Wang Qun, Gao Shan, Luo Yu, Kang Qian-Yan
Department of Ophthalmology, The First Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
Environment and Genes Related to Diseases Key Laboratory of Education Ministry, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
Exp Ther Med. 2017 Jul;14(1):600-608. doi: 10.3892/etm.2017.4528. Epub 2017 May 31.
As a Traditional Chinese Medicine, compound anisodine (CA) has previously been shown to regulate the vegetative nervous system, improve microcirculation and scavenge reactive oxygen species, and has been commonly utilized as a neuroprotective agent to treat ischemic optic neuropathy and choroidoretinopathy. The present study aimed to investigate the neuroprotective effects of CA on the proliferation and calcium overload of hypoxia-induced rat retinal progenitor cells (RPCs) and brain neural stem cells (BNSCs) harvested from neonatal Sprague-Dawley rats. Cells were treated with CA at 0.126, 0.252, 0.505 or 1.010 g/l for four hours prior to or after hypoxia (<1% oxygen) for four h, followed by re-oxygenation for four hours; a normal control group and a CA-untreated hypoxia model group were also included. An MTT assay demonstrated that the cell viability was markedly improved following treatment with 0.126-1.010 g/l CA, compared with that in the hypoxia model group (P<0.05). Bromodeoxyuridine (BrdU) immunocytochemical staining and flow cytometry indicated that after culture in hypoxia for 4 h, the number of BrdU RPCs and BNSCs was significant decreased, as well as the cell population in S+G2 phase of the cell cycle, which was significantly attenuated by treatment with 1.010 g/l CA for 4 h prior to hypoxia (P<0.05). Furthermore, laser scanning confocal microscopy showed that the intracellular calcium concentration in hypoxia-cultured RPCs and BNSCs was markedly increased, which was attenuated by 0.126-1.010 g/l CA in a concentration-dependent manner (P<0.05). Furthermore, western blot analysis demonstrated that after hypoxia, the protein levels of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were upregulated in RPCs and BNSCs, whereas phosphorylated extracellular signal-regulated kinase (phospho-ERK 1/2) and Cyclin D1 were downregulated; of note, treatment with 1.010 g/l CA significantly attenuated these changes (P<0.05). The results of the present study suggested that CA may improve the proliferation and inhibit calcium overload in hypoxia-induced RPCs and BNSCs by altering the protein levels of Cyclin D1 as well as signaling through the p-ERK1/2/HIF-1α/VEGF pathway.
作为一种传统中药，复方樟柳碱（CA）此前已被证明可调节自主神经系统、改善微循环并清除活性氧，并且已被广泛用作神经保护剂来治疗缺血性视神经病变和脉络膜视网膜病变。本研究旨在探讨CA对从新生Sprague-Dawley大鼠分离的缺氧诱导的大鼠视网膜祖细胞（RPC）和脑神经元干细胞（BNSC）增殖及钙超载的神经保护作用。在缺氧（<1%氧气）4小时之前或之后，用0.126、0.252、0.505或1.010 g/l的CA处理细胞4小时，随后再进行4小时的复氧；还设置了正常对照组和未用CA处理的缺氧模型组。MTT分析表明，与缺氧模型组相比，用0.126 - 1.010 g/l CA处理后细胞活力显著提高（P<0.05）。溴脱氧尿苷（BrdU）免疫细胞化学染色和流式细胞术表明，在缺氧培养4小时后，BrdU RPC和BNSC的数量以及细胞周期S + G2期的细胞群体显著减少，而在缺氧前用1.010 g/l CA处理4小时可显著减轻这种减少（P<0.05）。此外，激光扫描共聚焦显微镜显示，缺氧培养的RPC和BNSC细胞内钙浓度显著升高，而0.126 - 1.010 g/l CA以浓度依赖性方式减轻了这种升高（P<0.05）。此外，蛋白质印迹分析表明，缺氧后，RPC和BNSC中缺氧诱导因子（HIF）-1α和血管内皮生长因子（VEGF）的蛋白质水平上调，而磷酸化细胞外信号调节激酶（磷酸化ERK 1/2）和细胞周期蛋白D1下调；值得注意的是，用1.010 g/l CA处理可显著减轻这些变化（P<0.05）。本研究结果表明，CA可能通过改变细胞周期蛋白D1的蛋白质水平以及通过p-ERK1/2/HIF-1α/VEGF途径的信号传导来改善缺氧诱导的RPC和BNSC的增殖并抑制钙超载。
