The arterial rings and VSMCs were isolated or extracted as previously described (23 (link)). Calcification was induced by adding inorganic phosphate (Na2HPO4, Sigma, USA) to normal medium at a concentration of 2.6 mM (calcification medium). To evaluate the impact of BHB on calcification, BHB (Sigma, USA) was administrated into a calcification medium at a concentration of 4.0 mM (BHB intervention medium) (24 (link)). The arterial rings were cultured for 14 days, while VSMCs were cultured for 7 days, with the medium for both refreshed every 2 to 3 days (23 (link)).
To evaluate whether autophagy inhibition could reverse the protective effects of BHB on VC, the autophagosome inhibitor 3-methyladenine (Selleck, USA), at a concentration of 20 μM, and the autophagic flux inhibitor chloroquine (Selleck, USA), at a concentration of 10 μM, were administrated to the calcification models with BHB intervention (25 (link)).
To evaluate whether autophagy inhibition could reverse the protective effects of BHB on VC, the autophagosome inhibitor 3-methyladenine (Selleck, USA), at a concentration of 20 μM, and the autophagic flux inhibitor chloroquine (Selleck, USA), at a concentration of 10 μM, were administrated to the calcification models with BHB intervention (25 (link)).
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