Study Of Morroniside Protecting Nerve Cell From Apoptosis In Rats With Focal Cerebral Ischemial/Reperfusion

Ischemic cerebrovascular disease is a kind of common critical illness in clinic, and leading to high mortality and morbidity. Timely thrombolysis therapy is the precondition and foundation of the success for the remedy of cerebral ischemia. But cerebral ischemia-reperfusion results in a delayed neuronal damage, leads to the apoptosis of nerve cell. At the same time, activated genes and proteins related to apoptosis can start and promote the generation of apoptosis. Currently, many studies showed that the major damage mechanisms of neuron apoptosis include mitochondrion impairment, calcium overload, increased levels of oxygen radicals and so on. The previous study in our laboratory indicated that morroniside which was isolated from Cornus can inhibit calcium overload and apoptosis of neuroblastoma SH-SY5Y cells. Furthermore, Morroniside have shown neuroprotective function by inhibiting nerve cell apoptosis after cerebral ischemia-reperfusion 72 hours in Wistar rat. But the protective effects of morroniside in SD rat after cerebral ischemia-reperfusion 7 days have never been reported.AimTo observe the anti-apoptosis effects of morroniside in focal cerebral ischemia-reperfusion model of SD rat, and offer a new neuroprotective drug for the therapy of ischemic cerebrovascular disease. MethodsThe animal model was made by the occlusion of middle cerebral artery with suture embolus, ischemia for 30 minutes, and reperfusion for 7 days. The rats were randomly divided into sham operated group, model group and morroniside-low(30mg/kg), morroniside-middle(90mg/kg), morroniside-high(270mg/kg), positive control drug(vitamin E). To examine the apoptosis in rat cortex ischemic penumbra, we used terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) in rats on the seventh day after the operation. The activity of enzymes related to apoptosis including Cleaved caspase-3 and Cleaved caspase-9 in ischemia hippocampus of rat brain were detected with spectrophotometer. The content of Bcl-2,Bax,Cyt-c and the ratio of Bcl-2/Bax were determined by western blot. The expression of Cleave caspase-3,Bax,Cyt-c in ischemia hippocampus of rat brain were measured by immunohistochemistry method.Results1,Compared with vehicle group, morronside significantly decrease the development of apoptosis in nerve cell after cerebral ischemia-reperfusion 7 days,and it showed a clear dose dependent relationship(P<0.05).2,Cerebral ischemia-reperfusion injury may upset the balance of Bcl-2 and Bax which belong to Bcl-2 family, and morroniside can degrade the content of Bax, enhance the expression of Bcl-2, make the ratio of Bcl-2/Bax raise(P<0.05).3,Morroniside can significantly degrade the level of Cyt-c in cortex and ischemia hippocampus of rat brain, and it showed a clear dose dependent relationship(P<0.05).4,Compared with vehicle group, morroniside significantly decreased the content of enzymes related to trigger and execute apoptosis (P<0.05). ConclusionMorroniside can decrease the development of nerve cell apoptosis,further protecting cerebral ischemia-reperfusion injury. The probalble mechanisms of morroniside neuroprotective function are drawing back the balance between Bcl-2 and Bax; inhibiting the expression of Cyt-c and it's release from mitochondrion; decreasing the activity of enzymes related to trigger and execute apoptosis.