CsA对树鼩海马微灌流谷氨酸和钙所致线粒体应激的影响

Effect of CsA on mitochondria stress after microperfusing glutamate and calcium into tree shrews' hippocampus

目的:观察环孢菌素A(CsA)对树鼩海马由谷氨酸(Glu)及钙(Ca2+)引起微环境改变所致线粒体应激的影响,并探讨其分子机制。方法:单泵等速微灌流系统行树鼩海马Glu及Ca2+微灌流,24 h后免疫组化法检测海马神经元细胞色素C(Cyt C)蛋白表达;低温差速离心分离海马神经元线粒体和胞质部分,免疫印迹(West-ern blotting)法检测Cyt C在胞内表达空间分布;实时荧光定量PCR技术检测海马caspase-3及caspase-9 mRNA的含量。微灌流Glu和Ca2+溶液后6 h于舌下iv CsA40 mg/kg BW,24 h后观察上述指标的改变。结果:树鼩海马微灌流Glu和Ca2+溶液后24 h,海马神经元Cyt C表达增强,而线粒体Cyt C含量显著下降,同时胞质部分可见CytC;海马组织caspase-3、caspase-9mRNA明显升高;微灌流后6 h静脉注射CsA组,海马神经元Cyt C表达显著减少,而线粒体Cyt C含量则显著增加,胞质部分未见Cyt C;海马组织caspase-3、caspase-9 mRNA降低。结论:海马微环境中Glu与Ca2+的大量堆积,可促进线粒体Cyt C释放,激活caspase级联反应而导致线粒体应激;CsA的神经保护效应可与其抑制线粒体通透性转导孔(MPT)开放,防止Cyt C释放及减少caspase-3和caspase-9的活化有关。

AIM： To observe the changes of glutamate and calcium within the hippeeampal microenvironment in mitochondrial stress. METHODS： A lateral hippocampus was microperfused with glutamate and calcium chloride solution by a kind of single - pumped push - pull perfusion system in Tree Shrews. At 24 h, the expression of cytochrome C （Cyt C）was observed by immunochemistry. Also, the hippocampus was removed, then mitochondria and cytoplasmic frag- ment were divided by low temperature centrifugation and the distribution of cytochrome C was assessed through Western blotting. The relative amounts of caspase - 3 and caspase - 9 mRNA were evaluated by real time fluorescence polymerase chain reaction. In the treated group, cyclosporin A （CsA,40 mg/kg） was intravascularly injected at 6 h after perfusion of glutamate- calcium chloride solutions into the hippocampus and inspected the above - mentioned items at 24 h. RESULTS ： In the glutamate - calcium group, compared with the control group, cytochrome C immunoreactivity increased and the content of hippeeampal mitochondrial cytochrome C decreased. Also, the cytochrome C was detected in cytosol. Cyclosporin A treatment at 6 h after microperfusion, the cytochrome C expression weakened and no Cyt C in cytosol fraction was observed. By real time PCR, in relation to the control group, the caspase - 3 and caspase - 9 mRNA was higher in the glutamate -calcium group. Cyclosporin A treatment cut down both caspase -3 and caspase -9 mRNA contents. CONCLUSION： The accumulation of glutamate and calcium may promote Cyt C release, caspase cascade activation and the mitochondrial stress. The neuroprotection of CsA may results from uniquely inhibiting the mitochondrial permeability transition pore, and preventing Cyt C release and caspase activation.