Study On The Effect And Mechanism Of Codonopsis Pilosula Polysaccharide On The Senescent Vascular Endothelial Cells Induced By Active Oxygen

Objective:Experiment ObjectivesTo study the protective effect of Codonopsis pilosula polysaccharides on EA.hy926 cells stimulated by low concentration of hydrogen peroxide,and to explore the difference and significance of epigenetic genes expression of EA.hy926endothelial cells during senescence.Methods:EA.hy926 cells were cultivated in vitro and H₂O₂ was used to make the model,and then vitamin E was used as the positive control group.The EA.hy926 cells were randomly divided into six groups,normal group and model group cells with low serum medium containing 2%fetal bovine serum,vitamin E group with low serum medium containing100?g/ml vitamin E.Codonopsis pilosula polysaccharides of low,medium and high groups were added with low serum medium which was contained Codonopsis pilosula polysaccharides in 50,100,200?g/ml.After 30 min cultivated in incubator,low serum medium containing 60?mol/L H₂O₂ was added in model group,vitamin E group and Codonopsis pilosula polysaccharides groups.The MTT method was used to detect the inhibition rate of cell proliferation,cell morphology was observed by optical microscope,and SA-beta galactosidase staining method was used to detect cells for the state of senescence and cell cycles were detected by flow cytometry.Besides,Colorimetry assay was used to detected the content of nitric oxide?NO?in cell culture medium and the content of SOD and MDA in cells.Enzyme-linked immunosorbent assay?ELISA?was used to detect the cell culture medium for the levels of endothelin-1?ET-1?,inducible nitric oxide synthase?i NOS?and endothelial nitric oxide synthase?e NOS?.DCFH-DA Fluorescent chromogenic method was used for the levels of intracellular reactive oxygen species?ROS?.What's more,DNA methylation of DNA methylation kit was used to detect the methylation level of DNA.And the epigenetic correlation protein DNMT1,JMJD3,EZH2 and Bmi-1 were detected by Protein imprinting method Western blot.Real-time quantitative PCR was uesd to detected epigenetic related genes DNMT1,JMJD3,EZH2 and Bmi-1.Results:?1?Detection of cell viability by MTT:Compared with the normal group,the survival rate of cells in H₂O₂ model group was significantly decreased?P<0.05?.Besides,compared with the H₂O₂ model group,the survival rate of cells in vitamin E group and the codonopsis pilosula polysaccharide groups were obviously increased,with statistical significance?P<0.05?.?2?Observation of cell morphology under inverted microscope:Compared with the normal group,the number of cells in H₂O₂ model group was significantly reduced,the cell morphology was irregular,and some exfoliated cells can be observed.Compared with the H₂O₂ model group,vitamin E group and Codonopsis pilosula polysaccharide intervention groups showed different degrees of improvement in cell morphology,and the number of cells was relatively high.?3?Detection of senescence level of cells by SA-?-galactosidase staining:Compared with the normal cells,the number of Senescent chromocyte cells in the H₂O₂ model group was obviously increased?P<0.05?.The number of Senescent chromocyte cells in the vitamin E group and codonopsis pilosula polysaccharide intervention groups were relatively few compared with the H₂O₂ model group?P<0.05?.?4?Detection of cell cycle by flow cytometry:The ratio of G1 phase in H₂O₂ model group was obviously higher than that in the normal cell group?P<0.05?.Compared with H₂O₂ model group,the ratio of G1 phase in vitamin E and codonopsis pilosula polysaccharide intervention groups was decreased?P<0.05?.?5?Determination of NO,SOD and MDA content in cells and Serum by Enzymatic colorimetry:Compared with normal group,the content of No and SOD in cell culture medium of H₂O₂ model group decreased significantly,while the expression of MDA in cells increased?P<0.05?.And the contents of NO and SOD expression in vitamin E group and codonopsis pilosula polysaccharide groups cells were increased than that in H₂O₂ model group,and the expression of MDA was decreased,with the difference of statistically significant?P<0.05?.?6?Determination of ET-1,i NOS and e NOS content in cultured cells by ELISA:The content of ET-1,i NOS in the cell culture medium of H₂O₂ model group was increased significantly compared with normal group,but the content of e NOS expression is significantly decreased?P<0.05?.Compared with H₂O₂ model group,the ET-1 and i NOS in serum of vitamin E group and codonopsis pilosula polysaccharide groups was reduced,and the expression of e NOS was increased,and the difference was significant?P<0.05?.?7?Detection of ROS content in cells by immunofluorescence:Compared with normal group,the content of ROS in the H₂O₂ model group increased significantly?P<0.05?.The contents of ROS in vitamin E and codonopsis pilosula polysaccharide intervention groups were lower than that in H₂O₂ model group?P<0.05?.?8?Detection of methylation level of extracted DNA by DNA methylation quantitative Kit:The cell DNA methylation level in H₂O₂ model group was obviously decreased compared with normal group?P<0.05?.And that in vitamin E and codonopsis pilosula polysaccharide high and medium dose groups were higher than that in H₂O₂ model group?P<0.05?.?9?Detection of the expression of epigenetic related protein DNMT1,JMJD3,EZH2 and Bmi-1 by Western blot:Compared with the normal cell group,the expression of DNMT1,EZH2 and Bmi-1 in the H₂O₂ model group was decreased,while the expression of JMJD3was increased?P<0.05?.The expression of DNMT1,EZH2,Bmi-1 protein in vitamin E group and codonopsis pilosula polysaccharide high and medium dose group was increased than that in H₂O₂ model group,while the expression of JMJD3 was decreased relatively?P<0.05?.?10?Detection of the relative expression of the epigenetic related gene DNMT1,JMJD3,EZH2,Bmi-1mRNA by Real-time quantitative PCR:compared with the normal cells,the expression of DNMT1,EZH2 and Bmi-1 in the H₂O₂ model group was relatively decreased,while the expression of JMJD3 was increased?P<0.05?.Compared with H₂O₂ model group,the expression of EZH2 and Bmi-1 mRNA in vitamin E and codonopsis pilosula polysaccharide intervention group was increased?P<0.05?,DNMT1 mRNA expression in vitamin E group and codonopsis pilosula polysaccharide high dose group was higher?P<0.05?,while the expression of JMJD3 mRNA in vitamin E group and high dose group was decreased relatively?P<0.05?.Conclusion:?1?According to the periodic inspection and oxidative damage index test,it can be concluded that codonopsis pilosula polysaccharide can scavenge free radicals,reduce oxidative damage and protect vascular endothelial cells,weaken the function of stress senescence induced by oxidation,and slow down the aging process of endothelial cells.?2?Based on the detection of epigenetic related proteins by Western blot technology and the detection of epigenetic related genes by real-time quantitative PCR technique,it was found that the degree of methylation and the expression of epigenetic related proteins and genes in aging EA.hy926 cells were changed.?3?The codonopsis pilosula polysaccharide has a function of delaying the aging of endothelial cells,and its mechanism is related to oxidative stress and epigenetic inheritance.