Leptin在脑缺血性损伤中的神经保护作用和机制探讨
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摘要
脑缺血后梗死的病理变化为能量缺乏、脑水肿、炎症、自由基损伤、兴奋性氨基酸毒性作用和凋亡级联的启动等,寻找有效的神经保护因子,对脑缺血损伤的主要环节进行干预,减轻神经损伤、恢复神经功能一直是人们研究的热点。
瘦素(Leptin)是一种中枢性能量代谢调节因子,在炎症反应和创伤修复方面也具有重要功能,最近有关其神经保护作用的研究引起广泛关注,但是研究者对其作用机制还知之甚少。为了解Leptin在脑缺血再灌注损伤中的作用及其病理生理机制,本研究拟建立小鼠大脑中动脉栓塞(MCAO)模型、原代培养神经元、星形胶质细胞缺血缺氧/复氧损伤模型,探讨Leptin对脑缺血性损伤后神经元、星形胶质细胞功能状态的调节及其作用机制。本课题包括以下两个部分工作:
第一部分Leptin在小鼠脑缺血再灌注损伤中的神经保护作用和机制探讨
目的:通过小鼠局灶性脑缺血再灌注损伤模型,对Leptin的神经保护作用及机制进行探讨。
方法:用线栓法制作小鼠右侧大脑中动脉栓塞/再灌注(MCAO/R)模型。①检测~(125)I-Leptin在组织中的摄入和代谢情况;②激光多普勒监测大脑中动脉栓塞前后血流变化,小鼠神经功能评分、TTC染色测定脑梗死体积、HE染色检测组织病理学变化;③免疫组化检测脑组织中GFAP阳性星形胶质细胞和NSE阳性神经元;④检测梗死侧脑组织匀浆中LD/LDH、SOD/MDA、NO/NOS含量的动态变化;⑤TUNEL染色检测神经元凋亡,RT-PCR和免疫组化方法检测脑组织中凋亡相关基因bcl-2、bax、caspase-3的表达;⑥免疫组化方法检测脑组织中神经营养因子NGF、BDNF的表达。
结果:
1.小鼠局灶性脑缺血模型建立后血流量下降67%左右,再灌后开始恢复,再灌注24h后Leptin干预组血流量恢复情况较模型组明显改善(P＜0.01);
2.Leptin干预能够明显改善神经功能评分(P＜0.05),缩小脑梗死体积(P＜0.01),呈时间和剂量依赖性;
3.MCAO/R术后脑组织中的LD、LDH、MDA、NO和NOS含量明显升高、SOD含量明显降低(与假手术比较,P＜0.05);Leptin干预能够使LD、LDH、MDA、NO和NOS这几项损伤因子水平显著降低、SOD含量明显升高(与模型组比较,P＜0.05);
4.Leptin干预组神经细胞变性、坏死程度减轻,组织充血及水肿程度减轻;NSE表达明显增强(P＜0.001),GFAP阳性星形胶质细胞形态接近正常,GFAP表达明显增强(P＜0.01);
5.Leptin干预组脑组织缺血半影区TUNEL阳性细胞数明显少于模型组(P＜0.01);脑组织匀浆bcl-2 mRNA表达显著高于模型组,bax、caspase-3mRNA表达显著低于模型组(P＜0.01);免疫组织化学结果显示脑缺血半影区Bcl-2阳性细胞光密度值明显高于模型组,caspase-3阳性细胞光密度值明显低于模型组(P＜0.05);
6.免疫组织化学结果显示,Leptin干预组半影区NGF蛋白表达明显高于假手术组(P＜0.01),BDNF蛋白表达较假手术组和模型组显著增加(P＜0.01)。
结论:
1.外源性Leptin能够通过血脑屏障,在小鼠局灶性脑缺血再灌注损伤发挥改善神经功能、缩小脑梗死体积、减少神经元坏死等神经保护作用;
2.Leptin在小鼠脑缺血再灌注损伤后可显著降低缺血侧脑组织匀浆中LD、LDH、MDA、NO和NOS水平,升高SOD水平,在阻断脂质过氧化、促进自由基清除,维持内环境的稳定方面具有积极作用;
3.Leptin在脑缺血再灌注损伤中可通过上调抑凋亡基因bcl-2表达、下调促凋亡基因bax、caspase-3表达,减少神经细胞凋亡,改善卒中的预后;
4.Leptin在脑缺血再灌注损伤中能够上调神经营养因子NGF、BDNF蛋白表达,改善神经功能。
第二部分脑缺血性损伤中Leptin通过星形胶质细胞对神经元所发挥的作用
目的:通过神经元和星形胶质细胞缺血缺氧/复氧损伤模型,探讨Leptin-星形胶质细胞-神经元三者在脑缺血性损伤中的关系。
方法:①双重免疫荧光检测Leptin对MCAO/R小鼠脑组织缺血半影区星形胶质细胞和神经元的影响;②体外培养、纯化鉴定SD乳鼠神经元、脑皮质星形胶质细胞,换用无血清低糖培养基在5%CO_2+95%N_2密闭盒中孵育,建立缺血缺氧/复氧损伤模型,RT-PCR鉴定星形胶质细胞中Leptin受体Ob-Ra、Ob-Rb的表达;③RT-PCR和Western Blot方法检测Leptin对受损的星形胶质细胞凋亡相关基因bcl-2、bax、caspase-3表达的影响;④细胞毒性实验(MTT)检测星形胶质细胞和神经元存活率;⑤测定星形胶质细胞培养液中LDH、MDA的含量及神经元培养液中LDH漏出率;⑥采用Annexin V-FITC试剂盒检测星形胶质细胞和神经元凋亡;⑦免疫细胞化学方法检测星形胶质细胞胶质纤维酸性蛋白质(GFAP)的表达;⑧ELISA检测星形胶质细胞培养上清NGF表达。
结果:
1.模型组半影区星形胶质细胞过度增生,神经元较少;Leptin干预组半影区星形胶质细胞形态基本正常,GFAP表达较对照组明显增强,神经元数量较模型组明显增多;
2.Leptin对缺血缺氧/复氧损伤星形胶质细胞的影响:①与损伤组比较,Leptin干预组星形胶质存活率显著升高,细胞培养上清中LDH、MDA水平显著降低(P＜0.05);②与损伤组比较,Leptin干预组凋亡率显著降低(P＜0.01),抑凋亡基因bcl-2 mRNA和蛋白表达水平显著上调(P＜0.01),促凋亡基因bax、caspase-3 mRNA和蛋白表达水平显著下调(P＜0.01),呈剂量依赖性;③Leptin干预组细胞培养液中NGF水平较缺血缺氧损伤组明显升高(P＜0.05);
3.Leptin与星形胶质细胞培养液均可明显提高缺血缺氧/复氧损伤神经元存活率、降低LDH漏出率、减少神经元凋亡(P＜0.05),二者叠加后效果增强。
结论:
1.Leptin能够提高神经元、星形胶质细胞对缺血缺氧/复氧损伤的耐受性;
2.Leptin能够减少缺血缺氧/复氧损伤神经元、星形胶质细胞凋亡,后者与凋亡相关基因bcl-2表达上调、caspase-3表达下调有关;
3.Leptin能够促进星形胶质细胞生成神经营养因子NGF;
4.Leptin既可直接保护受损神经元,还可以间接通过星形胶质细胞对神经元发挥保护作用,二者叠加后效果增强。
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.
PartⅠProtective effect of leptin on cerebral ischemia/reperfusion injury in mice
Objective: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.①The intake of ~(125)I-Leptin were determined by counting theγvalue in defferent tissues.②The 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.③The expression of GFAP~+ astrocytes and NSE~+ neuron were detected by immunohistochemistry.④The 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-3.⑥Immunohistochemistry 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.
PartⅡEffects of leptin on neuron in cerebral ischemia injury via astrocytes
Objective: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%CO_2+95%N_2 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:①Compared 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).②Compared 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).③The 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.
瘦素(Leptin)是一种中枢性能量代谢调节因子,在炎症反应和创伤修复方面也具有重要功能,最近有关其神经保护作用的研究引起广泛关注,但是研究者对其作用机制还知之甚少。为了解Leptin在脑缺血再灌注损伤中的作用及其病理生理机制,本研究拟建立小鼠大脑中动脉栓塞(MCAO)模型、原代培养神经元、星形胶质细胞缺血缺氧/复氧损伤模型,探讨Leptin对脑缺血性损伤后神经元、星形胶质细胞功能状态的调节及其作用机制。本课题包括以下两个部分工作:
第一部分Leptin在小鼠脑缺血再灌注损伤中的神经保护作用和机制探讨
目的:通过小鼠局灶性脑缺血再灌注损伤模型,对Leptin的神经保护作用及机制进行探讨。
方法:用线栓法制作小鼠右侧大脑中动脉栓塞/再灌注(MCAO/R)模型。①检测~(125)I-Leptin在组织中的摄入和代谢情况;②激光多普勒监测大脑中动脉栓塞前后血流变化,小鼠神经功能评分、TTC染色测定脑梗死体积、HE染色检测组织病理学变化;③免疫组化检测脑组织中GFAP阳性星形胶质细胞和NSE阳性神经元;④检测梗死侧脑组织匀浆中LD/LDH、SOD/MDA、NO/NOS含量的动态变化;⑤TUNEL染色检测神经元凋亡,RT-PCR和免疫组化方法检测脑组织中凋亡相关基因bcl-2、bax、caspase-3的表达;⑥免疫组化方法检测脑组织中神经营养因子NGF、BDNF的表达。
结果:
1.小鼠局灶性脑缺血模型建立后血流量下降67%左右,再灌后开始恢复,再灌注24h后Leptin干预组血流量恢复情况较模型组明显改善(P＜0.01);
2.Leptin干预能够明显改善神经功能评分(P＜0.05),缩小脑梗死体积(P＜0.01),呈时间和剂量依赖性;
3.MCAO/R术后脑组织中的LD、LDH、MDA、NO和NOS含量明显升高、SOD含量明显降低(与假手术比较,P＜0.05);Leptin干预能够使LD、LDH、MDA、NO和NOS这几项损伤因子水平显著降低、SOD含量明显升高(与模型组比较,P＜0.05);
4.Leptin干预组神经细胞变性、坏死程度减轻,组织充血及水肿程度减轻;NSE表达明显增强(P＜0.001),GFAP阳性星形胶质细胞形态接近正常,GFAP表达明显增强(P＜0.01);
5.Leptin干预组脑组织缺血半影区TUNEL阳性细胞数明显少于模型组(P＜0.01);脑组织匀浆bcl-2 mRNA表达显著高于模型组,bax、caspase-3mRNA表达显著低于模型组(P＜0.01);免疫组织化学结果显示脑缺血半影区Bcl-2阳性细胞光密度值明显高于模型组,caspase-3阳性细胞光密度值明显低于模型组(P＜0.05);
6.免疫组织化学结果显示,Leptin干预组半影区NGF蛋白表达明显高于假手术组(P＜0.01),BDNF蛋白表达较假手术组和模型组显著增加(P＜0.01)。
结论:
1.外源性Leptin能够通过血脑屏障,在小鼠局灶性脑缺血再灌注损伤发挥改善神经功能、缩小脑梗死体积、减少神经元坏死等神经保护作用;
2.Leptin在小鼠脑缺血再灌注损伤后可显著降低缺血侧脑组织匀浆中LD、LDH、MDA、NO和NOS水平,升高SOD水平,在阻断脂质过氧化、促进自由基清除,维持内环境的稳定方面具有积极作用;
3.Leptin在脑缺血再灌注损伤中可通过上调抑凋亡基因bcl-2表达、下调促凋亡基因bax、caspase-3表达,减少神经细胞凋亡,改善卒中的预后;
4.Leptin在脑缺血再灌注损伤中能够上调神经营养因子NGF、BDNF蛋白表达,改善神经功能。
第二部分脑缺血性损伤中Leptin通过星形胶质细胞对神经元所发挥的作用
目的:通过神经元和星形胶质细胞缺血缺氧/复氧损伤模型,探讨Leptin-星形胶质细胞-神经元三者在脑缺血性损伤中的关系。
方法:①双重免疫荧光检测Leptin对MCAO/R小鼠脑组织缺血半影区星形胶质细胞和神经元的影响;②体外培养、纯化鉴定SD乳鼠神经元、脑皮质星形胶质细胞,换用无血清低糖培养基在5%CO_2+95%N_2密闭盒中孵育,建立缺血缺氧/复氧损伤模型,RT-PCR鉴定星形胶质细胞中Leptin受体Ob-Ra、Ob-Rb的表达;③RT-PCR和Western Blot方法检测Leptin对受损的星形胶质细胞凋亡相关基因bcl-2、bax、caspase-3表达的影响;④细胞毒性实验(MTT)检测星形胶质细胞和神经元存活率;⑤测定星形胶质细胞培养液中LDH、MDA的含量及神经元培养液中LDH漏出率;⑥采用Annexin V-FITC试剂盒检测星形胶质细胞和神经元凋亡;⑦免疫细胞化学方法检测星形胶质细胞胶质纤维酸性蛋白质(GFAP)的表达;⑧ELISA检测星形胶质细胞培养上清NGF表达。
结果:
1.模型组半影区星形胶质细胞过度增生,神经元较少;Leptin干预组半影区星形胶质细胞形态基本正常,GFAP表达较对照组明显增强,神经元数量较模型组明显增多;
2.Leptin对缺血缺氧/复氧损伤星形胶质细胞的影响:①与损伤组比较,Leptin干预组星形胶质存活率显著升高,细胞培养上清中LDH、MDA水平显著降低(P＜0.05);②与损伤组比较,Leptin干预组凋亡率显著降低(P＜0.01),抑凋亡基因bcl-2 mRNA和蛋白表达水平显著上调(P＜0.01),促凋亡基因bax、caspase-3 mRNA和蛋白表达水平显著下调(P＜0.01),呈剂量依赖性;③Leptin干预组细胞培养液中NGF水平较缺血缺氧损伤组明显升高(P＜0.05);
3.Leptin与星形胶质细胞培养液均可明显提高缺血缺氧/复氧损伤神经元存活率、降低LDH漏出率、减少神经元凋亡(P＜0.05),二者叠加后效果增强。
结论:
1.Leptin能够提高神经元、星形胶质细胞对缺血缺氧/复氧损伤的耐受性;
2.Leptin能够减少缺血缺氧/复氧损伤神经元、星形胶质细胞凋亡,后者与凋亡相关基因bcl-2表达上调、caspase-3表达下调有关;
3.Leptin能够促进星形胶质细胞生成神经营养因子NGF;
4.Leptin既可直接保护受损神经元,还可以间接通过星形胶质细胞对神经元发挥保护作用,二者叠加后效果增强。
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.
PartⅠProtective effect of leptin on cerebral ischemia/reperfusion injury in mice
Objective: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.①The intake of ~(125)I-Leptin were determined by counting theγvalue in defferent tissues.②The 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.③The expression of GFAP~+ astrocytes and NSE~+ neuron were detected by immunohistochemistry.④The 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-3.⑥Immunohistochemistry 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.
PartⅡEffects of leptin on neuron in cerebral ischemia injury via astrocytes
Objective: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%CO_2+95%N_2 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:①Compared 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).②Compared 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).③The 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.
引文
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24 Guo Z,Jiang H,Xu X,et al.Leptin-mediated cell survival signaling in hippocampal neurons mediated by JAK STAT3 and mitochondrial stabilization.J Biol Chem,2008,283(3):1754-1763.
25 Kinzig KP,Scott KA,Hyun J,et al.Altered hypothalamic signaling and responses to food deprivation in rats fed a low-carbohydrate diet.Obes Res,2005,13(10):1672-1682.
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