Production of nitric oxide (NO) by nitric oxide synthase (NOS) has been confirmed as a major mechanism in physiological and pathological processes in the central nervous system. 1, 2) In cerebral ischemia, because of the different cellular sources and the different stages of the ischemic process, the role of NO can be protective or destructive. 3) Studies have shown that NO produced by endothelial nitric oxide synthase (eNOS) plays a prominent role in maintaining cerebral blood flow and

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... ng neuronal injury as well as inhibiting platelet and leukocyte adhesion, and thus protects against stroke. [4] [5] [6] Consequently, the protective role of NO in brain ischemia underlines the necessity for selective therapeutic approaches to augment eNOS. Recently, several therapeutic modalities to upregulate and/or activate eNOS have been proved that it might mediate NO-dependent stroke-protective effects, 7) such as statins could improve stroke outcome through the eNOS-dependent mechanism. 8-10) Particularly, an extract of Ginkgo biloba, which has been reported to be able to alleviate cerebrovascular problems, can significantly enhance the level of eNOS mRNA as well. 11) Many therapeutics and remedies of traditional Chinese medicine are proved effective for stroke through years of practice. Refined Qing Kai Ling (RQKL) is an improved injection derived from the combination of effective and safety components in Qing Kai Ling injection, which has clinically been practiced as a useful injection to treat acute stroke. 12) We have previously demonstrated that some components of RQKL could relieve the damage of vascular endothelial cell and inhibit the process of inflammation, offering the neuroprotection effect in middle cerebral artery occlusion (MCAO) model of rats. 13) Herein, we investigated whether its cerebroprotective effects are correlated with the expression of eNOS under ischemic condition. MATERIALS AND METHODS Animal Model All experiments were performed on male SD rats (Experimental Animal Company of Beijing Vital, Certificate No. 19-053), weighing 280 to 320 g. The animal experiments were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines. Focal cerebral ischemia was induced by the filament model according to the method of Longa et al. 14) In short, animals were anesthetized with 10% chloral hydrate (0.35 g/kg, intra-peritoneally (i.p.)). After median incision of the neck skin, the left external carotid artery (ECA) was carefully dissected. An 18.5Ϯ0.5 mm length of nylon suture (dϭ0.25 mm, with one end rounded by heat and dϽ0.3 mm) was introduced into transected lumen of ECA and gently advanced into the internal carotid artery to block the origin of the left middle cerebral artery. Afterward, retracted soft tissues were replaced, wounds were sutured, and the rats were put back into their cages. Body temperature was kept at 37°C with a heat lamp and a piece of heating pad during operation until animal regained consciousness. After awakening, a brief behavioral assessment was performed on rats. The rats without right forelimb paresis were regarded unsuccessful MCAO, and these animals were excluded from further study. Thereafter, rectal temperature of rats with right forelimb paresis was checked every 10 to 15 min during the following 2 h and, if necessary, it was corrected to 37°C by placing a heating pad under the cage. Sham-operated animals underwent identical procedures except for only brief insertion of the filament into the ECA stump. Sham-operated animals served as controls in all experiments mentioned. Refined Qing Kai Ling (RQKL) is an improved injectable multi-component preparation derived from Qing Kai Ling, which could offer the neuroprotection effect in middle cerebral artery occlusion (MCAO) model of rats by relieving the damage of vascular endothelial cell as well as inhibiting the process of inflammation. Herein, we observed whether RQKL could exert influence on the expression of endothelial nitric oxide synthase (eNOS), as a mechanism of its protective effects against ischemia. Sprague-Dawley rat model of focal cerebral ischemia was established by permanent filament occlusion of the left middle cerebral artery. We found that the administration of RQKL could reduce the ischemic infarct size as well as neurological deficit of model rats. Furthermore, it was showed that the eNOS level was consistently increased in endothelium of blood vessels of the ischemic penumbra after 2 to 72 h of permanent MCAO, and the expression of eNOS increases more in animals treated with RQKL. Our results suggested that eNOS levels in penumbral zone were enhanced after permanent focal ischemia, and RQKL could stimulate postischemic eNOS expression, which may be an important mechanism in RQKL's protection against cerebral ischemia.