【作者】 陈宏；

【导师】 陈建宗；

【作者基本信息】 第四军医大学 ， 中西医结合临床， 2007， 硕士

【摘要】 帕金森病(Parkinson’s disease, PD)是常见的老年神经系统退行性疾病,以中脑黑质致密带多巴胺(Dopamine, DA)能神经元变性、丢失及支配纹状体神经的DA水平的下降为主要特征。其致病因素及潜在发病机制还不明确,内源性和外源性神经毒素的损害部分说明这一发病过程。多项证据表明,环境中的杀虫剂、金属、碳氢化合物等被疑为参入PD发病的起始阶段。百草枯(1,1,-dimethyl-4,4,-bypiridinium, Parquat, PQ)是一种广泛使用的非选择性的除草剂,它与已知神经毒物1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP)的活性代谢产物1-甲基-4-苯基-吡啶(1-methyl-4-pheny1-pyridium, MPP+)的化学结构极其相似,这种结构的相似性预示了它可能是PD的病原学潜在因素之一。大量流行病学研究显示长期接触PQ明显增加了PD的发病危险。研究发现PQ选择性的破坏实验动物的黑质纹状体DA能神经元。人参皂甙是来源于人参根部的药理活性成分。研究发现:通过提高胆碱能神经活力,人参皂甙Rg1和Rb1均可以逆转东莨菪碱诱导的健忘症,发挥神经营养和神经保护作用。其中人参皂甙Rg1还具有提高免疫力,延缓衰老等功效,其神经保护作用日益受到人们的关注。PC12细胞来源于大鼠肾上腺嗜铬瘤细胞,具有与DA能神经元的合成物、转运系统的类似特征。而且,PC12细胞合成的递质、表达的受体接近中脑DA能神经元,是DA能神经元体外研究理想模型。然而,人参皂甙Rg1对PQ诱导的DA能神经元的损伤是否具有保护作用,目前尚未见报道。在我们的实验中,我们利用PQ诱导的PC12细胞的损伤作为PD的体外模型来研究人参皂甙Rg1可能的神经保护作用及相关机制。实验一目的:筛选出PQ诱导PC12细胞损伤的有效浓度、时间及人参皂甙Rg1对该浓度PQ损伤PC12细胞的有效保护浓度。方法:PQ损伤作用设空白对照组及100、200、400、600、800、1000μmol/L六个PQ浓度组,各浓度设12、24、36、48 h四个干预时间。人参皂甙Rg1保护作用设空白对照组、PQ组(终浓度800μmol/L)、预处理组:加PQ(终浓度800μmol/L)前分别加入浓度为5、10、20μmol/L的人参皂甙Rg1预孵育24 h。采用MTT比色法和乳酸脱氢酶(LDH)释放量测定法对各浓度进行筛选。结果:经MTT比色法检测,PQ对PC12细胞具有损伤作用,其诱导的细胞活力的降低具有剂量和时间依赖性。与空白对照组相比,800μmol/L PQ处理24 h后细胞活力为44.8±6.9%(P<0.05)。而经不同浓度的人参皂甙Rg1预处理后,我们发现5、10、20μmol/L人参皂甙Rg1预处理组与PQ组(49.5±4.2%)相比,可减少细胞损伤,细胞活力分别上升为55.7±2.8%、71.2±3.8%、84.4±3.2%(P<0.05)。空白对照组LDH(单位为:U/mg prot)的释放量为89.5±3.1,PQ组LDH释放量明显增高为180.9±3.8(与空白对照组相比P<0.05),人参皂甙Rg1(5、10、20μmol/L)保护组抑制了PQ所引起的LDH释放量的升高(分别为160.8±3.8、145.8±4.4、118.3±4.0,与PQ组相比P<0.05)。结论:PQ对PC12细胞具有损伤作用,且这种损伤具有剂量和时间依赖性;人参皂甙Rg1对PQ所引起的PC12细胞损伤具有保护作用。实验二目的:探讨人参皂甙Rg1对PQ诱导PC12细胞凋亡的影响。方法:MTT比色试验检测细胞活性;FCM检测细胞凋亡比例;Hoechst 33258染色观察细胞核形态的改变。结果:MTT法检测显示,5、10、20μmol/L人参皂甙Rg1可抑制800μmol/L PQ作用24 h所诱导的PC12细胞活性的降低,与PQ组(46.4±3.6%)相比,人参皂甙Rg1预处理后细胞活力分别上升为53.6±3.3%、73.2±3.1%、82.2±2.6%(P<0.05);FCM结果显示,PQ处理后,凋亡细胞的比例(48.9%)与空白对照组(12.8%)相比是增加的,而经5、10、20μmol/L人参皂甙Rg1预处理后,凋亡细胞比例分别降至39.8%,20.1%,12.3%;正常细胞核经Hoechst 33258染色后显示为较大的圆形,形态规则,染色均匀弥散。经PQ处理24 h后,多数细胞显示胞核凝集,且可见DNA荧光碎片。与PQ组相比,5、10、20μmol/L人参皂甙Rg1预处理可抑制PQ诱导的细胞损伤,显著减少胞核凝集。结论:人参皂甙Rg1可抑制PQ诱导的PC12细胞调亡。实验三目的:探讨人参皂甙Rg1对PQ诱导PC12细胞损伤的抗氧化作用机制。方法:比色法进行谷胱甘肽还原酶(GSH)、超(过)氧化物歧化酶(SOD)的活性测定。结果:PQ诱导的PC12细胞,SOD、GSH(单位均为:U/mg prot)活性较空白对照组有显著的下降(SOD的活性从130.51±5.99下降到53.12±2.67,GSH的活性从107.87±4.84下降到49.50±2.54,与空白对照组相比P<0.05)。使用5、10、20μmol/L人参皂甙Rg1预处理后,SOD、GSH活性同PQ组相比有显著的升高(SOD的活性分别为80.58±3.36、105.08±4.92、118.35±3.69;GSH的活性分别为61.53±3.90、72.90±4.32、87.39±4.54,与PQ组相比P<0.05)。结论:人参皂甙Rg1可明显抑制PQ诱导的PC12细胞SOD、GSH的活性的下降,从而表现较强的抗氧化活性。实验四目的:探讨人参皂甙Rg1对PQ诱导PC12细胞调亡作用机制。Rh123染色检测细胞线粒体膜电位(MMP);Caspase-3活性检测;免疫细胞化学染色测Cyt C、Bcl-2表达;Western blotting方法检测凋亡相关蛋白Bcl-2、Bax表达。结果:Rh123染色表明空白对照组细胞线粒体显示明亮绿色,荧光强度较强,表现较高的膜电位水平。PQ作用24 h后,Rh123染色荧光强度明显减弱,表明细胞MMP显著下降。分别加入5、10、20μmol/L人参皂甙Rg1后,与PQ组相比荧光强度增强,MMP显著提高;PQ组Caspase-3(单位为:pmol.min-1.mg-1)活性显著增强(与空白对照组相比P<0.001),与PQ组(1600.6±55.7)相比,经人参皂甙Rg1预处理后,Caspase-3活性分别降至1219.7±28.7、1083.8±82.7、925.0±29.1(P<0.001);免疫细胞化学染色显示正常组细胞形态良好,Cyt C、Bcl-2的免疫细胞化学染色呈淡棕黄色,均匀分布于胞质及突起内。PQ处理后部分细胞变圆,皱缩,Cyt C在细胞内表达增强,染色呈深棕黄色。PC12细胞的Bcl-2表达应激、代偿性增强,染色细胞呈深棕黄色,阳性染色分布于胞质和突起中,着色细胞数目多。人参皂甙Rg1预处理组的Cyt C免疫细胞化学染色明显减弱,而Bcl-2得到表达增加,细胞着色进一步加深,细胞形态也有改善。PQ组细胞Cyt C表达呈阳性细胞数(50.3%±1.6%)明显增多,5、10、20μmol/L人参皂甙Rg1预处理则明显减少Cyt C表达呈阳性细胞数(44.9±3.5%、42.0±2.6%、28.0±2.1%),较PQ诱导组下降明显(P<0.05); Western blotting检测Bcl-2、Bax蛋白表达,结果表明PQ处理后胞浆内Bcl-2、Bax表达增加。人参皂甙Rg1预处理后Bcl-2表达进一步增加,而Bax的表达减少,同时,Bax/Bcl-2的比率显著降低。结论:人参皂甙Rg1对PQ诱导的PC12细胞损伤具有保护作用;机制可能与其抑制PQ引起的细胞MMP下降,Caspase-3激活,调节凋亡相关蛋白Cyt C及Bcl-2、Bax在胞浆中表达有关。更多还原

【Abstract】 Parkinson’s disease(PD)is an age-related progressive neurodegenerativeisease which defining pathological features are selective loss of dopaminergiceurons in substantia nigra pars compacta and subsequent decrease of dopamineevels in the striatum,the main target innervated by these neurons.Although thetiology of PD remains unclear,both exogenous and endogenous neurotoxicubstances are known to provide partial explanation of these processes.Severalactors are suspected to participate in the onset of PD that includesnvironmental exposure to pesticides,metals and hydrocarbons.Paraquat(1,1，-dimethyl-4,4，-bypiridinium,Parquat PQ),a nonselectiveerbicide widely used in agriculture,has extremely similar chemical structureith MPP+(1-methyl-4-pheny1-pyridium,MPP+),the active metabolite ofPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)which is a knowneurotoxin.The structural similarity between MPP+and PQ confers to theseindings a potential interest in the study of the etiological causes of PD.pidemiological studies have found that there is an association between the use of PQ in agriculture and incidence of PD. Furthermore, research using animal models has also indicated the neurotoxicity induced by PQ in nigrostriatal dopaminergic cells.Ginsenosides, the pharmacologically active components found in ginseng, and may modulate neurotransmission. Both Rg1 and Rb1 are capable of partially reversing scopolamine induced amnesia by improving cholinergic activity and having partial neurotrophic and neuroprotective effects. Ginsenosides Rg1 has a lot of beneficial effects, such as improving learning and memory, enhancing immunity, and delaying apolexis. It seems to be an interesting drug for the neuroprotective effect of Ginsenosides Rg1.PC12 cells, the rat adrenal pheochromocytoma cell line, which possesses dopamine synthesis, metabolism and transporting systems. The membrane receptors and synthesizing transmitters of PC12 cell close to dopaminergic neurons in midbrain. Therefore, it has been used as a cellular model of PD. In this study, we investigated whether Ginsenosides Rg1 can protect against PQ-induced apoptosis in cultured PC12 cells as model of PD in vitro, and related mechanisms of the neuroprotective effect.Experiment 1Objective: To sieve the effective concentration of PQ inducing injury and the protective concentration of Ginsenosides Rg1 to the injury in PC12 cells. Methods: Setting control group and 100, 200, 400, 600, 800, 1000μmol/L PQ groups, PC12 cells were respectively exposed to various concentrations of PQ for 12, 24, 36, 48 h; Ginsenosides Rg1 protective groups were set control,PQ group(800μmol/L),preventive groups: pretreated with Ginsenoside Rg1(final concentration: 5, 10, 20μmol/L) for 24 h, then PQ(final concentration: 800μmol/L) was added for an additional 24 h. The cell viability was measured by MTT chromometry and LDH-release assay. Results: After exposed to a range of concentrations of PQ for various periods of time, there was a dose- and time-dependent decrease in cell viability as measured by MTT assay. In the experimental group treated with 800μmol/L PQ for 24 h, cell viability was reaching 44.8±6.9% compared with control group (P<0.05). PC12 cells were incubated with different concentrations of Ginsenoside Rg1 for 24 h then exposed to 800μmol/L PQ for another 24 h. Compared with control group, it is observed that pretreatment with Ginsenoside Rg1(5, 10, 20μmol/L) caused a significant decrease in the level。of cell death compared with PQ-treated cells(49.5±4.2%). After incubated with Ginsenoside Rg1, cell viability was reaching 55.7±2.8%、71.2±3.8%、84.4±3.2%, respectively(P<0.05). Different concentrations of Ginsenoside Rg1 had protective effects on the PQ damage, in certain scope the protective effects potentized along with the increase of Ginsenoside Rg1 concentration. Conclusion: PQ can injure PC12 cells, and the injury is in a dose and time dependent manner. In certain scope Ginsenoside Rg1 has protective effects on the injury induced by PQ in PC12 cells, which potentize along with the increase of Ginsenoside Rg1 concentration.Experiment 2Objective: To explore the neurotoxic effect of PQ on PC12 cells and observe the protective effect of Ginsenoside Rg1 against the apoptosis induced by PQ. Methods: MTT assay was used to detect the cell viability; FCM was used to detect the apoptosis ratio; Hoechst 33258 staining was employed to observe morphological changes of the cell nuclear. Results: MTT assay showed that 5, 10, 20μmol/L Ginsenoside Rg1 inhibited the decrease of cell viability induced by 800μmol/L PQ for 24 h. Compared with PQ-treated cells (46.4±3.6%), after pretreatment with Ginsenoside Rg1, cell viability was reaching 53.6±3.3%, 73.2±3.1%, 82.2±2.6%(P<0.05), respectively; FCM results indicated that, after PQ treatment, the percentage of apoptotic cells (48.9%) was increased compared with control (12.8%), but was dropped respectively to 39.8%, 20.1%,and 12.3% with 5, 10, 20μmol/L Ginsenoside Rg1 pretreatment; Hoechst 33258 staining demonstrated nuclear condensation, one of the typical hallmarks of apoptosis. Nuclei of normal cells appeared with regular contours and were round and large in size, which showed a homogeneous and diffused staining. While exposed to PQ for 24 h, most cells exhibited an asymmetric and fluorescent fragment could be seen in some cells in PQ group. 5, 10, 20μmol/L Ginsenoside Rg1 significantly inhibited PQ-induced cell damage, and condensed nuclei decreased markedly compared with PQ-induced group. Conclusion: Ginsenoside Rg1 can relieve the apoptosis induced by PQ in PC12 cells.Experiment 3Objective: To explore the related mechanisms of the protective effect of Ginsenoside Rg1 on PQ-induced apoptosis in PC12 cells. Methods: The active of superoxide dismutase(SOD),γ-glutamylcysteinylglycine(GSH) were (unit:U/mg prot) in PQ-induced cells decreased markedly from 130.51±5.99 to 53.12±2.67 and 107.87±4.84 to 49.50±2.54, respectively, compared with controls. However, the activities of SOD, GSH increaseed by 80.58±3.36, 105.08±4.92, 118.35±3.69,and 61.53±3.90, 72.90±4.32, 87.39±4.54 with 5, 10, 20μmol/L Ginsenoside Rg1 treatment, severally(P<0.05). Conclusion: The mechanisms of the protective effect of Ginsenoside Rg1 was partially dependent on antioxidative stress effects, inhibiting decrease the activation of SOD, GSH in PC12 cells induced by PQ.Experiment 4Objective: To explore the related mechanisms of the protective effect of Ginsenoside Rg1 on PQ-induced apoptosis in PC12 cells. Methods: Mitochondrial membrane potential(MMP) was detected by Rh123 staining; Caspase-3 activity was measured with a colorimetric Caspase-3 assay kit; the expression of pro-apoptotic protein Cyt C and Bcl-2 in cytosol was observed by immunocytochemical staining, and the expression of Bcl-2, Bax was observed by Western blotting. Results: Control cells exhibited numerous brightly staining mitochondria that emitted green fluorescence,which was indicative of normal high membrane potential, PQ treatment induced a transition in mitochondria permeability and a significant loss of membrane potential. Ginsenoside Rg1 treatment inhibited the collapse of mitochondrial membrane potential with increasing dosage, as indicated via the reappearance of brightly green mitochondrial staining; A significant increase in Caspase-3 (unit: pmol.min-1.mg-1) activity was induced after PQ exposure compared with controls (P<0.001), while this activation was reduced by 5, 10, 20μmol/L Ginsenoside Rg1 incubation. After pretreatment with Ginsenoside Rg1, the Caspase-3 activity reached to 1219.7±28.7, 1083.8±82.7, 925.0±29.1(P<0.001), respectively, compared with group treated with PQ alone (1600.6±55.7); In control group, the staining cells were light-coloured and their morphous was more integrity. After incubating with PQ for 24 h, the expression of Cyt C and Bcl-2 increased in PC12 cells. Most cells were stained and their morphous changed. The staining cells were dark yellowish-brown and the positive stain distributed in cytoplasm. Immunocytochemical staining indicated that 5, 10, 20μmol/L Ginsenoside Rg1 downregulated the over-expression of Cyt C and significantly further enhanced anti-apoptotic Bcl-2 protein levels in the cytosol induced by PQ and improved the morphous of PC12 cells. Western blotting revealed an increased expression of Bcl-2, Bax in PQ treated group. Treatment of PC12 cells with Ginsenoside Rg1(5, 10, 20μmol/L) for 24 h, significantly further enhanced anti-apoptotic Bcl-2 protein levels in PC12 cells as compared with controls. Protein levels of pro-apoptotic Bax were significantly reduced, and the Bax/Bcl-2 ratio was significantly decreased in PC12 cells. Conclusion: Apoptosis of PC12 cells was induced by PQ, and this effect could be attenuated by Ginsenoside Rg1. The possible mechanism may be through maintaining MMP, inhibiting Caspase-3 activity and regulating the expression of pro-apoptotic protein protein Cyt C, Bcl-2 and Bax in cytosol.更多还原