[Inhibitory mechanism of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells].

This study aimed to observe the inhibitory effect of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells(VSMCs) and elucidate the molecular mechanism of icariin in inhibiting endoplasmic reticulum stress(ERS)-mediated atherosclerotic calcification, so as to provide new ideas for exploring the anti-atherosclerotic mechanism of Epimedii Folium. The VSMCs in rat thoracic aorta were subjected to adherent culture and then treated with the complete calcification DMEM containing high glucose and hydrogen peroxide(H_2O_2) for three weeks. The resulting calcified VSMCs were divided into different treatment groups. Icariin was added one week after calcification induction for protecting the VSMCs, whose viability was then detected using cell counting kit-8(CCK-8). Alizarin red-S staining was conducted to observe the calcification degree. The activity of alkaline phosphatase(ALP) in VSMCs was measured using the disodium phenyl phosphate substrate and the calcium content was measured by arsenazo Ⅲ method. The mRNA expression levels of ossification-related factors including osteocalcin(OC), osteopontin(OPN), Runt-related transcription factor 2(Runx2), and type Ⅰ collagen(Col Ⅰa) were detected by real-time PCR. Western blot was carried out to determine the protein expression levels of α-smooth muscle actin(α-SMA), Runx2, activating transcription factor 4(ATF4), and eukaryotic translation initiation factor(eIF)-2α. The results showed that H_2O_2 significantly induced the calcification of VSMCs, increased the ALP activity and calcium content in VSMCs, promoted OC, OPN, Runx2, and Col Ⅰa mRNA expression and Runx2 protein expression, and reduced α-SMA protein expression. The ATF4 protein expression and eIF2α phosphorylation were also elevated significantly. Icariin reversed the calcification of VSMCs induced by H_2O_2, inhibited ALP activity and calcium content in VSMCs, down-regulated the mRNA expression levels of OC, OPN, Runx2 and Col Ⅰa and Runx2 protein expression, and relatively up-regulated the expression of α-SMA. The expression of ATF4 and phosphorylation of eIF2α also declined significantly. All these have demonstrated that icariin inhibited VSMCs calcification by down-regulating the ossification-related factors and lowering ALP activity and calcium content in VSMCs. Besides, the down-regulation of Runx2 expression and the inhibition of ATF4 and eIF2α-mediated cellular calcification pathway in ERS might also be involved in such calcification-suppressing process.