人参皂苷Rg1对局灶性脑缺血再灌注损伤大鼠海马p-ERK1/2与p-JNK表达的影响

目的探讨人参皂苷Rg1抗脑缺血再灌注（ischemia reperfusion，I/R）损伤大鼠海马神经元凋亡的可能机制。方法成年健康雌性SD大鼠120只随机分为脑缺血再灌注模型组（模型组）、人参皂苷Rg1低（10 mg/kg）、中（20 mg/kg）、高剂量（40 mg/kg）组及假手术组，每组18只。各组均腹腔注射给药，假手术组及模型组腹腔注射等量生理盐水，每天1次，连续7天，末次给药后30 min，大鼠右侧大脑中动脉阻塞（middle cerebral artery occlusion， MCAO）2 h再灌注24 h制备I/R模型。以Longa EZ法评定神经功能，尼氏染色、TUNEL染色观察海马锥体神经细胞的损伤情况，并计算神经细胞凋亡率。采用Western blot法检测细胞外信号调节蛋白激酶1/2（ extracellular signal-regulated kinase 1/2， ERK1/2）及磷酸化细胞外信号调节蛋白激酶1/2（phosphorylated extracellular signal-regulated kinase 1/2，p-ERK1/2）、c-Jun氨基末端激酶（c-Jun N-terminal kinases， JNK）及磷酸化c-Jun氨基末端激酶（phosphorylated c-Jun N-terminal kinases， p-JNK）表达。结果与假手术组比较，模型组神经功能评分、细胞凋亡率、p-JNK、p-ERK1/2蛋白表达升高（P<0.05，P<0.01），锥体细胞存活数减少（P<0.01）；与模型组比较，人参皂苷Rg1各剂量组神经功能评分、细胞凋亡率降低（P<0.05，P<0.01），人参皂苷Rg1中、高剂量组大鼠锥体细胞存活数增加，海马CA1区p-JNK蛋白表达降低，p-ERK1/2表达升高（P<0.05，P<0.01）。假手术组海马CA1区有3－4层锥体细胞，排列整齐、紧密，高倍镜下细胞核大而圆，有1~2个核仁。脑组织缺血损伤后，海马区神经细胞受损严重，CA1区失去正常结构，细胞排列散乱，细胞数量减少。部分神经元皱缩，核固缩、深染，呈三角形、长条形、梭形或不规则形，核染色聚集，核仁不清晰。与人参皂苷Rg1低剂量组比较，人参皂苷Rg1中、高剂量组神经功能评分、细胞凋亡率及p-JNK蛋白表达降低（P<0.05，P<0.01），锥体细胞存活数增加，p-ERK1/2表达升高（P<0.05，P<0.01）。人参皂苷Rg1中、高剂量能够改善缺血神经细胞形态，减少神经细胞的丢失，其中，高剂量组作用强于低剂量组。JNK 蛋白条带分为两个亚带，JNK1是分子量为46 kD的蛋白，JNK2分子量为54 kD。ERK蛋白条带也分为两个亚带，ERK1是分子量为44 kD的蛋白，ERK2分子量为42 kD的蛋白。结论人参皂苷Rg1对I/R大鼠的保护作用与抑制海马神经元凋亡，调节p-JNK及p-ERK1/2表达水平有关。

英文摘要:

Objective To study the possible anti-apoptotic mechanism of ginsenoside Rg1 on the apoptosis of hippocampal neuron after cerebral ischemia/reperfusion （I/R） injury rats. Methods Totally 120 healthy male adult SD rats were randomly divided into the cerebral I/R model group （the model group）， the low dose ginsenoside Rg1 group （10 mg/kg）， the middle dose ginsenoside Rg1 group （20 mg/kg）， the high dose ginsenoside Rg1 group （40 mg/kg）， and the sham-operation group， 18 in each group. Rats received medication by peritoneal injection. Equal volume of normal saline was peritoneally injected to rats in the sham-operation group and the model group， once daily， for 7 successive days. The cerebral I/R injury model was prepared by 2-h middle cerebral artery occlusion （MCAO） followed by 24-h reperfusion. Rats in the sham-operation group received the same surgical procedure without the carotid arteries occluded. The neurofunction was assessed using Longa EZ method. The injury of hippocampal pyramidal cells was observed by Nissel staining and TUNEL assay. The nerve cell apoptosis rate was calculated. The protein expression levels of extracellular signal-regulated kinase 1/2 （ERK1/2）， phosphorylated extracellular signal-regulated kinase 1/2 （p-ERK1/2）， c-Jun N-terminal kinases （JNK）， and phosphorylated c-Jun N-terminal kinase （p-JNK） were detected using Western blot. Results Compared with the sham-operation group， the score of neurofunction， the apoptosis rate， the expression levels of p-JNK and p-ERK1/2 increased， the survived number of pyramidal cells decreased in the model group （P<0.05，P<0.01）. Compared with the model group， the score of neurofunction and the apoptosis rate decreased in each ginsenoside Rg1 group （P<0.05， P<0.01）. The survived number of pyramidal cells increased in the high and middle dose ginsenoside Rg1 groups， the expression of p-JNK in the hippocampal CA1 region decreased， and the expression level of p-ERK1/2 increased （P<0.05， P<0.01）. Compared with the low dose ginsenoside Rg1 group， the score of neurofunction， the apoptosis rate， the p-JNK protein expression decreased， the survived number of pyramidal cells increased， the expression of p-ERK1/2 increased in the high and middle dose ginsenoside Rg1 groups （P<0.05，P<0.01）. Three to four layers of pyramidal cells were arranged tightly and compactly in the hippocampal CA1 region of the sham-operation group. The nucleus was big and round under high power lens， with 1－2 kernel. After cerebral I/R injury， the hippocampal nerve cells were severely injured. Normal structure was lost in the CA1 region， with disarranged cell line and reduced cell amount. Partial neurons were shrunken， and the kernel was condensed and darkenedly stained. They were in triangular， long strip， fusiform， or irregular shape. The staining of nucleus was clustered and the kernel was not clear. Ginsenoside Rg1 （20 and 40 mg/kg） could improve the morphology of ischemic nerve cells， reduce their loss. Of them， stronger effects were shown in the high dose ginsenoside Rg1 group than in the middle dose ginsenoside Rg1 group. The JNK protein band was divided into two subzones， JNK1 （46 kD） and JNK2 （54 kD）. ERK protein band was also divided into two subzones， ERK1 （44 kD） and ERK2 （42 kD）. Conclusion The protective effect of ginsenoside Rg1 on cerebral I/R injury was correlated with inhibiting the apoptosis of hippocampal neurons， regulating the expression levels of p-ERK1/2 and p-JNK.

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