| Objective: The incidence of vascular calcification in patients with chronic kidney disease(CKD)is 10~30 times that of the general population.There was study found that acidosis environment can inhibit vessels and soft tissue calcification in renal failure rats,but the mechanism remains unclear.Activation of T cell nucleus factor was initially found in T cells because of its immune regulating function,research in the late showed that NFAT play an important role in osteoblast differentiation and vascular calcification.This study intends to observe in the inhibition of high phosphorus acid induced rat vascular smooth muscle cells calcification NFATc1 transcription factors play a role in the processMethods: 1 Animals Experiments 1.1 Preparation 24 male SD rats of clean level and 5-weeks-old were randomly divided into 4 groups: control group,chronic renal failure group,vascular calcification group,acid intervention calcification group.1.2 Model preparation judgment method rats were raised 5 weeks(adaptive feeding 1 week,model preparation + postoperative recovery 1 week,accept stimulation 3 weeks,a total of 5 weeks),serum creatinine(Scr)and urea nitrogen(BUN)level were tested,at the same time,p H and HCO3-concentration were tested.1.3 Detection of rat aorta using immunohistochemical staining method to detect the expression of Runx2 and NFATc1 in rat aorta;Using von Kossa calcification staining to observe vascular calcification;With reference to the method of literature,detection of calcium content in rat aorta tissue.2 Cells Experiments 2.1 Cell group VSMCs were randomly divided into 3 groups: normal control group,high phosphorus + p H7.4 group,high phosphorus + p H7.1 group.Using hydrochloric acid and sodium bicarbonate medium p H value,once every 24 hours in liquid.2.2 Using alizarin red staining methods test VSMCs calcium deposition;test calcium content in cells.2.3 Western Blot method to detect protein expression VSMCs purpose intervention after 4d cells extracted proteins,determination of NFATc1 and Runx2 protein expression.Turn the cell protein electrophoresis separation and membrane,closed,successively adding a diluent resistant(NFATc1 1:1000.Runx2 1:500 and GAPDH 1:50 00),two diluent(1:50 00),enhancement,analysis,after analysis with grey value.3 Statistical methods Using SPSS 21.0 statistical software for statistical analysis,if accord with normal distribution of measurement data,then adopt (?)±s said that,if the two independent sample mean,then analyzed by t test,and multiple sample mean compared with single factor analysis of variance,between two groups to compare with the S-N-K inspection.If P<0.05,the difference was statistically significant.Results: 1 1 of Chronic renal failure and vascular calcification group rats died respectively,and the other two groups all survived,and put all the rats to death on the 36 th day to take aim tissues.2 Effects of acidosis environment on vascular calcification in chronic renal failure rats: von Kossa staining results showed that there was no calcification in the control group and chronic renal failure group,aortic calcification can found in vascular calcification group,acid intervention can inhibit vascular calcification.The calcium content determination results consistent with the results of von Kossa staining results 3 Effects of asidosis on the expression of Runx2 in chronic renal failure rats:immunohistochemical score resuts showed that the expression of Runx2 in vascular calcification group was higher than the control group and chronic renal failure group.Acidosis intervention group can inhibit Runx2 expression.4 Effects of asidosis on the expression of NFATc1 in chronic renal failure rats: immunohistochemical score resuts showed that the expression of NFATc1 in vascular calcification group was higher than the control group and chronic renal failure group.Acidosis intervention group can inhibit NFATc1 expression.5 The correlation of NFATc1 and Runx2 expression in calcified vascular: correlation analysis showed that NFATc1 expression was positively related with Runx2 expression(r=0.877,P=0.877)in calcified rats blood vessels of rats.6 The effects of acidic environment on VSMCs calcification induced by high phosphorus of the rats: compared with normal control group,the high phosphorus+p H7.4 group seen more large orange calcium salt deposits.Compared with high phosphorus + p H7.4 group,high phosphorus+p H7.1 group seen less calcium salt deposition.Calcium content results similar to calcium staining.7 The effects of acidic environment on phenotype transformation of VSMCs induced by high phosphorus:Western Blot results showed that Runx2 expression level of high phosphorus+p H7.4 group was higher than normal control group,and acid intervention can inhibit Runx2 expression in VSMCs.ALP activity test results showed that the ALP activity of high phosphorus + p H7.1 group was obviously lower than high phosphorus + p H7.4 group(P< 0.05).8 The effects of acidic environment on NFATc1 expression in VSMCs induced by high phosphorus and the correlation of NFATc1 and Runx2 expression and ALP activity: Western Blot results showed that NFATc1 protein expression level of VSMCs of high phosphorus+p H7.4 group was higher than normal control group,and that acid intervention can inhibit NFATc1 protein expression in VSMCs.Correlation analysis results showed that expression NFATc1 and Runx2 expression in VSMCs were positively correlated(r=0.801,P=0.801),the expression of NFATc1 and activity of ALP also positively correlated(r=0.698,P=0.698).Conclusion: Acidic environment can inhibit vascular calcification,and its possible mechanism is by inhibiting NFATc1 expressiom and then inbibit vascular calcification. |
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