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BACKGROUND: Vascular endothelial cell apoptosis participates in cerebral ischemia/reperfusion injury, and the method of Qi-supplementation and blood-activation has remarkable neuroprotective effects against cerebral ischemia/reperfusion injury.OBJECTIVE: To explore the molecular mechanism that Dingnaofang (Chinese herbs for supplementing Qi and activating blood circulation) inhibits vascular endothelial cell apoptosis induced by cerebral ischemia/reperfusion injury.DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at the Department of Central Laboratory, Third Xiangya Hospital, Central South University, China, between October 2007 and December 2008.MATERIALS: Dingnaofang consisted of Huangqi (Milkvetch Root; Radix Astragali), Chuanxiong (Szechwan Lovage Rhizome; Rhizoma Chuanxiong), Yinxingye (ginkgo leaf; Folium Ginkgo), Dilong (earthworm; Pheretima), Danggui (Chinese Angelica; Radix Angelicae Sinensis), Tianqi (Radix Notoginseng), and Gancao (Radix Glycyrrhizae; Radix Glycytthizae), with a proportion of 5: 2: 2: 1: 1: 1: 1. METHODS: A total of 130 Sprague Dawley rats were randomly divided into sham-surgery (n = 10), cerebral ischemia/reperfusion (n = 40), cerebral ischemia pretreatment (n = 40) and Dingnaofang pretreatment groups (n = 40). Middle cerebral artery occlusion was used to induce cerebral ischemic injury. The bilateral common carotid artery in the cerebral ischemia pretreatment group was blocked for 10 minutes on days 7, 3 and 1 prior to ischemia/reperfusion injury, while rats in Dingnaofang pretreatment group were intragastrically administrated with 4 g Dingnaofang 1 week prior to cerebral ischemia once per day, for 7 successive days.MAIN OUTCOME MEASURES: Apoptosis ratios in vascular endothelial cells were measured using Hoechst 33258 staining and flow cytometry; apoptosis was detected by monitoring DNA gradient bands and the activation of caspase-3, 8, 9 and Bid using Western blot.RESULTS: Following cerebral ischemia/reperfusion injury, the number of apoptotic vascular endothelial cells in the middle cerebral artery significantly increased (P < 0.01); however, cerebral ischemia pretreatment and Dingnaofang pretreatment groups significantly reduced apoptosis induced by cerebral ischemia/reperfusion injury (P < 0.01). In particular, DNA gradient bands were not observed following Dingnaofang pretreatment. At 24 hours after cerebral ischemia/reperfusion injury, cleaved fragments of caspase-3, 8 and 9 were detected at 11 kD (P 11), 20 kD (P 20) and 10 kD (P 10), respectively, following Western blot. Bid was also cleaved into its truncated form (tBid; 15 kD). Gray scale analysis indicated that P 11, P 20, P 10 and tBid band values in the Dingnaofang pretreatment group were significantly less than in the cerebral ischemia/reperfusion and cerebral ischemia pretreatment groups (P < 0.01 or P < 0.05). CONCLUSION: Dingnaofang inhibits vascular endothelial cell apoptosis induced by cerebral ischemia/reperfusion injury via inhibition of apoptotic signal transduction pathways.