| Objective: The mortality rate of maintaining hemodialysis(MHD)patients is rising year by year,which cardiovascular disease is one of the main causes of death in patients with MHD and vascular calcification is the most common pathological manifestations.The mortality caused by vascular calcification accounts for about 30% of the total mortality of MHD patients.There are many risk factors of vascular calcification in MHD patients,such as hyperlipidemia,high phosphorus,age,anemia,and long dialysis age,ect.However,the traditional risk factors can not completely explain vascular calcification in MHD patients.Based on this,our previous study found that the acidic environment inhibits VSMCs calcification and the basic environment promoted VSMCs calcification.MHD patients due to 3 times per week dialysis,so that the p H after dialysis wave in 7.50-7.52.However,the effect of intermittent alkali stimulation on VSMCs calcification and the possible mechanism is not yet entirely clear.So this study will explore the effects and possible mechanism of intermittent alkali stimulation on VSMCs calcification by simulating the specific p H changes in patients with MHD.Methods:1 Primary culture and experimental group of VSMCsAdventitia stripping and then incised along blood vessels,sterile gauze wipe membrane using tissue explant method of primary cultured VSMCs,and were identified.After natural purification,the 4 generation cells were selected to carry out the experiment.The VSMCs were randomly divided into 6 groups:normal control group,high phosphorus+p H7.4 group,high phosphorus group+p H7.5,high phosphorus group+p H7.6,high phosphorus group+p H7.7and verapamil intervention group.After the intervention of 4d,m RNA and protein were extracted from each group,RNA and protein levels were detected,and the calcification was detected after 14 d intervention.2 Culture and experimental group of aortic ringsIsolated rat thoracic aortic rings,the aortic rings were randomly divided into 6 groups: normal control group,high phosphorus+p H7.4 group,high phosphorus+p H7.5 group,high phosphorus group+p H7.6,high phosphorus group+p H7.7 and verapamil intervention group.After 14 days intervention,the expression of LTCC ?3,SM22 and Runx2 were detected by immunohistochemistry.3 RT-PCR and Western blotting assay for the expression of each target gene and protein After 4 days intervention,extracted m RNA and protein from each group.Detected the expression of LTCC ?3 subunit and Runx2 by RT-PCR and Blot Western.4 Immunohistochemical method for detection of protein expression in aortic ringsFrom paraffin sections(3?m)conventional dewaxing and EDTA pressure cooker boiled antigen repair,3% H2O2 to eliminate endogenous peroxidase,goat serum blocking,drop one plus anti: LTCC ?3(1:50),Runx2(1:100),SM22?(1:100),keep 4? for the night;drops and the second generation of biotin labeled second antibody liquid and horseradish peroxidase(HRP)markers chain enzyme egg white element working liquid,DAB coloration,under the microscope,distilled water to stop the reaction,and hematoxylin staining,obvious brown yellow granules positive while coloring is negative.5 Determination of VSMCs calcification and ALP activityCalcium content was determined by BCA method after 14 days,and the content of calcium(mg/g protein)was standarded by protein content.Alizarin red staining was used to detect calcification: cells were cultyred in 14 days,alizarin red dye in 37? incubator for 30 min and the judgment standard of calcium salt deposition is orange.ALP activity assay: the activity of ALP was measured by the ALP activity kit,and the results was standarded by protein content.6 Determination of aortic rings calcificationAortic rings calcium content determination: vascular rings cultured for 14 days,then used Ketone of o-cresol complexing colorimetric method measured calcium content,and used mg/g as a unit.VonKossa staining:Vascular ring were conventional dewaxing and dehydration intervention in 14 days.Calcification of silver nitrate staining,add 5% silver nitrate solution,ultraviolet irradiation 60 min,5% sodium thiosulfate solution fixing,fuchsine redyeing,calcium salt deposition was dyed dark brown.7 Determination of calcium ion concentration in VSMCsAfter 4 days of cell culture,calcium ion probe was used to detect the concentration of calcium ion in VSMCs.8 Statistical analysisData analyses were conducted using SPSS 17.0 software.All results were expressed as mean ± standard deviation.Differences among groups were determined by analysis of variance(ANOVA),and the Student-Newman-Keuls method was determined by post-hoc testing.P<0.05 denoted a statistically significant difference.Results:1 Effects of intermittent alkaline environment on VSMCs calcification induced by high phosphorusVSMCs alizarin red staining and the calcium content determination results are consistent.Compared with the normal control group,calcium deposition of high phosphorus+p H7.4 group was increased(P<0.05).Compared with high phosphorus+p H7.4 group,calcium deposition of high phosphorus+p H7.5 group was increased(P<0.05).Compared with high phosphorus+p H7.5 group,calcium deposition of high phosphorus+p H7.6group was increased(P<0.05).Compared with high phosphorus+p H7.6 group,calcium deposition of high phosphorus+p H7.7 group was increased(P<0.05).Compared with high phosphorus+p H7.7 group,calcium deposition of verapamil intervention group was decreased(P<0.05).2 Effects of intermittent alkaline environment on aortic rings calcification induced by high phosphorusVascular rings VonKossa staining and the calcium content determination results are consistent.Compared with the normal control group,calcium deposition of high phosphorus+p H7.4 group was increased(P<0.05).Compared with high phosphorus+p H7.4 group,calcium deposition of high phosphorus+p H7.5 group was increased(P<0.05).Compared with high phosphorus+p H7.5 group,calcium deposition of high phosphorus+p H7.6group was increased(P<0.05).Compared with high phosphorus+p H7.6 group,calcium deposition of high phosphorus+p H7.7 group was increased(P<0.05).Compared with high phosphorus+p H7.7 group,calcium deposition of verapamil intervention group was decreased(P<0.05).3 Effects of intermittent alkali stimulation on the expression of Runx2 VSMCs and ALP activity induced by high phosphorusRT-PCR and Western blot results showed that compared with the normal control group,Runx2 expression of high phosphorus+p H7.4 group was increased(P<0.05).Compared with high phosphorus+p H7.4 group,Runx2 expression of high phosphorus+p H7.5 group was increased(P<0.05).Compared with high phosphorus+p H7.5 group,Runx2 expression of high phosphorus+p H7.6 group was increased(P<0.05).Compared with high phosphorus+p H7.6 group,Runx2 expression of high phosphorus+p H7.7 group was increased(P<0.05).Compared with high phosphorus+p H7.7 group,Runx2 expression of verapamil intervention group was decreased(P<0.05).ALP activity test results show that the intermittent alkali stimulation increased ALP activity(P<0.05),and verapamil inhibits ALP activity(P<0.05).4 Effects of intermittent alkali stimulation on the expression of Runx2 and SM22 in vascular rings induced by high phosphorusImmunohistochemical results show that compared with the normal control group,Runx2 expression of high phosphorus+p H7.4 group was increased(P<0.05),and SM22?was decreased(P<0.05).Compared with high phosphorus+p H7.4 group,Runx2 expression of high phosphorus+p H7.5 group was increased(P<0.05),and SM22?was decreased(P<0.05).Compared with high phosphorus+p H7.5 group,Runx2 expression of high phosphorus+p H7.6group was increased(P<0.05),and SM22?was decreased(P<0.05).Compared with high phosphorus+p H7.6 group,Runx2 expression of high phosphorus+p H7.7 group was increased(P<0.05),and SM22?was decreased(P<0.05).Compared with high phosphorus+p H7.7 group,Runx2 expression of verapamil intervention group was decreased(P<0.05),and SM22?was increased(P<0.05).ALP activity test results show that the intermittent alkali stimulation increased ALP activity(P<0.05),and verapamil inhibits ALP activity(P<0.05).5 Effects of intermittent alkali stimulation on the expression of LTCC ?3subunit and the effects of calcium ion on the VSMCs induced by high phosphorusRT-PCR and Western blot results showed that compared with the normal control group,the expression of LTCC ?3 in high phosphorus+p H7.4 group was increased(P<0.05).Compared with high phosphorus+p H7.4 group,the expression of LTCC ?3 in high phosphorus+p H7.5 group was increased(P<0.05).Compared with high phosphorus+p H7.5 group,the expression of LTCC ?3 in high phosphorus+p H7.6 group was increased(P<0.05).Compared with high phosphorus+p H7.6 group,the expression of LTCC ?3 in high phosphorus+p H7.7 group was increased(P<0.05).Compared with high phosphorus+p H7.7 group,the expression of LTCC ?3 in verapamil intervention group was decreased(P<0.05).Calcium fluorescence probe results show that the intermittent alkali stimulation increased calcium ion concentration in VSMCs,calcium ion concentration in verapamil intervention group was decreased(P<0.05).6 Effect of intermittent alkali stimulation on the expression of LTCC ?3subunit in vascular rings induced by high phosphorusImmunohistochemical results showed that compared with the normal control group,the expression of LTCC ?3 in high phosphorus+p H7.4 group was increased(P<0.05).Compared with high phosphorus+p H7.4 group,the expression of LTCC ?3 in high phosphorus+p H7.5 group was increased(P<0.05).Compared with high phosphorus+p H7.5 group,the expression of LTCC ?3 in high phosphorus+p H7.6 group was increased(P<0.05).Compared with high phosphorus+p H7.6 group,the expression of LTCC ?3 in high phosphorus+p H7.7 group was increased(P<0.05).Compared with high phosphorus+p H7.7 group,the expression of LTCC ?3 in verapamil intervention group was decreased(P<0.05).Conclusion:Intermittent alkaline stimulation promotes vascular calcification induced by high phosphorus in VSMCs.The mechanism may be related to the up regulation of the expression of LTCC ?3 by intermittent alkaline stimulation,which increases the influx of calcium ions and promotes the transformation of VSMCs into the chondrogenic phenotype. |
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