Study On The Mechanism Of Uremic Toxin Indophenol Sulfate Mediated Calcification Of Vascular Smooth Muscle Cells Through Notch1 Signal Pathway

Objective: To study the effect of uremic toxin Indoxyl sulfate(IS)on calcification of human aortic smooth muscle cells(HAVSMC),and explore the relationship between it and Notch1 signal pathway and its mechanism.Methods: HAVSMC was cultured in vitro and divided into groups according to the experimental requirements.(1)Detect the expression of Notch1 signal pathway related molecules: set the time gradient(0 h,12 h,24 h,48 h)and concentration gradient(0 mol/L,250 mol/L,500 mol/L,1000 mol/L),use IS to interfere with HAVSMC,and detect the expression of Notch1 protein and m RNA.According to the previous experiment,the time threshold and concentration threshold of IS action were selected to interfere with HAVSMC and detect the expression of RBP-JK and MSX2 protein downstream of Notch 1 signal pathway.(2)To study the role of aromatic hydrocarbon receptor(AHR)and organic anion transporter(OAT)in IS and Notch 1 signal pathway: divide HAVSMC into 6 groups,namely control group,IS group,OAT inhibitor,IS+OAT inhibitor,AHR inhibitor,IS+AHR inhibitor,and IS+AHR inhibitor,and detect the expression of Notch 1 protein.(3)To study the effect of IS on the phenotypic transformation of HAVSMC: HAVSMC was divided into control group and intervention group(500 mol/L of IS for 48 hours),and the phenotypic marker of smooth muscle cells α-smooth muscle actin(α-smooth muscle actin,α-SMA)and the expression of Runt-related transcription factor 2(RUNX2)protein and m RNA.(4)Detection of the effect of IS on the calcification of HAVSMC: HAVSMC was divided into 6 groups,namely control group,IS group,OAT inhibitor,IS+OAT inhibitor,AHR inhibitor,IS+AHR inhibitor,and alizarin red staining was used to observe the level of cell calcification in each group.(5)To explore the effect of IS on p53 expression: set the concentration gradient and time gradient of the action of IS,and intervene with OAT inhibitor and AHR inhibitor to detect the expression of p53 protein.Results:(1)IS inhibited the expression of Notch1 signal pathway related molecules.(2)OAT and AHR are involved in the process of IS reducing the expression of Notch 1 protein.(3)IS results in HAVSMC α-The expression of SMA decreased and the expression of RUNX2 increased.(4)IS promoted the calcification of HAVSMC,but the addition of OAT inhibitor and AHR inhibitor weakened the calcification level.(5)IS promotes the expression of p53 protein,and OAT and AHR also participate in this process.Conclusion:Through OAT and AHR,IS can reduce the expression of Notch1 signal pathway related molecules,increase the expression of p53,promote the osteogenic transformation of HAVSMC,and then cause calcification.