-density lipoprotein. LDL was placed in 10μmol / L CuSO4 solution in 37℃for 24 h, after that it was placed in PH7.4 PBS for 24 h for dialysis. Ox-LDL was obtained after filtration, 4℃preservation. UV spectrophotometry was used to monitor the process and the level of oxidation. Coomassie brilliant blue law was used to determine its concentration.2 Culture and divide ECV304 cellsThe ECV304 cells were cultured in DMEM containing 15% calf serum. They were subculture by Trypsin digestion method. Cells were randomly divided into normal control group, injury group, 100μg/ml Equisetum extract group, 50μg/ml Equisetum extract group, 25μg/ml Equisetum extract group. Observe cells growth situation.3 Observation and detectionMorphological changes of cells were observed under inverted light microscopes. The activity of NO in the culture media and activity of NOS in ECV304 cells were measured sepretallly by nitrate reductase assay and chemical colorimetry assay. The content of IL-8 was measured by radio immunoassay. The rate of apoptosis and Caspase-3 protein related in ECV304 cells were detected by flow cytometry. Caspase-3 mRNA,iNOS mRNA, IL-8 mRNA,Lox-1 mRNA,Nox4 mRNA,Bax mRNA and Bcl-2 mRNA expression of ECV304 cells were detected by semi-quantitative RT-PCR. iNOS protein related in ECV304 cells were detected by western blotting.Results:The First partThe average level of Quercetin: 1.69 mg/g, the average level of ferulic acid: 1.23 mg/g, the average level of Equisetum extract: 2.92 mg/g. The purity of Equisetum extract is 63.28%.The Second part1 The isolation and oxidation of LDLThe Electrophoresis of LDL showed that it was a single strap and in the same line with human blood serum. The result of ox-LDL also was a single strap and its speed was almost twice times than LDL which showed the isolation and oxidation of LDL had succeeded. The curve plot of oxidization of LDL demonstrates that LDL was completely oxidized around 10 hours.2 Inverted microscope observationsUnder inverted microscope, the cells in control group grew well, there was no dead cells.The cells in injury group had obvious morphological changes: shrinkage, smaller and varied round, loose connections, and more loss cells. The conditions in Equisetum extract was better than injury group, expecially in 50μg/ml Equisetum extract group.3 detections related to inflammatory reaction3.1 The effects of Equisetum extract on the contents of NO in the culture mediaThe contents of NO in injury group was lower than that in control group (P<0.01). the expression of NO in Equisetum extract groups was increased compared with that in injury group (P<0.01).3.2 The effects of Equisetum effective parts on the activity of NOS and iNOS mRNA expression,iNOS protein expression in ECV304 cellsThe activity of NOS in injury group was lower while iNOS was higher than that in control group (P<0.01). the activity of NOS in Equisetum extract groups was increased while iNOS was decreased compared with that in injury group (P<0.01). The activity of iNOS and iNOS mRNA in injury group was higher than that in control group (P<0.01). the activity of iNOS and expression of iNOS mRNA in Equisetum extract groups was decreased compared with that in injury group (P<0.01).3.3 The effects of Equisetum extract on the content of IL-8 in the culture media and expression of IL-8 mRNAThe content of IL-8 and expression of IL-8 mRNA in injury group were higher than that in control group (P<0.01). the content of IL-8 and expression IL-8 mRNA in Equisetum extract groups was decreased compared with that in injury group (P<0.01).3.4 The effects of Equisetum extract on the expression of lox-1 mRNAThe expression of lox-1 mRNA in 50μg/ml Equisetum extract group was expecially decreased compared with that in injury group(P<0.01).3.5 The effects of Equisetum extract on the expression of Nox4 mRNAThe expression of Nox4 mRNA in 50μg/ml Equisetum extract group was expecially decreased compared with that in injury group(P<0.01).4 detections related to apoptosis4.1 The effects of Equisetum extract on the apoptosis rate in ECV304 cellsThe apoptosis rate in injury group was higher than that in control group (P<0.01) and it was decreased evidently in Equisetum extract group compared with the injury group (P<0.01). 4.2 The effects of Equisetum extract on the expression of Bax mRNA ,Bcl-2 mRNA in ECV304 cellsThe expression of Bax mRNA in injury group was higher than that in control group (P<0.01). Compared with the injury group, the expressions in Equisetum extract group was markedly lower (P<0.01). The trend of Bcl-2 mRNA was contrary to the above. The differences of Bax/Bcl-2 were more obvious among groups, the ratio was the highest in injury group (P<0.01), the ratio in Equisetum extract groups were significantly lower than that in injury group (P<0.01).4.3 The effects of Equisetum extract on expression of Caspase-3 protein and Caspase-3 mRNA in ECV304 cellsThe expression of Caspase-3 protein and Caspase-3 mRNA in injury group were higher than that in control group (P<0.01). And both were decreased evidently in Equisetum extract groups (P<0.01).Conclusion: Through this process, we got highly purified of total flavone and phenolic acid, which reached standard of effective part. Through the intervention of Equisetum extract in cell experiments we confirmed that Equisetum extract had a depressant effect on apoptosis and inflammatory reaction of ECV304 cells induced by ox-LDL. Equisetum extract played a role in anti-oxidative damage and remission inflammatory reaction through inhibiting gene expression of IL-8,iNOS,Bax,Caspase-3,Lox-1 and Nox4, which may be its mechanisms in anti-AS. |
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