Abstract
To observe the protein expression of Notch1 in the cultured calcified human heart valve interstitial cells (hVICs) in vitro and related mechanisms. hVICs were divided into two groups: control hVICs were cultured in conventional media for 14 days and calcified hVICs were cultured with calcification inducers: β-glycerophosphate (500 μl), ascorbic acid (200 μl), dexamethasone(100 μl) for 7 days. The calcified hVICs were further divided into calcified hVICs group and inhibited calcified hVICs by adding specific Notch1 inhibitor DAPT (50 μmol/L(4 μl/hole))groups and cultured for another 7 days. Inflammatory response of all groups were induced by lipopolysaccharide (LPS) for 8 to 12 hours. Western blot was used to detect the protein expression of Notch1, phosphorylation nuclear transcription factor κB (p-NF-κB), bone morphogenetic protein-2/4(BMP-2/4). ELISA was applied to detect the content of BMP-2 secretion of the groups. Von Kossa staining was used to observe of cellular calcification. (1)Von Kossa staining is positive in the induced calcification group, the expression of Notch1, p-NF-κB, BMP-2 and BMP-4 is significantly higher in the induced calcification group than in the control group (all P<0.05). The expression of BMP-2 is significantly higher in the induced calcification group than in control group ((88.23±3.28) pg/ml vs. (25.41±3.68) pg/ml, P=0.02). (2) After treatment with DAPT, the calcification and the expression of Notch1, p-NF-κB, BMP-2 and BMP-4 were significantly decreased compared to calcification group (all P<0.05). The expression of BMP-2 is (26.74±4.62) pg/ml in the calcification inhibition group and (80.41±2.96) pg/ml in calcified control group (P=0.02). Upregulated Notch 1 expression promotes BMP-2/4 secretion in LPS stimulated hVICs, and contributes to osteogenic changes in hVICs. Inhibiting Notch1 can decrease the BMP-2/4 secretion and calcification in hVICs, which may serve as a novel therapeutic option for treating calcific valve disease.
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