Protective Effects Of Leptin Against Cerebral Ischemia/reperfusion-induced Injury And The Involved Mechanisms

Background Stroke is an acute central nervous system disease characterized by the suddenly loss of blood circulation to the brain, resulting in a injury of neurologic function. Ischemic stroke is the leading causes of death worldwide, which accounts for about 4/5 of stroke including ischemic and hemorrhagic stroke, with high rate of incidence, mutilation and mortality. On the beginning of stroke fast and effective measures can decrease the mortality and recurrence rate. Until now few therapies have been effective other than fibrinolytic therapy, including urokinase (UK), the tissue plasminogen activator (t-PA), recombinant tissue-type plasminogen activator (rt-PA),single chain urokinase-type plasminogen activator (sCUPA) and anisoylated plasminogen streptokinase activator complex (APSAC). Despite the fact that efficient treatments has been carried out by a large number of clinical trials to cure ischemic stroke, but the result is not of optimistic. Acute ischemic stroke are instigated by a number of physiologic factors, including overflow of the reactive oxygen species(ROS), calcium overload, excitotoxic and inflammation. Therefore, many researchers have been contributing themselves to elucidate the pathogenesis of ischemic stroke and explore new neuroprotective treatments.Leptin is a peptide hormone produced by adipose tissue and acts in brain centers to control critical physiological functions. Leptin receptors (ObRs) belong to cytokine receptor superfamily. Alternative splicing of the ObRs gene are classified as six leptin receptor forms (ObRa to ObRf). The long isoform ObRb is essential for mediating leptin’s intracellular signal transduction. Up to now, the effect of leptin in the cerebral ischemia/reperfusion injury is still unclear. Therefore, the present research was designed to elucidate the role of leptin in ischemic stroke, and to explore the molecular mechanisms involved in neuroprotection finding a new target for stroke therapy.Methods Forty adult male C57BL/6 mice (six to seven weeks of age), were randomly saperated into five groups (n=8): SHAM, MCAO+NS (equivalent dose of 0.9% saline administered i.c.v.) or MCAO+Leptin, 0.5, 1.0, 2.0μg/10g of leptin, respectively, at 30 min before MCAO by i.c.v. injection. The reversible ischemia procedure(occlusion-reperfusion) was induced by MCAO according to the method described by Longa et al. The 2,3,5-triphenyl four azole nitrogen chloride (TTC) was performed measured the infarct volume at 24 h after reperfusion, to conclude the most effective concentration of leptin. Seventy adult male C57BL/6 mice were randomly saperated into three teams (n=24): SHAM, MCAO+Leptin (1.0μg/10g,i.c.v.) or MCAO+NS.Post-stroke motor behavior coordination was assessed throught the means of the rotarod, adhesive removal and balance beam walk test. After the operation, one section was treated with Nissl staining for histological assessment of damage. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)was used to observe the neurocell apoptosis. Antibody against cleaved caspase-3 immunofluorescence was performed, and western blot analysis was used to measure the protein expression levels of Bcl-2, Bax and PGC1-α.Results1. Compared with the sham group, after 24h reperfusion, the infarct volume of the vehicle group was larger, the infarct volume of the leptin (1.0μg/10g)-treated groups was expressively smaller than that of vehicle control ones. Middle dose of leptin attenuate cerebral ischemic injury in mice is obvious.2. This impairment was significantly alleviated by treatment with leptin using the rotarod test, adhesive removal and balance in 3, 4 days after that. Leptin therapy after surgery significantly improved tMCAO mouse movement, sensory disturbances, ataxia(P <0.05). These results suggest that leptin reduces cerebral ischemia-reperfusion induced damage and promotes recovery of neurological function injury.3. Compared with the sham group, there is a significant increase in TUNEL positive cell in MCAO+NS in cerebral ischemia penumbra, meanwhile cleaved caspase-3 protein significantly increased, decreased Bcl-2/Bax relative expression (P<0.01). The expression of this apoptosis indexs of Bcl-2/Bax are increased significantly (P <0.01) after 24 h ischemia-reperfusion in MCAO+Leptin, meanwhile cleaved caspase-3 protein significantly decreased.4. Compared with the sham group, the PGC1-α expression in ischemic mice significantly reduced. Leptin treatment (MCAO+Leptin) significantly increased PGC1-α expression compared with model group (P <0.01). It indicates that leptin may reduced the level of mitochondrial apoptosis by increasing PGC1-α expression, thereby reduced cerebral ischemia and reperfusion-induced cell damage.Conclusion1. Leptin haves an important neuroprotective role to cerebral ischemia-reperfusion induced damage and promotes recovery of neurological function defect.2. Leptin can inhibit the apoptosis of ischemia reperfusion induced injury, the mechanism of it may be up-regulated apoptotic inhibitory protein Bcl-2 expression and down-regulated pro-apoptotic protein Bax expression, providing a new clinical ischemic therapy.3. Leptin may increase the expression of PGC1-α to decrease neuronal apoptosis mitochondria, reducing brain damage.