尼莫地平对脑缺血大鼠自噬的影响
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摘要
目的:研究尼莫地平对大鼠脑缺血自噬的影响及治疗作用。方法:将90只SD大鼠随机均分为大脑中动脉局灶性线栓法(MCAO)模型组(缺血模型组)、尼莫地平组、假手术组,每组30只。根据Zea-Longa评分选取实验用模型鼠。制备MCAO大鼠模型,采用蛋白质印迹法检测LC3-Ⅱ、Beclin-1、m-TOR/p-mTOR蛋白表达。应用TTC染色观察脑组织梗死体积,HE染色观察脑组织细胞损伤状况。制备星形胶质细胞氧糖剥夺(OGD)模型,将细胞分为空白对照组、OGD模型组、尼莫地平组,采用蛋白质印迹法检测LC3-Ⅱ、Beclin-1、m-TOR/p-mTOR蛋白,CCK8实验检验星形胶质细胞活性。结果:与假手术组相比,缺血模型组LC3-Ⅱ、Beclin-1蛋白以及m-TOR/p-mTOR蛋白表达明显增加(P均<0.05)。与缺血模型组相比,尼莫地平组LC3-Ⅱ、Beclin-1蛋白表达明显降低,m-TOR蛋白表达明显增加(P <0.05),脑组织梗死体积和细胞死亡数明显降低(P均<0.05)。在OGD试验中,与OGD模型组相比,尼莫地平组细胞活性升高,LC3-Ⅱ、Beclin-1蛋白表达降低(P <0.05)。结论:尼莫地平可抑制脑缺血大鼠自噬发生,降低自噬对脑组织的损伤。
Objective: To study the effect of nimodipine on autophagy in rats with cerebral ischemia and its therapeutic effect. Methods: Ninety SD rats were randomly divided into nimodipine group,sham operation group,and middle cerebral artery focal line suture method(MCAO) model group(ischemic model group),30 in each group. Experimental model rats were selected according to the Zea-Longa score. The MCAO rat model was prepared and the expression level of LC3-Ⅱ,Beclin-1,m-TOR/p-m TOR proteins were detected by Western blotting. The infarct volume of brain tissue was observed by TTC staining,and the damage of brain tissue was observed by HE staining. The astrocyte oxygen glucose deprivation(OGD) model was constructed,and the cells were divided into blank control group, OGD model group and nimodipine group. LC3-Ⅱ, Beclin-1, m-TOR/p-m TOR protein expression were determined by Western blotting; astrocyte activity was tested by CCK8 assay. Results:Compared with the sham operation group,the ischemic model group showed higher expression of LC3-Ⅱ,Beclin-1 and m-TOR/p-m TOR protein(P < 0.05). Compared with the ischemic model group,the nimodipine group showed lower protein expression of LC3-Ⅱ,Beclin-1 and higher expression of m-TOR(P < 0.05),with decreased infarct volume of brain tissue and the number of cell death.In the OGD test,compared with the OGD model group,the cell activity in the nimodipine group was increased,and the expression of LC3-Ⅱand Beclin-1 protein was decreased significantly(P < 0.05).Conclusion: Nimodipine could inhibit autophagy and reduce the damage of brain tissue in rats with cerebral ischemia.
Objective: To study the effect of nimodipine on autophagy in rats with cerebral ischemia and its therapeutic effect. Methods: Ninety SD rats were randomly divided into nimodipine group,sham operation group,and middle cerebral artery focal line suture method(MCAO) model group(ischemic model group),30 in each group. Experimental model rats were selected according to the Zea-Longa score. The MCAO rat model was prepared and the expression level of LC3-Ⅱ,Beclin-1,m-TOR/p-m TOR proteins were detected by Western blotting. The infarct volume of brain tissue was observed by TTC staining,and the damage of brain tissue was observed by HE staining. The astrocyte oxygen glucose deprivation(OGD) model was constructed,and the cells were divided into blank control group, OGD model group and nimodipine group. LC3-Ⅱ, Beclin-1, m-TOR/p-m TOR protein expression were determined by Western blotting; astrocyte activity was tested by CCK8 assay. Results:Compared with the sham operation group,the ischemic model group showed higher expression of LC3-Ⅱ,Beclin-1 and m-TOR/p-m TOR protein(P < 0.05). Compared with the ischemic model group,the nimodipine group showed lower protein expression of LC3-Ⅱ,Beclin-1 and higher expression of m-TOR(P < 0.05),with decreased infarct volume of brain tissue and the number of cell death.In the OGD test,compared with the OGD model group,the cell activity in the nimodipine group was increased,and the expression of LC3-Ⅱand Beclin-1 protein was decreased significantly(P < 0.05).Conclusion: Nimodipine could inhibit autophagy and reduce the damage of brain tissue in rats with cerebral ischemia.
引文
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[2]Klionsky DJ,Emr SD.Autophagy as a regulated pathway of cellular degradation[J].Science,2000,290(5497):1717-1721.
[3]Alirezaei M,Kemball CC,Flynn CT,et al.Short-term fasting induces profound neuronal autophagy[J].Autophagy,2010,6(6):702-710.
[4]Ecker N,Mor A,Journo D,et al.Induction of autophagic flux by amino acid deprivation is distinct from nitrogen starvation-induced macro autophagy[J].Autophagy,2010,6(7):879-890.
[5]Rabinstein AA,Lanzino G,Wijdicks EF.Multidisciplinary management and emerging therapeutic strategies in aneurysmal subarachnoid haemorrhage[J].Lancet Neurol,2010,9(5):504-519.
[6]Scheller C,Richter HP,Engelhardt M,et al.The influence of prophylactic vasoactive treatment on cochlear and facial nerve functions after vestibular schwannoma surgery:a prospective and open-label randomized pilot study[J].Neurosurgery,2007,61(1):92-97.
[7]胡明,刘昌勤,冯作化.尼莫地平预处理和梗塞后治疗对大鼠脑缺血损伤保护作用的比较研究[J].脑与神经疾病杂志,2005,13(3):182-185.
[8]Bonova P,Burda J,Danielisova V,et al.Development of a pattern in biochemical parameters in the core and penumbra during infarct evolution after transient MCAOin rats[J].Neurochem Int,2013,62(1):8-14.
[9]Wang W,Xu J,Li L,et al.Neuroprotective effect of morroniside on focal cerebral ischemia in rats[J].Brain Res Bull,2010,83(5):196-201.
[10]Zhang T,Wang H,Li Q,et al.Modulating autophagy affects neuroamyloidogenesis in an in vitro ischemic stroke model[J].Neuroscience,2014,263:130-137.
[11]Yan L,Sadoshima J,Vatner DE,et al.Autophagy in ischemic preconditioning and hibernating myocardium[J].Autophagy,2009,5(5):709-712.
[12]Qin AP,Liu CF,Qin YY,et al.Autophagy was activated in injured astrocytes and mildly decreased cell survival following glucose and oxygen deprivation and focal cerebral ischemia[J].Autophagy,2010,6(6):738-753.
[13]Jatana M,Giri S,Ansari MA,et al.Inhibition of NF-κB activation by 5-lipoxygenase inhibitors protects brain against injury in a rat model of focal cerebral ischemia[J].J Neuroinflammation,2006,3:12.
[14]Pandey S,Chandravati.Autophagy in cervical cancer:an emerging therapeutic target[J].Asian Pac J Cancer Prev,2012,13(10):4867-4871.
[15]Kapuy O,Bnhegyi G.Depletion of luminal pyridine nucleotides in the endoplasmic reticulum activates autophagy with the involvement of m TOR pathway[J].Biomed Res Int,2013,2013:942431.
[16]Wei K,Wang P,Miao CY.A double-edged sword with therapeutic potential:an updated role of autophagy in ischemic cerebral injury[J].CNS Neurosci Ther,2012,18(11):879-886.