Bai Xiao-Jun, Liu Yan, Gao Shan-Shan, Zhang Wei-Ping
Department of Cardiology, the First Affiliated Hospital of Xi'an Jiaotong University Xi'an 710061, China.
Zhongguo Zhong Yao Za Zhi. 2021 Sep;46(17):4497-4503. doi: 10.19540/j.cnki.cjcmm.20210603.406.
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.
本研究旨在观察淫羊藿苷对氧化应激诱导的主动脉血管平滑肌细胞（VSMCs）钙化的抑制作用，并阐明淫羊藿苷抑制内质网应激（ERS）介导的动脉粥样硬化钙化的分子机制，为探索淫羊藿叶抗动脉粥样硬化机制提供新思路。取大鼠胸主动脉VSMCs进行贴壁培养，然后用含高糖和过氧化氢（H₂O₂）的完全钙化DMEM处理3周。将所得钙化VSMCs分为不同处理组。钙化诱导1周后加入淫羊藿苷以保护VSMCs，然后使用细胞计数试剂盒-8（CCK-8）检测其活力。进行茜素红-S染色以观察钙化程度。使用磷酸苯二钠底物测定VSMCs中碱性磷酸酶（ALP）的活性，并用偶氮胂Ⅲ法测定钙含量。通过实时PCR检测骨钙素（OC）、骨桥蛋白（OPN）、Runt相关转录因子2（Runx2）和Ⅰ型胶原（ColⅠa）等骨化相关因子的mRNA表达水平。进行蛋白质免疫印迹法以测定α-平滑肌肌动蛋白（α-SMA）、Runx2、激活转录因子4（ATF4）和真核翻译起始因子（eIF）-2α的蛋白表达水平。结果显示，H₂O₂显著诱导VSMCs钙化，增加VSMCs中ALP活性和钙含量，促进OC、OPN、Runx2和ColⅠa mRNA表达及Runx2蛋白表达，并降低α-SMA蛋白表达。ATF4蛋白表达和eIF2α磷酸化也显著升高。淫羊藿苷逆转了H₂O₂诱导的VSMCs钙化，抑制VSMCs中ALP活性和钙含量，下调OC、OPN、Runx2和ColⅠa的mRNA表达水平及Runx2蛋白表达，并相对上调α-SMA的表达。ATF4的表达和eIF2α的磷酸化也显著下降。所有这些表明，淫羊藿苷通过下调骨化相关因子并降低VSMCs中ALP活性和钙含量来抑制VSMCs钙化。此外，Runx2表达的下调以及对ERS中ATF4和eIF2α介导的细胞钙化途径的抑制也可能参与了这种钙化抑制过程。
