Liu Kun, Wang Xiu-Juan, Li Yan-Ning, Li Bin, Qi Jin-Sheng, Zhang Jing, Wang Yu
Department of Biochemistry, Hebei Key Laboratory of Medical Biotechnology, Hebei Medical University, Shijiazhuang, Hebei 050017, China.
Chin Med J (Engl). 2016 Feb 20;129(4):442-7. doi: 10.4103/0366-6999.176076.
Claudin-5, claudin-9, and claudin-11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeability, which is the initiating process and pathological basis of cardiovascular disease. Although tongxinluo (TXL) has satisfactory antianginal effects, whether and how it modulates claudin-5, claudin-9, and claudin-11 in hypoxia-stimulated human cardiac microvascular endothelial cells (HCMECs) have not been reported.
In this study, HCMECs were stimulated with CoCl2to mimic hypoxia and treated with TXL. First, the messenger RNA (mRNA) expression of claudin-5, claudin-9, and claudin-11 was confirmed. Then, the protein content and distribution of claudin-9, as well as cell morphological changes were evaluated after TXL treatment. Furthermore, the distribution and content histone H3K9 acetylation (H3K9ac) in the claudin-9 gene promoter, which guarantees transcriptional activation, were examined to explore the underlying mechanism, by which TXL up-regulates claudin-9 in hypoxia-stimulated HCMECs.
We found that hypoxia-suppressed claudin-9 gene expression in HCMECs (F = 7.244; P = 0.011) and the hypoxia-suppressed claudin-9 could be reversed by TXL (F = 61.911; P = 0.000), which was verified by its protein content changes (F = 29.142; P = 0.000). Moreover, high-dose TXL promoted the cytomembrane localization of claudin-9 in hypoxia-stimulated HCMECs, with attenuation of cell injury. Furthermore, high-dose TXL elevated the hypoxia-inhibited H3K9ac in the claudin-9 gene promoter (F = 37.766; P = 0.000), activating claudin-9 transcription.
The results manifested that TXL reversed the hypoxia-suppressed claudin-9 by elevating H3K9ac in its gene promoter, playing protective roles in HCMECs.
紧密连接蛋白5、紧密连接蛋白9和紧密连接蛋白11在内皮细胞中表达以构成紧密连接，它们的缺乏可能导致高通透性，这是心血管疾病的起始过程和病理基础。尽管通心络（TXL）具有令人满意的抗心绞痛作用，但它是否以及如何调节缺氧刺激的人心脏微血管内皮细胞（HCMECs）中的紧密连接蛋白5、紧密连接蛋白9和紧密连接蛋白11尚未见报道。
在本研究中，用氯化钴刺激HCMECs以模拟缺氧，并给予TXL处理。首先，确认紧密连接蛋白5、紧密连接蛋白9和紧密连接蛋白11的信使核糖核酸（mRNA）表达。然后，评估TXL处理后紧密连接蛋白9的蛋白质含量和分布以及细胞形态变化。此外，检测紧密连接蛋白9基因启动子中保证转录激活的组蛋白H3K9乙酰化（H3K9ac）的分布和含量，以探讨TXL上调缺氧刺激的HCMECs中紧密连接蛋白9的潜在机制。
我们发现缺氧抑制HCMECs中紧密连接蛋白9基因的表达（F = 7.244；P = 0.011），而TXL可逆转缺氧抑制的紧密连接蛋白9（F = 61.911；P = 0.000），这通过其蛋白质含量变化得到验证（F = 29.142；P = 0.000）。此外，高剂量TXL促进缺氧刺激的HCMECs中紧密连接蛋白9的细胞膜定位，减轻细胞损伤。此外，高剂量TXL升高了紧密连接蛋白9基因启动子中缺氧抑制的H3K9ac（F = 37.766；P = 0.000），激活紧密连接蛋白9的转录。
结果表明，TXL通过升高其基因启动子中的H3K9ac来逆转缺氧抑制的紧密连接蛋白9，对HCMECs起保护作用。
