左旋多巴活化NMDAR1/ERK1/2信号对单眼剥夺弱视大鼠视皮质神经细胞保护作用的研究

The protective effect of the activation of NMDAR1/ERK1/2 signal pathway induced by levodopa on visual cortical neurons in monocular deprivation rats

目的探讨左旋多巴活化NMDA受体1（NMDAR1）/ERK1/2（丝裂原活化蛋白激酶）信号对单眼剥夺弱视大鼠视皮质神经细胞的保护作用。 方法实验研究。54只SD大鼠根据随机数字表分为9组：正常对照组、弱视模型（MD）组、低剂量左旋多巴治疗组、高剂量左旋多巴治疗（H-左旋多巴＋MD）组、高剂量左旋多巴＋MK801组、高剂量左旋多巴＋PD98059组、MK801组、PD98059组、二甲基亚砜溶剂对照组，每组6只。通过右眼缝合制备弱视大鼠模型，左旋多巴灌胃治疗，MK801、PD98059通过侧脑室注射，免疫印迹法检测N-甲基-D-天冬氨酸受体（NR1）、磷酸化丝裂原活化蛋白激酶（p-ERK1/2）、丝裂原活化蛋白激酶（ERK1/2）、神经生长因子（NGF）、即刻早期基因c-FOS的表达，Nissl染色检测神经元细胞形态学变化，核苷酸末端转移酶介导的dUTP缺口标记（TUNEL）法检测神经元细胞凋亡，免疫组织化学法检测神经元细胞半胱氨酸天冬氨酸蛋白酶-3（caspase-3）、NGF和c-FOS的表达。采用单因素或双因素方差分析联合LSD-t检验进行组间比较。 结果正常对照组小鼠神经元尼氏小体完整，形态清晰；MD组视皮质区神经元尼氏小体体积缩小，神经元丢失和核固缩。与对照组相比，MD组神经细胞凋亡显著增多（23.09±2.00、2.20±0.35，t=12.120，P=0.000），Caspase-3阳性细胞增多（22.70±1.50、3.30±0.54，t=12.120，P=0.000），NGF（0.31±0.04、0.74±0.09，t=7.674，P=0.000）和c-FOS（0.25±0.03、0.57±0.07，t=5.919，P=0.000）表达降低，NGF（8.30±0.82、35.18±2.01，t=12.370，P=0.0000）和c-FOS（10.84±1.02、35.68±2.55，t=9.056，P=0.0001）阳性细胞数减少，视皮质区NR1（0.40±0.05、0.55±0.07，t=4.533，P=0.001）、p-ERK1/2（0.68±0.17、0.88±0.11，t=0.920，P=0.142）的表达降低；左旋多巴治疗后，神经细胞形态恢复正常，尼氏小体染色清晰；有效缓解神经细胞凋亡（13.07±1.47、23.09±2.00，t=8.698，P=0.000），减少Caspase-3阳性细胞率（17.05±1.11、22.70±1.50，t=3.019，P=0.015）；NR1（0.75±0.09、0.40±0.05，t=8.528，P=0.001）、p-ERK1/2（2.13±0.26、0.68±0.17，t=3.488，P=0.008）的表达显著升高；NGF（18.07±0.87、8.30±0.82，t=8.18，P=0.0000）和c-FOS（19.78±0.91、10.84±1.02，t=6.543，P=0.0001）阳性细胞率增加。MK-801或PD98059干预处理后，有效拮抗左旋多巴治疗作用。MK801、PD98059处理下调NR1（0.53±0.06、0.95±0.12，t=5.647，P=0.005）及下游p-ERK1/2（1.52±0.18、2.58±0.30，t=3.091，P=0.013）的表达，并进一步促进MD小鼠尼氏小体体积缩小、细胞核固缩、神经元凋亡、Caspase-3表达，并抑制NGF和c-FOS的表达；二甲基亚砜溶剂未见明显作用。 结论左旋多巴通过活化NMDA-ERK1/2 MAPK信号对大鼠视皮质神经元细胞的保护作用。

Objective To investigate the protection effects of the activation of NMDAR I（NMDA receptor 1）/ERK1/2 signal pathway on visual cortex nerve calls induced by levodopa in amblyopia rats. Methods SD rats of SPF grade, were randomly divided into for groups of 9, group A. Control, B. MD group, C. L-levodopa（20 mg· kg-1）±MD group, D. H-levodopa （80 mg kg-）±MD group, E. H-levodopa±MD±MK801 group, F. H-levodopa±MD±PD98059 group, G. MD±MK801 group, H. MD±PD98059 group, I. MD±DMSO group. Amblyopia rats were made by suture of the right eye. Levodopa was used to treatment amblyopia by garage, and intervened by intracerebroventricular injection of MK801 and PD98059 respectively. The expression of NR1, p-ERKI/2, ERK1/2, NGF and c-FOS were detected by Western blotting. Nissl staining was used to detect morphological changes of neurons. Neuronal apoptosis was detected by TUNEL method, and detected the expression of Caspase-3, NGF and c-FOS by immunohistochemical staining. One/Two way Chi-square analysis was used for data analysis. LSD-t test was used as comparison between every two groups. Results The morphology of Nissl bodies in neurons was complete and clear in A group, and the size of Niss[ bodies got smaller, and caused karyopyknosis and loss of neurons in visual cortex of B group. Compared with A group, the apoptosis of visual cortical neurons（23.09±2.00 vs. 2.20±0.35, t=12.120, P= 0.000） and the number of Caspase-3 postre ceils （22.70± 1.50 vs. 3.30±0.54, t=12.120, P=0.000）were significantly increased, the expression of NGF（0.31 ±0.04 vs. 0.74±0.09, t=7.674, P=0.O00） and c-FOS（0.25± 0.03 vs. 0.57±0.07, t=5.919, P=O.000） and the rats of NGF（8.30±0.82 vs. 35.18±2.01, t=12.37, P=O.0000） and c-FOS （10.84± 1.02 vs. 35.68±2.55, t=9.056, P=0.O001） postive cell were decreased significantly in B group. After treatment with levodopa, the morphology of neurons recovered, the apoptosis of visual cortical neurons relieved, the expression of NR1（0.75±0.09 vs. 0.40±0.05, t=8.528, P=O.001） and p-ERK1/2（2.13±0.26 vs. 0.68±0.17, t=3.488, P=O.O08） were increased significantly, and the rats of NGF （18.07±0.87 vs. 8.30± 0.82, t=8.18, P=0.0000） and c-FOS （19.78 ±0.91 vs. 10.84± 1.02, t=6.543, P=0.0001） postive cells were significantly increased. MK-801 or PD98059 intervention could effectively attenuate the effect of levodopa. It could effectively down-regulated the expression of NR1 （0.53±0.06 vs. 0.95±0.12, t=5.647, P=O.O05） and p-ERK1/2（1.52±O.18 vs. 2.58±0.30, t=3.091, P--0.013） interference with MK801 or PD98059 in MD rats. MK-801 or PD98059 intervention further promote the Nissl body volume reduced, neurons karyopyknosis, the apoptosis of visual cortical neurons and Caspase-3 expression, and restrain the expression of NGF and c-FOS. Conclusion Levodopa played a protective role in visual cortex nerve cells of amblyopia rats at least nartiallv throuzh activation of NMDA-ERK1/2 siznal nathwav.