The Neuroprotective Effect Of Leonurine And The Underlying Mechanism In Experimental Ischemic Stroke

Objective: Ischemic stroke is one of the three leading causes of death in the world, with high rate of morbidity, disability and mortality. Brain tissue injuries secondary to ischemia aggravated the illness and inhibited the recovery of patients. Despite advances in the understanding of the pathophysiology of cerebral ischemia in recent years, therapeutic options remain limited. After ischemia, damnification of the neuronal cell has two forms necrosis and apoptotic. As the"energy center"of the cell, the mitochondrial play the important role in the two forms. Uncoupling Protein 4 (UCP4) is a kinds of transporters located in the inner membrane of mitochondrion and expressed predominantly in brain. Actived UCP4 can dissociate oxidative phosphorylation from respiration. Overexpression of UCP4 can regulate the mitochondrion membrane potential and calcium homeostasis, reduce oxidative stress and suppress apoptosis. Oxidative stress is the imbalance state of antioxidant defense mechanism and the production of free radicals. Reactive oxygen species (ROS) is predominantly produced in mitochondria. Oxygen free radicals resulted from oxidative stress are directly implicated in the initiation of apoptosis. Although the possible mechanisms modulating apoptosis during ischemia are not well-understood, recent animal studies and clinical observation suggests that the balance between anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax plays a major role in regulating apoptosis. Leonurine is an alkaloid present in Herba Leonuri. Leonurine have uterotonic action and antiplatelet aggregation activities. Previous researches show that Leonurine has cardioprotective effects on ischemic myocardium by increasing anti-oxidant, anti-apoptotic ability and protecting mitochondrial function. The purpose of this study is to evaluate the potential neuroprotective effect of Leonurine and underlying mechanisms after cerebral ischemia induced in male adult Sprague-Dawly rats by permanent middle cerebral artery occlusion (MCAO).Methods: Healthy, male Sprague-Dawley rats were subjected to modified permanent MCAO as described by Longa previously. Rats were randomly assigned to four groups: Sham operated group(Sham), stroke with vehicle group (Vehicle), stroke with low-dose (7.5 mg/kg per day) leonurine group (Leo-L) and stroke with high-dose (15 mg/kg per day) leonurine group (Leo-H). 2 h after MCAO, neurological deficit was evaluated and leonurine was administered, then once daily on the following days. At 24 h, 72 h and 7 d after ischemia neurological deficit was evaluated, brain water content was measured by wet-dry method, infarct size were analyzed with 2, 3, 5- triphenyltetrazolium chloride(TTC)staining; immunohistochemistry, western blot and reverse transcription–polymerase chain reaction(RT-PCR)were used to analyze the expression of UCP4, Bcl-2 and Bax; activites of superoxide dismutase (SOD) and catalase (CAT), and malondiadehyde (MDA) content were measured by spectrophotometer to evaluate the oxidation reaction in brain tissue.Results:1 Rats in Sham group had a neurological score of zero at all time points. Rats in Vehicle group, high dose group and low dose group performed a right palsy. Neurological deficit score in high dose group was significantly decreased compared with Vehicle group (P < 0.05). There was no significant difference in the neurological deficit score between Vehicle group and low dose group (P > 0.05).2 The brain water content was reduced in Leo-H group compared with that in Vehicle group and Leo-L group from 24 h to 7 d (P < 0.05), and also significantly lower in Leo-L group than in Vehicle group (P < 0.05) respectively at 72 h and 7 d.3 Infarct size was decreased in Leo-H group compared with that in Vehicle group from 24 h to 7 d (P < 0.05), but no statistical significance was observed between Leo-L group and Vehicle group(P > 0.05). 4 High dose of leonurine significantly upregulated the protein and mRNA levels of UCP4 and Bcl-2, and downregulated Bax at these time points (P < 0.05). Low dose of leonurine also upregulated the protein and mRNA levels of UCP4 and Bcl-2 and downregulated Bax, but the trend is unstable.5 High dose of leonurine enhanced the activities of SOD and CAT compared these in Vehicle group at these time points (P < 0.05). MDA levels were significantly lower in the Leo-H group than in Vehicle group at all these time points (P < 0.05). The effect of low dose of leonurine is unstable.Conclusions: Leonurine administration protacted ischemic cortex through increasing SOD, CAT, UCP4 and Bcl2 activities and decreasing Bax, MDA expression.