| Stroke is cerebrovascular injury that has been reported to be the third leading cause of death and the first leading cause of disability in the world. Pathophysiological responses in the brain following stroke are highly complex and involve multiple mechanisms, including excitotoxicity, free radical damage, metabolic acidosis, intra-cellular calcium overload, mitochondria injury, apoptosis, ion unbalance and inflammation. Studies of monotherapy approaches for saving neuronal function have yet to produce any clinically effective neuroprotectants. Currently, there is only one FDA approved treatment for stroke which is recombinant tissue plasminogen activator. This treatment has a narrow therapeutic window of6hours after ischemic stroke and can adversely cause the production of oxygen free radicals and intracranial hemorrhage. These limitations result in only2-3%of all stroke victims as being candidates for this therapy as many patients do not arrive at the hospital in time to receive treatment, are not properly diagnosed, or do not know that they have had a stroke within this6hour time period. It is essential to develop a safe and valid drug to use in acute cerebral ischemia and subsequent functional recovery.8-O-acetyl shanzhiside methylester (ND01), is an iridoid glucoside, is isolated from the leaves of Lamiophlomis rotata (Benth.) Kudo., is a Chinese folk medicinal plant in Xi-zang (Tibet). For thousands years, Lamiophlomis rotata has been used as one of the traditional drugs with the effects of alleviating pain, detumescence, hemostasis, reinforcing marrow and promoting blood circulation to remove blood stasis. The present study was designed to investigate:(i)the protective effects of ND01on cultured rat cortical neurons subjected to oxygen-glucose deprivation-induced injury and the potential mechanism on mitochondrial energy metabolism;(ii) the effects of ND01on rat cerebral ischemia model and its potential neuroprotective mechanism, including globe cerebral ischemia injury model, cerebral ischemia and reperfusion injury model and middle cerebral artery occlusion (MCAO) model;(iii) the effect of ND01on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro and angiogenesis after cerebral ischemia and reperfusion (I/R) injury in vivo and its potential mechanism.I ND01attenuates apoptosis and ameliorates mitochondrial energy metabolism in rat cortical neurons exposed to oxygen-glucose deprivationCulture rat cortical neurons are exposed to oxygen-glucose deprivation for3h followed by12h incubation with neurobasal medium. The results show that ND01treatment obviously attenuates apoptosis and ameliorates mitochondrial energy metabolism in rat cortical neurons by increasing cell survival rate, mitochondrial respiratory enzymes activities, mitochondrial respiratory control ratio and adenosine triphosphate (ATP) content, and by attenuating lactate dehydrogenase (LDH) leakage, intracellular Ca2+level and caspase-3activity in a concentration-dependent manner. These findings indicate that ND01has potential against cerebral ischemic injury, and its protective effect on oxygen-glucose deprivation-induced injury might be due to the suppression of intracellular Ca2+elevation and caspase-3activity, and improvement of mitochondrial energy metabolism.II Neuroprotective efficacy of ND01:in rat model of globe cerebral ischemia injuryMale Sprague-Dawley rats subjected to90min of bilateralis common carotid artery occlusion and22.5h of reperfusion. Rats received an intravenous bolus injection of ND01at30min after reperfusion. The results showed that ND0110mg/kg and20mg/kg significantly reduced neuron cell necrosis and apoptosis in a dose-dependent manner compared with the vehicle-treated animals. It suggests that ND01has neuroprotective efficacy in rat model of globe cerebral ischemia injury.Ⅲ Neuroprotective efficacy and therapeutic window of ND01:in rat model of cerebral ischemia injuryMale Sprague-Dawley rats subjected to1.5h of middle cerebral artery occlusion (MCAO) and22.5h of reperfusion. In the dose-response study, ND01at doses of10.20,40and80mg/kg administered intravenously30min after ischemia and reperfusion (I/R) decreased neurological deficit scores, attenuated the decrease of viable neuron cells in ischemic zone. In the therapeutic time-window study, ND01at a dose of20mg/kg significantly decreased neurological deficit scores, attenuated the decrease of viable neuron cells in ischemic zone even after delayed administration intravenous at1h,3h and5h but not7h after I/R. In the long-term study. ND01at a dose of20mg/kg, a bolus injection intravenous at30min after I/R, significantly decreased neurological deficit scores and reduced cerebral infarction within14days after I/R. It suggests that ND01exerts potent and long-term neuroprotective effects with an appropriate dose-response curve and a favorable therapeutic time-window in cerebral I/R rats.IV Neuroprotective efficacy of NDO1:in rat model of MCAO Male Sprague-Dawley rats subjected to MCAO, ND01at doses of5,10and20mg/kg administered intravenously30min after ischemia, the results indicates that ND01decreasea neurological deficit scores, reduces infarct volume, attenuates cerebral edema in a dose-dependent manner compared with the vehicle-treated animals. It suggests that ND01exerts potent neuroprotective effect in rat model of MCAO.V ND01increases in vitro proliferation, migration, and tube formation of HUVECsHUVECs were grown in M199medium. Cell proliferation was determined by SRB assay. Cell migration assay was performed using scarification, collagen matrigel three dimensional cultured HUVECs to determine tube formation. The results showed that NDOl treatment obviously increases proliferation, migration and tube formation of HUVECs in vitro. It suggests that ND01increases angiogenesis in vitro.VI ND01increases angiogenesis and improves functional recovery after strokeMale Sprague-Dawley rats were subjected to1h of MCAO and reperfusion and were treated with or without different doses (5and10mg/kg) of ND01, starting24h after I/R and daily for14days. Neurological functional tests were performed, and cerebral Evans blue extravasation was measured. Angiogenesis and angiogenic factor expression were measured by immunohistochemistry and Western blot, respectively. ND01significantly promoted angiogenesis in the ischemic brain, and improved functional outcome after stroke. NDO1also significantly increased vascularization compared with the vehicle-treated animals. NDO1increased the expression of VEGF, Angl, phosphorylation of Tie2and Akt. VEGF, the Angl/Tie2axis, and Akt pathways appear to mediate NDO1-induced angiogenesis.ConclusionThese data suggested that NDO1provided significant neuroprotective effects on cerebral ischemia injury and significantly improved functional outcome and increased angiogenesis after cerebral ischemia injury. |
PDF Full Text Request