-actin was used as a control. vascular endothelial growth factor (VEGF), inflammatory protein such as intercellular adhesion molecule-1 (CAM-1), tight junction proteins such as ZO-1, and Occludin and the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/AKT were decided in the ischemic brains by Western blot, respectively. Results The treatment of AG extract significantly decreased the volumes of brain infarction, and edema in MACO-induced ischemic rats. AG extract decreased the increase of BBB permeability, and neuronal death and inhibited the activation of astrocytes and microglia in ischemic brains. AG extract also significantly increased the expression of Ang-1, Tie-2, VEGF, ZO-1 and Occludin through activation of the PI3K/Akt pathway. AG extract significantly increased the expression of ICAM-1 in ischemic brains. Conclusions Our results indicate that this hairy root of AG has a neuroprotective effect in ischemic stroke. NAKAI (Umbelliferae; Angelica Gigantis Radix), known as Korean angelica, (AG) root is a herbal medicine for the treatment of various circulatory disorders with female afflictions such as dysmenorrhea, amenorrhea, menopause, abdominal pain, migraine and arthritis [15]. AG has biological activities such as anti-cancer [15-18], anti-platelet aggregation [16], neuroprotection [17], anti-inflammatory, anti-oxidant [18] and anti-osteoclastogenesis [19] with several coumarin derivates including decursin decursinol, decursinol angelate, nodakenin, nodakenetin and umbelliferone [20,21]. In Oriental medicine, the root of AG is able to divide two parts, root body and hairy root according to their efficacy on tonify blood and promote blood circulation. For example, the body root has been used for blood deficiency syndrome, and the hairy root has been used for blood stasis. However, the effect of AG extract around the BBB permeability and GSK-2881078 angiogenesis with vascular stabilization has not been investigated. Stroke is an inflammatory disease caused by the extravasation of blood in the brain. Therefore, in this study, we evaluate the effect of the hairy root of AG on blood stasis and inflammation in ischemic brain through improving the blood disability. For this, we GSK-2881078 investigated the expression of angiogenesis-induced proteins, such as VEGF and Ang-1/Tie-2, and tight junction molecules such as Occludin and ZO-1 with the BBB permeability in transient middle artery cerebral occlusion (tMCAO)-induced ischemic stroke in rats, and investigated its action mechanism around the PI3K/Akt signaling pathway. Methods Preparation of AG extract (AG) roots were purchased from a medicinal materials company (Kwangmyungdang Medicinal Herbs, Ulsan, Republic of Korea) and authenticated by Y. K. Park, a botanist ITGAL in the Department of Herbology, College of Oriental Medicine, Dongguk GSK-2881078 University (DUCOM), Republic of Korea. AG extract was prepared by the following procedure. The roots were boiled in distilled water for 3 h, filtered through a two-layer mesh and Whatman No. 1 paper, and concentrated under vacuum. The final yield of concentrated extract was 29.1% of the dried powder. AG extract was stored at 4C, and dissolved in saline prior to use. Animals Male Sprague-Dawley (SD) rats weighing an average of 280??10 g (Orient Bio Inc., Gyeonggi-do, Rep. of Korea) were used in the experiments. The animals were housed under controlled environmental conditions at an ambient heat of 23??1C, relative humidity of 50??10% and 12 h light/dark cycle with free access to food and water. All animals were handled according to the animal welfare guidelines issued by the Korean National Institute of Health and the Korean Academy of Medical Sciences for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee of Dongguk University. Preparation of ischemic stroke rat model The ischemic stroke rat model was prepared by transient middle cerebral artery occlusion (tMCAO) and reperfusion following a standard procedure [22]. Rats were anesthetized with 4% isoflurane and maintained using 1% isoflurane in a mixture of 30% oxygen and 70% nitrous oxide, during the surgical procedure. Rectal heat was measured with a rectal probe and was kept at 37C using a heating pad (FHC Inc., ME, USA). The left common carotid artery (CCA) was uncovered and separated carefully from the vagus nerve and ligated at the more proximal side through a right paramedian incision. The external carotid artery (ECA) was ligated. The occipital artery and the pterygopalatine artery were coagulated. Ischemia was produced by advancing the tip of a rounded 3-0 nylon suture into the ICA through the ECA. After placement, the.
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