【作者】 包翠芬；

【导师】 秦书俭；

【作者基本信息】 辽宁中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 目的:明确人参皂苷Rg1对脑缺血再灌注损伤的保护作用,进一步探讨人参皂苷Rg1调控JNK及其效应分子的表达、抑制神经元凋亡、发挥神经保护作用的机制。材料与方法:健康SD大鼠(250～300g)随机分为假手术组、单纯缺血再灌注组(以下简称:模型组)、模型+人参皂苷Rg1治疗组(以下简称:人参皂苷Rg1组)、模型+尼莫地平1 mg/kg阳性药物治疗组(以下简称:阳性药物对照组),其中人参皂苷Rg1组又分为3个亚组(10、20、40 mg/kg组),每组35只。实验模型采用阻断大鼠大脑中动脉致局灶性脑缺血再灌注(MCAO)模型,分别选用栓塞2小时后再灌注4小时、22小时作为观察时间。人参皂苷Rg1各组于术前5天至取材当日分别注射10、20、40 mg/kg的人参皂苷Rg1,阳性药物对照组给予1 mg/kg的尼莫地平,假手术组及模型组给予等体积等渗生理盐水。MCAO术后24小时,通过神经功能评分观察各实验组大鼠神经功能缺损症状的改善情况,动物处死后采用TTC染色观察大鼠脑梗死体积变化,利用干湿重法观察大鼠脑水肿情况,采用HE染色观察大鼠右侧海马CA1区的病理学变化,采用电镜技术观察CA1区神经元超微结构的改变,利用TUNEL法检测CA1区神经元凋亡率,采用Nissl染色观察CA1区神经元尼氏体的变化,计数存活神经元数量,采用免疫组织化学及免疫印迹方法对CA1区的线粒体途径(Bcl-2、Bax)及死亡受体途径(FAS、FAS-L)的凋亡相关蛋白表达情况进行检测,并利用细胞图像分析系统对其进行定量分析。于MCAO术后6小时,采用免疫组织化学及免疫印迹方法对CA1区的p-JNK蛋白表达及其下游效应分子c-jun的活化情况进行检测,并利用细胞图像分析系统对其进行定量分析。结果:1.人参皂苷Rg1对脑缺血再灌注的保护作用(1)假手术组、模型组、人参皂苷Rg1 10、20、40 mg/kg组和阳性药物对照组神经功能评分分别为0.00±0.00、2.80±0.58、1.96±0.74、1.52±0.82、1.24±0.72、1.44±1.00;与模型组比较,人参皂苷Rg1各组神经功能缺损症状显著改善(P＜0.05);Rg110 mg/kg组大鼠神经功能缺损症状虽然减轻,但与阳性药物对照组比较仍有显著性差异(P＜0.05),而20、40 mg/kg组与阳性药物对照组比较无统计学差异(P＞0.05);Rg1组间比较有显著性差异(P＜0.05),其中以Rg1 40 mg/kg组改善神经功能缺损症状的程度最为明显。(2)假手术组、模型组、人参皂苷Rg1 10、20、40 mg/kg组和阳性药物对照组脑梗死体积百分比分别为(0.00±0.00)%、(36.20±5.77)%、(27.86±3.72)%、(20.24±3.37)%、(15.72±2.83)%、(18.08±3.78)%;Rg1各组脑梗死体积显著缩小,与模型组比较有显著性差异(P＜0.05);Rg1 10 mg/kg组大鼠脑梗死体积虽然减少,但与阳性药物对照组比较仍有显著性差异(P＜0.05),而20、40 mg/kg组与阳性药物对照组比较无统计学差异(P＜0.05);Rg1 20、40 mg/kg组与10 mg/kg组比较均有显著性差异(P＜0.05),其中以Rg1 40 mg/kg组脑梗死体积减少的程度最为明显。(3)假手术组、模型组、Rg1 10、20、40 mg/kg组和阳性药物对照组脑组织含水量分别为(77.77±1.50)%、(83.42±2.75)%、(81.78±1.10)%、(80.77±0.76)%、(78.80±0.78)%、(81.13±1.64)%;Rg1 20、40 mg/kg组脑组织含水量显著减少,与模型组比较有显著性差异(P＜0.05),其中以40 mg/kg组脑水肿减轻的程度最为明显;Rg1 10mg/kg组大鼠脑组织含水量虽然减少,但与模型组比较无显著性差异(P＞0.05)。(4) Rg1各组右侧海马CA1区细胞排列较整齐,间质水肿程度减轻,神经元胞体肿胀减轻,与模型组比较各项病理改变均有不同程度的改善。2.人参皂苷Rg1抑制脑缺血再灌注大鼠海马CA1区的神经元凋亡(1)模型组部分神经元坏死,也可见不同时期的神经元凋亡,有时可见凋亡小体形成;Rg1各组与模型组比较,粗面内质网肿胀及线粒体嵴断裂程度均有不同程度的减轻,可见少量早期凋亡细胞,中晚期凋亡细胞较少见。(2)假手术组、模型组、Rg1 10、20、40 mg/kg组和阳性药物对照组海马CA1区神经元凋亡率分别为(1.91±1.46)%、(22.80±5.15)%、(17.08±3.15)%、(14.45±3.22)%、(9.70±1.91)%、(12.42±1.93)%;Rg1各组海马CA1区的凋亡细胞数量显著降低,与模型比较有显著性差异(P＜0.05);Rg1 10、40 mg/kg组与阳性药物对照组比较均有显著性差异(P＜0.05),其中40 mg/kg组抑制凋亡的程度显著优于阳性药物对照组,而20mg/kg组与阳性药物对照组比较无统计学差异(P＞0.05)。(3)假手术组、模型组、Rg1 10、20、40 mg/kg组海马CA1区存活神经元计数分别为每高倍视野71.57±9.37、45.77±8.11、50.20±10.21、54.37±8.50、59.77±7.88、56.50±8.91;Rg1 20、40 mg/kg组神经元存活数量得到不同程度地提高,与模型组比较有显著性差异(P＜0.05),而Rg1 10 mg/kg组神经元存活数量虽然有所增高,但与模型组比较无显著性差异(P＞0.05);Rg1各组与阳性药物对照组比较无统计学差异(P＞0.05)。(4)假手术组、模型组、人参皂苷Rg1 10、20、40 mg/kg组和阳性药物对照组海马CA1区Bcl-2蛋白表达含量分别为1.54±0.06、1.39±0.76、1.72±0.21、2.76±0.15、4.31±0.89、3.11±0.19,Bax蛋白表达含量分别为0.30±0.07、4.31±0.55、2.21±0.12、1.80±0.20、0.93±0.08、1.57±0.09;免疫组织化学与免疫印迹结果显示Rg1 20、40mg/kg组Bcl-2蛋白表达量显著增强,Rg1各组Bax蛋白含量显著降低,Bcl-2/Bax比值显著提高,与模型组比较有显著性差异(P＜0.05)。(5)人参皂苷Rg1 10、20、40 mg/kg组和阳性药物对照组海马CA1区FAS蛋白表达含量分别为0.28±0.05、3.01±0.24、1.76±0.12、1.51±0.60、1.31±0.13、1.53±0.15,FAS-L蛋白表达含量分别为0.14±0.02、0.86±0.19、0.72±0.06、0.62±0.16、0.29±0.09、0.52±0.18;Rg1各组FAS、FAS-L平均灰度明显增高(表达量减少),蛋白含量显著降低,与模型组比较有显著性差异(P＜0.05),其中40 mg/kg组FAS、FAS-L蛋白含量最低;10 mg/kg组与阳性药物对照组比较有显著性差异(P＜0.05),而20、40 mg/kg组与阳性药物对照组比较无统计学差异(P＞0.05)。3.人参皂苷Rg1抑制脑缺血再灌注大鼠p-JNK、p-c-jun表达:(1)假手术组、模型组、人参皂苷Rg1 10、20、40 mg/kg组和阳性药物对照组海马CA1区p-JNK蛋白表达含量分别为1.21±0.39、2.27±0.28、2.21±0.27、1.93±0.30、1.64±0.31、1.85±0.41;人参皂苷Rg1各组p-JNK蛋白含量显著降低,与模型组比较有显著性差异(P＜0.05),其中10 mg/kg组p-JNK蛋白含量最高,40 mg/kg组p-JNK蛋白含量最低;10、40 mg/kg组与阳性药物对照组比较有显著性差异(P＜0.05),而20 mg/kg组与阳性药物对照组比较无统计学差异(P＞0.05)。(2)假手术组、模型组、人参皂苷Rg1 10、20、40 mg/kg组和阳性药物对照组海马CA1区p-c-jun蛋白表达含量分别为0.51±0.07、2.02±0.17、1.21±0.11、0.87±0.22、0.69±0.07、0.80±0.18;模型组大鼠p-c-jun含量最高,假手术组p-c-jun含量最低;人参皂苷Rg1 20、40 mg/kg组p-c-jun蛋白含量显著降低,与模型组比较有显著性差异(P＜0.05),其中40 mg/kg组p-c-jun蛋白含量最低;人参皂苷Rg1 10 mg/kg组与阳性药物对照组比较有显著性差异(P＜0.05),而Rg1 20、40 mg/kg组与阳性药物对照组比较无统计学差异(P＞0.05)。结论:1.人参皂苷Rg1可以显著改善脑缺血再灌注大鼠的神经功能缺损症状。2.人参皂苷Rg1能显著降低脑缺血再灌注大鼠海马CA1区神经元凋亡率;进一步研究显示Rg1可能通过抑制脑缺血后海马CA1区的JNK及其效应分子c-jun的活化,进一步调节死亡受体、线粒体依赖的凋亡途径,抑制脑缺血再灌注后的神经元凋亡,降低缺血后迟发性神经元死亡,控制缺血半暗带向梗死区的发展,以发挥其对脑缺血再灌注损伤的保护作用。更多还原

【Abstract】 Purpose:To explore the roles and underlying mechanism of ginsenoside Rg1 in the protective effect on the injury caused by cerebral ischemic-reperfusion. Materials and methods:Healthy male Sprage-Dawl rats(250-300 g) were randomly divided into the sham-operative group(sham group),focal cerebral ischemia-reperfusion group(model group),Ginsenoside Rg1 treated group,and Nimodiping treated(1mg/kg) group(n=35 in each group).The Rg1 treated group was further divided into three subgroups according to the Rg1 dosage(10,20, 40mg/kg).The middle cerebral artery occlusion model(MCAO 6 h,24 h) was established by suture method.Rg1(10,20,40mg/kg respectively) was injected as follows:5 days before operation,at the day of operation,24 h after operation (q.d),positive control group(1mg/kg)was injected in Nimodiping group(1mg/kg), NS was injected in the same time point in Sham group and Model group.24 h after operation,the neurological function was evaluated by neurological deficit scoring.Then the rats were sacrificed and the volume change of cerebral infarction was examined by TTC staining,the moisture of brain tissue was detected by dry-wet weight assay,morphological changes in hippocampus CA1 zone were observed by HE staining,the neuron apoptosis status in hippocampus CA1 zone was detected by TUNEL assay,the ultramicrostructure of neurons in hippocampus CA1 zone was detected by transmission electric microscopy,the numbers of neuron was detected by nissle staining.The expression of Bcl-2,Bax,FAS and FAS-L were analyzed by immunohistochemistry and wstern blot.After 6h of focal cerebral ischemia,the expression of p-JNK and p-c-jun were detected by immunohistochemistry staining and wstern blot.Results:1.Compared with model group,the symptoms of neurological deficit were differentially improved in Ginsenoside Rg1 treated group after 24 h of MCAO. 40 mg/kg subgroup was more significant than 10,20 mg/kg subgroup(P＜0.05). Volumes of cerebral infarction were significantly decreased in Rg1 treated group compared with model group.40mg/kg subgroup was more evident(P＜0.05).Moisture of brain tissue was significantly decreased in 20,40 mg/kg subgroups after 24 h of MCAO.40mg/kg subgroup was more evident(P＜0.05),while the difference between 10mg/kg and model groups is not significant.Compared with model group, cells arranged in order,the extent of edema of interstitial and neurons were decreased in hippocampus CA1 zone in Ginsenoside Rg1 treated group after 24 h of MCAO.Rg1 40 mg/kg subgroup was more evident(P＜0.05),while the difference between 10 mg/kg and model groups is not significant.2.Compared with model group,apoptosis rate of neuron in hippocampus CA1 zone was significantly decreased in 20,40mg/kg subgroups after 24h of MCAO.40mg/kg subgroup was more evident(P＜0.05).Apoptotic neurons of different period were observed in hippocampus CA1 zone in model group,a few viable apoptotic cells were observed in hippocampus CA1 zone in Ginsenoside Rg1 treated group after 24 h of MCAO.Compared with model group,the extent of rER edema in hippocampus CA1 zone was decreased,and the extent of crista mitochondria fracture was improved in Ginsenoside Rg1 treated group after 24 h of MCAO.Compared with model group,neuron number was more in hippocampus CA1 zone in 20,40mg/kg subgroups after 24h of MCAO,while the difference between 10mg/kg and model groups is not significant.Immunohistochemistry revealed that Bcl-2 and Bax were expressed in the cytoplasm of the neurons.Compared with model group,Bcl-2 positive cells were significantly increased.However,Bax positive cells were significantly decreased in hippocampus CA1 zone after 24 h of MCAO in Rg1 treated group.Western blot analysis verified our results of immunohistochemistry.Compared with model group,immunostaining analysis revealed the expression of FAS and FAS-L in hippocampus CA1 zone was significantly decreased after 24h of MCAO in Rg1 treated group.Western blot analysis obtained the consistent results.The differences among 20,40mg/kg and model groups were significant,but 10mg/kg subgroup was not significant.3.Compared with model group,expression of p-JNK in hippocampus CA1 zone was significantly decreased in Rg1 treated group compared with model group.40mg/kg subgroup was more evident(P＜0.05).Western blot analysis revealed the same tendency as immunohistochemistry.Expression of p-c-jun in hippocampus CA1 zone was significantly decreased in Rg1 treated group compared with model group.40 mg/kg subgroup was more evident(P＜0.05).Western blot analysis obtained the consistent results.Conclusions,1.Ginsenoside Rg1 improved obviously the neurological deficit caused by cerebral ischemic-reperfusion.2.Ginsenoside Rg1 inhibited significantly the apoptosis of neurons in ischemic zone caused by cerebral ischemic-reperfusion.Further research revealed that Rg1 suppresses the phosphorylation of JNK and c-jun,inhibits the expression of Bax,FAS,FAS-L and promotes the expression of Bcl-2.更多还原