中西药合用对帕金森病大鼠神经细胞凋亡的影响

Effect of integrated Chinese-western medicine on nerve cell apoptosis of rats with Parkinson disease

目的:观察滋补肝肾、通络解毒中药与美多巴合用对帕金森病大鼠神经细胞凋亡的影响。方法:实验于2003-03/2004-06在上海中医药大学科研实验中心完成。①选用雄性Wistar大鼠58只,7～8周龄。将其中40只大鼠用于制备帕金森病模型(以6-羟基多巴胺注射于大鼠脑右侧黑质,10d后腹腔注射阿朴吗啡0.5mg/kg诱发大鼠向一侧旋转,以开始旋转至30min内旋转圈数平均每分钟超过7次者为合格帕金森病模型),造模成功18只,随机分为2组:模型组、中西药治疗组,每组9只;另纳入9只正常大鼠为正常对照组(只固定大鼠,不进行任何处理),9只大鼠注射含0.2g/L抗坏血酸的等量生理盐水(其余条件与造模手术相同大鼠)为假手术组。②然后,正常对照组、假手术组、模型组:灌胃生理盐水2mL。中西药治疗组:灌胃中西药混悬液2mL[滋补肝肾、通络解毒中药:汤药内含熟地、桑寄生、枸杞子、天麻、钩藤、僵蚕、丹参、莪术、白芍、生南星,按既定工艺煎煮成汤药,每毫升含原药材2.961g,由上海中医药大学附属龙华医院制剂室制备;蝎蜈胶囊内含全蝎、蜈蚣,0.3g/粒,由上海中医药大学附属龙华医院提供,批号:010813。按比例将胶囊混匀于汤药中,使浓度为3.006g/mL;含美多巴(上海罗氏制药公司产品,批号:052000,250mg/片)135mg/kg),1次/d,共45d。③实验结束后,每组取6只大鼠,处死,取出中脑组织,甲醛固定,梯度脱水,常规石蜡包埋,冠状切片,每块脑组织5张切片;每组取3只大鼠进行心脏灌注固定,后分离中脑黑质,制作电镜标本。用原位末端标记法染色和透射电镜(×9000)的方法观察中西药合用对神经细胞凋亡的影响。④组间比较采用方差分析。结果:造模失败22只,由于造模失败脱失22只,进入结果分析36只,每组9只。①模型组标本中均能检测到原位末端标记法染色阳性细胞核,细胞核呈棕褐色,但主要集中在损毁侧黑质区内,细胞核体积缩小,或明显皱缩,形态呈圆形或不规整。模型组神经细胞凋亡数明显多于正常对照组、假手术组和中西药治疗组[(37.83±6.46),(1.67±1.9),(1.83±1.92),(1.28±4.75)个,P<0.01]。中西药治疗组也能检测到凋亡细胞核,但与模型组比较,凋亡细胞数较少,单个散在分布。②电镜检查结果显示中西药合用组神经细胞病理变化明显比模型组轻,细胞膜稍有皱缩,细胞体积略缩小,线粒体等细胞器形态正常,细胞核稍呈皱缩状态,染色质有轻度凝聚成块并有边聚倾向,核膜基本完整。结论:中西药合用对帕金森病大鼠神经元细胞凋亡有明显抑制作用。

AIM： To observe the effect of combined Chinese herbs characterized by nourishing and tonifying the liver and kidney, cleanng meridians and neutralize poison with madopar on the nerve cell apoptosis of rats with Parkinson disease （PD）. METHODS： The experiment was completed in the Scientific Research Laboratory Center of Shanghai University of Traditional Chinese Medicine from March 2003 to June 2004, ① Altogether 58 male Wistar rats aged from 7-8 weeks were selected, and 40 of them were prepared for the PD models （the 6-hydroxy dopamine was injected into the black substance of right-side brain, followed by intraperitoneal injection of 0.5 mg/kg apomorphine （AOP） 10 days later, Afterwards, the rats were induced to rotate toward one side; more than 7 times per minute on average during 30 minutes lasting from the beginning of rotation representing qualified PD models）, and then the 18 successful PD models were divided randomly into the model group and integrated Chinese-western medicine group with 9 rats in each group. Another 9 rats were included in the normal control group （there were no other interferences except immobilizing the rats） and another 9 rats with injection of 0.2 g/L antiscorbic acid were included in the sham-operation group （the other conditions were exactly the same as the model group）. ② The rats in the normal control group, sham-operation group and model group were administered with 2 mL saline by garage; and the rats in the group of integrated Chinese-western medicine were administered with 2 mL suspension [the Chinese herbal formula to nourish and tonffy the liver and kidney, clear meridians and neutralize poison containing the following herbs： shudi （Radix Rehmanniae Peparata）, sangjisheng （Chinese taxillus leaf and twig）, gouqizi （Fructus Lycii）, tianma （Rhizoma Gastrodiae）, gouteng （Ramulus Uncariae cum Uncis）, jiangcan （Bombyx Batryticatus）, danshen （Radix Salviae Miltiorrhizae）, ezhu （Rhizoma Curcumae）, baishao （Radix Paeoniae alba） and shengnanxing （Fresh Rhizoma Arisaematis）. The decoction was prepared according to the designed process and contained crude herbs of 2.961 g/mL by the preparation room, Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine; the xiewu capsules containing quanxie （Scorpion） and wugong （centipede）, 0.3 g/capsule was provided by Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine （batch number： 010813）. The capsules were evenly mixed into. the decoction by proportion to make the concentration of 3.006 g/mL; the suspension containing madopar （manufactured by Shanghai Roche Pharmaceutical Company, the batch munber： 052000 and 250 mg/tablet） of 135 mg/kg]. The administration was done once a day, for 45 days in total. ③ After the experiment was over, 6 rats of each. group were decapitated, and the midbrain tissues were taken out for formalin fixation, gradient dehydration, routine paraffin imbedding and coronary slices, each piece of the brain tissue including 5 slices; three rats of each group were selected for heart perfusion fixation, followed by the isolation of the black substance of the middle brain for samples of electron microscope. Then the observation was made about the effect of integrated Chinese-Western medicine on nerve cells apoptosis by TUNEL staining and transmission electro-speculum （× 9 000）. ④ The analysis of variance was adopted for comparison among the different groups. RESULTS： Twenty-two rats failed in the modeling process, and 36 rats with 9 in each experiment group were done the result analysis： ① The TUNEL positive nucleus had been detected in all the rats in model group, which appeared brown-dark color but mainly centered on the black substance area of the damaged side, and the volume of nucleus diminished or obviously crimpled and appeared round or irregular shape. The cells apoptosis counting in the model group were significantly more than that in the normal control group, shaim-operation group and group of integrated Chinese-western medicine [（37.83 ±6.46）, （1.67 ±1.9）, （1.83 ±1,92）, and （1.28±4.75）, P 〈 0.01]. The apoptosis nucleus had also been detected in the group of integrated Chinese-western medicine; however there was less cells apoptosis counting when compared with the model group and distributed in scatter areas. ② The electron microscopic result showed that the group of integrated Chinese-western medicine had significantly milder condition than the model group, manifesting as the following aspects： The cellular membrane slightly crimpled; the cellular volume slightly diminished; the cell organ including mitochondrion appeared normal shape; the nucleus slightly crimpled; the chromatin appeared slight agglomeration and tendency to margin clustering and the nuclear membrane was basically complete. CONCLUSION： The integrated Chinese-western medicine can significantly inhibit the neuron cells apoptosis of the PD rats.