Calcification Of Vascular Smooth Muscle Cells Induced By High Phosphorus And Knockdown SET8

Backgroud and ObjectiveAt present,chronic kidney disease still has a high incidence rate,while the number of End stage renal disease(ESRD)patients is increasing gradually.Cardiovascular disease is the main cause of readmission and death of ESRD patients.Artery calcification is an independent predictor of all-cause and death in patients with cardiovascular disease(CVD).Prospective and cross-sectional studies of ESRD patients showed that the vascular calcification is a key link and independent risk factor for the increase of CVD incidence rate and mortality in ESRD patients.Therefore,it is of great significance to find out the factors and mechanism of intervening vascular middle membrane calcification,to prevent and delay the occurrence and development of vascular middle membrane calcification in ESRD patients,to reduce the mortality of ESRD patients,and to improve their quality of life.Hyperphosphatemia is the initiating factor among many factors that cause the calcification of vascular mesothelium.Hyperphosphatemia can induce the phenotype transformation and apoptosis of VSMCs in vascular mesothelium through various mechanisms,and then lead to the occurrence of vascular calcification.It is of great significance to find the mechanism of calcification induced by high phosphorus factors and take effective prevention and treatment measures for the corresponding targets to reduce the mortality of ESRD patients.The vascular middle membrane calcification is not a passive process,but a highly adjustable process mediated by cells.The apoptosis of vascular smooth muscle cells(VSMCs)is an important mechanism of vascular middle membrane calcification.SET8 is a lysine methyltransferase that can induce apoptosis.SET8 can methylate p53,twist,Wnt and other non histones,and affect the expression of corresponding genes through the regulation of transcription process,and then participate in the regulation of cell cycle,chromatin condensation and DNA replication.p53,the most important non histone substrate of SET8,is an important cell cycle and apoptosis regulator.At present,there are few studies on the role of SET8 in the calcification of vascular smooth muscle cells induced by high phosphorus at home and abroad.The purpose of this study is to investigate the effect of SET8 on VSMCs proliferation and apoptosis through p53 signaling pathway,to provide a theoretical basis for the prevention and development of vascular mesothelium calcification in advance,and to provide a new idea and target for the prevention and treatment of vascular mesothelium calcification in ESRD patients,so as to reduce cardiovascular events and all-cause mortality in ESRD patients.Materials and methods1.Primary culture of rat vascular smooth muscle cells(VSMCs):Select 100g-120g SD rats,take thoracic aorta for primary culture,3-4 generations of cells for experimental use,the third generation of VSMCs is covered with six pore plate,the cell fusion rate is 60%-80%,and then intervention can be carried out.2.Experimental group and intervention:In order to investigate the apoptosis and calcification of VSMCs induced by high phosphorus,VSMCs were randomly divided into normal group and high phosphorus group(10 mmol/L ?-glycerophosphate)and cultured for 4 days.In order to further explore the regulatory effect of SET8 on apoptosis,VSMCs were randomly divided into three groups:(1)SET8 shRNA group:transfection of SET8 specific shRNA.(2)Empty plasmid group:negative control shRNA was transfected;(3)normal control group:DMEM complete medium with 10%fetal bovine serum was added.3.RT-PCR and Western blot were used to detect the expression of SET8,p53,BCL2,Bax,Caspase3 genes and proteins in VSMCs after intervention.The expression of SET8 and caspase-3 was detected by immunofluorescence double staining.4.Alizarin red staining and calcium content were used to detect the biological indicators of VSMCs calcification after intervention.5.MTT and flow cytometry were used to detect the biological indexes of VSMCs proliferation and apoptosis.6.Spss19.0 statistical software was used for data processing.The normal distribution of measurement data was expressed as mean±standard deviation(x ±s).Single factor analysis of variance was used for comparison between multiple groups.SNK test was used for comparison between two groups.Pearson correlation analysis was used for correlation.P<0.05 was statistically significant.Results1.Apoptosis and calcification of vascular smooth muscle cells induced by high phosphorus.1.1.The primary cell identification of VSMCs:the cell SM22?immunohistochemical staining showed that the cytoplasm was brownish yellow,the actin in the cytoplasm was brownish yellow,distributed parallel along the long axis of the cell,the nucleus was blue,the SM22? staining was positive,indicating that the cell was vascular smooth muscle cell.1.2.The number of orange calcified nodules in the high phosphorus group was significantly higher than that in the normal group,and the calcium deposition was significantly increased(P<0.05).1.3.MTT method was used to detect the effect of high phosphorus on the proliferation of VSMCs.The results showed that compared with the normal group,the proliferation of VSMCs in the high phosphorus group decreased at 24h,36h and 48h,the difference was statistically significant(P<0.05).1.4.Effect of high phosphorus on apoptosis of VSMCs:flow cytometry showed that compared with the normal group,apoptosis of high phosphorus group increased significantly;correlation analysis showed that apoptosis and calcification were positively correlated,the difference was statistically significant(P<0.05).1.5.The effect of high phosphorus on the mRNA expression of VSMCs:the results of PCR showed that the relative mRNA expression of SET8 and Bcl-2 in high phosphorus group was significantly lower than that in normal group,while the relative mRNA expression of p5 3,Caspase3 and Bax was significantly higher(P<0.05).1.6.The effect of high phosphorus on the protein expression of VSMCs:Western blot showed that compared with the normal group,the relative protein expression of SET8 and Bcl-2 in the high phosphorus group was significantly reduced,while the relative protein expression of p53,Caspase3 and Bax was significantly increased(P<0.05).2.The role of interfering the expression of SET8 in calcification of vascular smooth muscle cells.2.1.After interfering with SET8,the expression of SET8 in SET8 shRNA group was significantly lower than that in empty plasmid group and normal control group(P<0.05).2.2.Calcification of VSMCs after interference with SET8:the orange calcified nodule in SET8 shRNA group was significantly higher than that in normal control group and empty plasmid group,and the calcium content of SET8 shRNA was significantly increased(P<0.05).2.3.MTT assay was used to detect the proliferative ability of VSMCs after interfering with SET8:after VSMCs were transfected with SET8 plasmid,the proliferative ability of SET8 shRNA group was significantly lower than that of normal control group and empty plasmid group at Oh,12h,24h,36h and 48h(P<0.05).2.4.Flow cytometry showed that the number of apoptotic cells in SET8-shrna group was significantly higher than that in normal control group and empty plasmid group(P<0.05).Correlation analysis showed that the apoptosis and calcification of VSMCs were positively correlated with the interference of SET8 gene expression(P<0.05).2.5.After interfering with the expression of SET8 gene,the mRNA expression level of Bcl-2 decreased,while that of p53,Caspase3 and Bax increased(P<0.05).2.6.After interfering with the expression of SET8 gene,the protein expression level of Bcl-2 decreased,and that of p53,Caspase3 and Bax increased(P<0.05).2.7.After interfering with the expression of SET8 gene,compared with the normal control group and the empty plasmid group,there was a small amount of SET8 expression in the cytoplasm of the SET8 shRNA group,and the fluorescence intensity was significantly reduced.There was a large amount of Caspase3 expression in the cytoplasm of the SET8 shRNA group,and the fluorescence intensity was significantly enhanced,the difference was statistically significant(P<0.05).Conclusions1.High phosphorus can reduce the expression of SET8 in vascular smooth muscle cells,induce apoptosis of vascular smooth muscle cells,and then cause vascular calcification.2.Interfering with SET8 expression can promote apoptosis of vascular smooth muscle cells,and then promote the occurrence of vascular calcification.