The Effect of Velvet Antler Proteins on Cardiac Microvascular Endothelial Cells Challenged with Ischemia-Hypoxia
The Effect of Velvet Antler Proteins on Cardiac Microvascular Endothelial Cells Challenged with Ischemia-Hypoxia
复制标题
鹿茸蛋白对缺血缺氧心肌微血管内皮细胞的影响
DOI：
10.3389/fphar.2017.00601
复制
发表时间：
2017-09-04
影响因子：
5.6
通讯作者：
Huang, Li
中科院分区：
文献类型：
--
作者：
Xiao, Xiang;Xu, Shuqiang;Huang, Li
鹿茸是一种具有再生能力的名贵中药。基于中医心肾相济的理论，VA已被用于治疗心脏疾病，包括缺血性心脏病，心力衰竭和心律失常。我们研究了VA蛋白对缺血缺氧（IH）的原代心脏微血管内皮细胞（CMECs）的影响，以探讨其在治疗缺血性心脏病中的作用和作用机制。以水为溶剂，采用超声波法和冷冻干燥技术提取鹿茸蛋白质（VA-pro）;然后通过Nano LC-MS/MS进行分析。此外，通过MTS测定、EdU测定、Annexin V-FITC/PI双染色测定和JC-1测定来评估VA-pro在细胞活力、增殖、凋亡和线粒体膜电位（MMP）中的作用，分别通过划痕试验和Transwell试验评价细胞迁移。检测细胞凋亡相关蛋白Akt和p-Akt的表达及基质胶中微管的形成。总共鉴定了386个VA-pro。结果表明，IH显著降低了CMEC的存活率（P < 0.001），并抑制了CMEC的DNA拷贝数，从而抑制了CMEC的增殖（P < 0.001）。对照组OD值为1.81 ± 0.08，IH组OD值为1.25 ± 0.03。缺氧46 h后，CMECs迁移能力降低75%（P < 0.001），肾小管形成能力和MMP也降低（P < 0.001）。VA-pro处理组CMECs的存活率和增殖能力均明显高于对照组（P < 0.001）。如0.5、1、2 mg/ml的OD值分别上升至1.56 ± 0.5、1.74 ± 0.1、1.65 ± 0.1。同样，经VA-pro处理后，CMECs的迁移能力（划痕试验P < 0.001，Transwell试验P < 0.05）和小管形成能力（P < 0.05）更好。同时，MMP的稳定性得到较好的保持（P < 0.001）。IH组CMECs凋亡率为50%（P < 0.001），而VA-pro组凋亡率明显降低（P < 0.001）。所有上述结果表明，1 mg/ml VA-pro产生最大结果。IH组促凋亡蛋白表达增加，而抗凋亡蛋白表达减少（P < 0.05），VA pro可逆转上述变化（P < 0.001）。提示VA-pro通过调节PI 3 K/Akt信号通路减轻IH诱导的CMEC损伤。
Velvet antler (VA) is a precious traditional Chinese medicine that is capable of repeated regeneration. Based on the Chinese medicine theory of coordination the heart and kidneys, VA has been employed to treat heart diseases, including ischemic heart disease, heart failure, and arrhythmia. We examined the effects of VA proteins on primary cardiac microvascular endothelial cells (CMECs) that were subjected to ischemia-hypoxia (IH) to investigate their effects on and mechanism of action in the treatment of ischemic heart disease. Velvet antler proteins (VA-pro) were extracted with water as the solvent, the ultrasonic wave method, and freeze-drying technology; then it was analyzed by Nano LC-MS/MS. In addition, the role of VA-pro in cell viability, proliferation, apoptosis, and mitochondrial membrane potential (MMP) were evaluated by the MTS assay, the EdU assay, the Annexin V-FITC/PI double-staining assay, and the JC-1 assay, respectively. Cell migration were evaluated by the scratch assay and the Transwell assay. The expression of apoptosis-associate proteins, Akt and p-Akt, and tube formation in Matrigel of CMECs were also detected. In total, 386 VA-pro were identified. Our results showed that IH significantly reduced the viability of the CMECs (P < 0.001) and suppressed copies of DNA to hold back CMEC proliferation (P < 0.001). The OD of control group was 1.81 ± 0.08 and IH group OD was 1.25 ± 0.03. After suffering with IH for 46 h, CMECs were 75% less likely to migrate (P < 0.001), and its tubule formation ability and MMP were also decreased (P < 0.001). VA-pro treatment resulted in an improvement in CMECs’ viability and proliferation (P < 0.001). Such as, the OD of 0.5, 1, and 2 mg/ml rose to 1.56 ± 0.5, 1.74 ± 0.1 and 1.65 ± 0.1, respectively. Similarly, CMECs’ migration (for the scratch assay P < 0.001, for the Transwell assay P < 0.05) and tubule formation (P < 0.05) ability were better after treated with VA-pro. At the same time, the stability of MMP was retained preferably (P < 0.001). 50% apoptosis was induced after CMECs were cultured in IH conditions (P < 0.001), while VA-pro decreased the number of apoptotic cells (P < 0.001). All above results showed that 1 mg/ml VA-pro produced maximum results. Furthermore, the expression of pro-apoptosis proteins was higher, but the expression of anti-apoptosis proteins was lower in the IH group (P < 0.05); VA-pro reversed these changes (P < 0.001). These findings suggest that VA-pro ameliorate CMEC injuries induced by IH via regulating the PI3K/Akt signaling pathway.