Protective mechanisms of picroside Ⅱ on aquaporin-4 expression in a rat model of cerebral ischemia/reperfusion injury

BACKGROUND： Aquaporin-4 （AQP-4） over-expression following cerebral ischemia results in cerebral edema. Picroside Ⅱ has been shown to exhibit a neuroprotective effect on neuronal apoptosis. However, few reports have addressed the neuroprotective mechanisms and therapeutic times following cerebral ischemic reperfusion injury. OBJECTIVE： To explore the neuroprotective effects and ideal treatment window for picroside Ⅱ treatment of middle cerebral artery occlusion and reperfusion injury in rats. DESIGN, TIME AND SETTING; A randomized, controlled, animal experiment was performed at Institute of Cerebrovascular Diseases, Qingdao University Medical College from September 2008 to May 2009. MATERIALS： Picroside II was purchased from Tianjin Kuiqing Medical Technology, China. METHODS： A total of 165 adult, healthy, male, Wistar rats were randomly assigned to sham-surgery （n = 15）, model （n = 75）, and treatment groups （n = 75）. Rats in the model and treatment groups underwent middle cerebral artery occlusion and reperfusion through the use of an intraluminal monofilament suture on the left external-internal carotid artery, The treatment group was injected with 1.0% picroside Ⅱ （10 mg/kg） into the tail vein, and the model and sham-surgery groups were injected with 0.1 mol/L phosphate buffered saline （250 μL）. MAIN OUTCOME MEASURES： Neurological functional scores were evaluated using the Longa＇s method; cerebral infarction volume was detected through the use of tetrazolium chlodde staining; cellular apoptosis was determined through the use of the in situ end-labeling method; aquaporin-4 expression was measured using fluorescence labeling analysis and reverse transcription polymerase chain reaction technique. RESULTS： At 0.5 hour following cerebral ischemic injury, neurological functional scores were low, and a small infarction focus was detected in the ischemic cortex of the model group. Along with prolonged ischemia and an increased number of apoptosis-positive cells, AQP-4 mRNA and protein expression was increased. At 1-2 hours after ischemia, neurological scores and infarction sizes were significantly increased in the model group. Apoptotic-positive cells were widespread in the ipsilateral cortex and stdatum. In addition, AQP-4 mRNA and protein expression levels were increased. Picroside II treatment significantly decreased neurological scores and infarction volume, and reduced AQP-4 mRNA and protein expression levels compared with the model group （P 〈 0.05 or P 〈 0.01）. At 1 hour after ischemia, the therapeutic effect of picroside Ⅱ was notable （P 〈 0.01）. CONCLUSION： Picroside Ⅱ played a protective role in cerebral ischemic reperfusion injury by inhibiting apoptosis and regulating AQP-4 expression. The best therapeutic time window was 1 hour after cerebral ischemic reperfusion.

Optimal therapeutic dose and time window of picroside II in cerebral ischemic injury

A preliminary study from our research group showed that picroside II inhibited neuronal apop- tosis in ischemic penumbra, reduced ischemic volume, and improved neurobehavioral function in rats with cerebral ischemia. The aim of the present study was to validate the neuroprotective effects of picroside II and optimize its therapeutic time window and dose in a rat model of cerebral ischemia. We found that picroside Ⅱ inhibited cell apoptosis and reduced the expression of neuron-specific enolase, a marker of neuronal damage, in rats after cerebral ischemic injury. The optimal treatment time after ischemic injury and dose were determined, respectively, as follows： （1） 2.0 hours and 10 mg/kg according to the results of toluidine blue staining; （2） 1.5 hours and 10 mg/kg according to early apoptotic ratio by flow cytometry; （3） 2.0 hours and 10 mg/kg according to immunohistochemical and western blot analysis; and （4） 1.5 hours and 10 mg/kg according to reverse transcription polymerase chain reaction. The present findings suggest that an intraperitoneal injection of 10 mg/kg picroside II 1.5-2.0 hours after cerebral ischemic injury in rats is the optimal dose and time for therapeutic benefit.

The effect of picroside II on the expressions of AQP4, MMP9 and COX2 in cerebral ischemic injury in rats

The aim is to study the neuroprotective effect and optimize the therapeutic dose and time window of picrosede II by orthogonal test in cerebral ischemic injury in rats. The forebrain ischemia models of rats were established by bilateral common carotid artery occlusion (BCCAO) methods. The successful models were randomly grouped according to orthogonal experimental design and treated by injecting picroside II intraperitonenally with different therapeutic dose at different ischemic time. The contents of aquaporins 4 (AQP4), matrix metalloproteinases 9 (MMP 9) and cyclooxygenase 2 (COX2) in brain tissue were determined by enzyme linked immunosorbent assay to evaluate the therapeutic effect of picroside II on cerebral ischemic injury. The results indicated that the best therapeutic time window and dose of picroside II in cerebral ischemic injury should be 1) ischemia 1.5 h with 20 mg/kg body weight according to the content of AQP4;2) ischemia 2.0 h with 20 mg/kg according to the content of MMP9;and 3) ischemia 1.5 h with 20 mg/kg according to the content of COX2 inbrain tissue. It is concluded that the optimized therapeutic dose and time window is injecting picroside II peritonenally with 10 - 20 mg/kg body weight at ischemia 1.5 - 2.0 h in cerebral ischemic injury according to the principle of lowest therapeutic dose with longest time window.