Study On The Protective Effect And Relative Mechanism Of Leptin On Cerebral Ischemia Injury

The neuropathologic change in cerebral ischemia injury include energy deficiency,hydropsia,inflammation,injury by free radicle,the toxicity of excitatory amino acids,and the occurrence of apoptosis cascade.So,development of new interventions geared toward a wider threrapeutic window is necessary to meet the large need for this important and undertreated disorder,to reduce ischemia brain damage and improve neurological function.Leptin acts on hypothalamic neuronal targets to regulate energy balance and neuroendocrine function.However,recent research has shown that leptin is also involved in neuroprotection,but few are known about its mechanism.We prepare to make the focal ischemia-reperfusion model by stuture occlusion of right middle cerebral artery(MCAO) in mice,and make the ischemia/hypoxia model in cultured neuron and astrocytes,aimed at investigation the protective effect and neuropathologic mechanism of leptin on cerebral ischemia injury.Our studies include the following two parts.Partâ… Protective effect of leptin on cerebral ischemia/reperfusion injury in miceObjective:To investigate the protective effect and neuropathologic mechanism of leptin on cerebral ischemia/reperfusion injury in mice.Methods:Transient focal cerebral ischemia injury model in mice was induced by occlusion of the right middle cerebral artery.â‘ The intake of ¹²⁵I-Leptin were determined by counting theγvalue in defferent tissues.â‘¡The volume of blood flow were monitoring by laser droppler perfusion imager,the infarct volume and neurological function scores were determined by the method of TTC staining and the Longa's score,the histopathological change was observed after HE staining.â‘¢The expression of GFAP⁺ astrocytes and NSE⁺ neuron were detected by immunohistochemistry.â‘£The levels of lactic acid(LD),lactate dehydrogenase (LDH),malondialdehyde(MDA),supreoxide dismutase(SOD),nitric oxide(NO), and nitric oxide synthases(NOS) in icchemia brain tissue were measured by colorimetry.⑤The effect of Leptin on brain tissue nerve cell apoptosis was detected by TUNEL staining,reverse transcription polymerase chain reaction(RT-PCR) and immunohistochemistry were employed to assess the mRNA and proteinum expression of bcl-2,bax and caspase-3.â‘¥Immunohistochemistry was employed to assess the proteinum expression of NGF and BDNF.Results:1.The volume of blood flow decreased about 67%after MCAO operation, recovered gradually after reperfusion,and there is distinct difference in leptin intervention group and ischemia groupt after 24h later(P＜0.01).2.Treatment with leptin could dose-dependently decrease neurological deficit induced by transient ischemia(P＜0.05),and reduce cerebral infarct volume (P＜0.01).3.After MCAO operation,the levels of LD,LDH,MDA,NO and NOS in ischemia brain homogenate increased significantly,the levels of SOD decreased significantly,compared with sham group(P＜0.05).Treatment with leptin could markedly decrease LD,LDH,MDA,NO and NOS levels,and increase SOD level in brain homogenate,compared with ischemia group(P＜0.05).4.The HE staining result showed that,the phenomenon such as neuron cell degeneration,necrosis,hyperemia and edema in ischemia cerebral tissue of Leptin intervention group were much less than ischemia group.The immunohistochemistry staining result showed that the expression of NSE and GFAP of Leptin intervention group increased significantly than that of ischemia group(P＜0.001 & P＜0.01).5.TUNEL staining result showed that positive cells in ischemia penumbra region of leptin intervention group were much less than that of ischemia group(P＜0.01). RT-PCR result showed that the mRNA expression of bcl-2 in leptin intervention group was much higher than ischemia group,and the mRNA expression of bax and caspase-3 was much lower than ischemia group(P＜0.01).The immunohistochemistry staining result showed that bcl-2 immune positive cells's optical density values in ischemia penumbra region of leptin intervention group was much more than that of ischemia group(P＜0.01),however,caspase-3 immune positive cells's optical density values in ischemia penumbra region of leptin intervention group was much less than that of ischemia group(P＜0.05). 6.Immunohistochemistry staining showed that NGF immune positive cell's optical density values in ischemia penumbra region of leptin intervention group and ischemia group were much higher than that of sham group(P＜0.01),and proteinum expression of BDNF in ischemia penumbra region of leptin intervention group increase significantly than that of sham and ischemia group (P＜0.01).Conclusion:1.Exogenous leptin could enter across the blood-brain barrier,and significantly improved the nerve function,reduced the infart volume,eased the damage of organization of mice with focal cerebral ischemia/reperfusion injury.2.Leptin could decrease significantly the LD,LDH,MDA and NO level and increase significantly the SOD level of injury brain homogenate,it suggested that leptin exert a role of protective effect in cerebral ischemia/reperfusion injury through inhibit lipid peroxidation,stabilize internal environment.3.Leptin could decrease significantly the apoptosis of nerve cells,and it could increase obviously the mRNA and proteinum expression of anti-apoptosis factor bcl-2,and decrease the mRNA and proteinum expression of promote-apoptosis factor caspase-3.4.Leptin could increase significantly the protein level of NGF and BDNF,and improve the nerve function.Partâ…¡Effects of leptin on neuron in cerebral ischemia injury via astrocytesObjective:To investigate the interaction of leptin-astrocytes-neuron in the ischemia/hypoxia/reoxygenation injury model of neuron and astrocytes.Methods:1.Double immunofluorescence staining detected the effect of leptin on focal ischemia injury cerebral astrocytes and neuron in mice with MCAO/R.2.The cerebral neuron and astrocytes isolated from neonatal SD rats were purified and identified,then incubated with 5%CO₂+95%N₂ in serum- and glucose-free medium to induce ischemic/hypoxia/reoxygenation injury.The expression of leptin receptors Ob-Ra and Ob-Rb in astrocytes were observed by RT-PCR.3.The effct of leptin on the expression of apoptosis factor bcl-2,bax,caspase-3 in injury astrocytes was detected by RT-PCR and Western Blot.4.The cellular viability of injury of neuron and astrocytes was detected by MTT assay.5.The supernatant contents of lactate dehydrogenase(LDH) activity and malonaldehyde(MDA) were analyzed with colorimetry.6.The apoptosis of neuron and astrocyte were detected with Annexin V-FITC kit.7.The level of glial fibrillary acidicprotein(GFAP) was detected with fluorescence immunocytochemistry.8.The supernatant contents of NGF were analyzed with ELISA.Results:1.The astrocytes excessive hyperplasia,and the number of neuron in ischemia penumbra region of ischemia group was less than leptin intervention group.In ischemia penumbra region of leptin intervention group,the morphology of astrocytes similar to normal,and the expression of GFAP and the number of neuron were much more than that of ischemia group.2.The effect of leptin on Ischemia/hypoxia/reoxygenation injury astroctyes:â‘ Compared with the ischemia group,the cellular viability of leptin intervention group increased significantly,and the LDH and MDA levels in supernatant contents of leptin intervention group decreased significantly(P＜0.05).â‘¡Compared with ischemia group,the astrocytes apoptosis of leptin intervention group significantly decreased(P＜0.01).The mRNA and proteinum expression level of anti-apoptosis factor bcl-2 in leptin intervention group was much higher than ischemia group(P＜0.01),and the mRNA and proteinum expression of bax and caspase-3 was much lower than ischemia group (P＜0.01).â‘¢The level of NGF in astrocytes supernatant contents of leptin intervention group increased obviously,compared with that of ischemia group (P＜0.05).3.Leptin and/or astrocyte conditioned medium(ACM) could increase the neuron viability,decrease the LDH level of neuron supernatant contents,and neuron apoptosis(P＜0.05).And the effect increased obviously while they interacted with each other.Conclusion:1.Leptin could improve the tolerance of neuron and astrocytes to ischemia/hypoxia/reoxygenation injury.2.Adding leptin could induce significant decrease of the apoptosis neuron and astrocytes,and relevant to increase of bcl-2 expression and decrease of MDA and LDH level.3.Leptin could induce higher level of NGF produced by injury astrocytes.4.Lepitn had directly neuroprotection,and also could exert its role indirectly via astrocytes in ischemia injury.And the effect of neuroprotection increased obviously while they interacted with each other.