Pretreatment with Dingnaofang reduces vascular endothelial cell apoptosis induced by cerebral ischemia/perfusion injury

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 SETFING： 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 Astragah）, Chuanxiong （Szechwan Lovage Rhizome; Rhizoma Chuanxiong）, Yinxingye （ginkgo leaf; Fofium 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 ischemiaJreperfusion 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.